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https://openalex.org/W2895967792	https://energyinformatics.springeropen.com/track/pdf/10.1186/s42162-018-0059-6	English	null	Empowering the selection of demand response methods in smart homes: development of a decision support framework	Energy Informatics	2,018	cc-by	15,162	© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Empowering the selection of demand response methods in smart homes: development of a decision support framework Dennis Behrens* , Thorsten Schoormann, Sebastian Bräuer and Ralf Knackstedt * Correspondence: dennis.behrens@ uni-hildesheim.de University of Hildesheim, Universitätsplatz 1, 31141 Hildesheim, Germany Abstract Demand Response (DR) facilitates the monitoring and management of appliances in energy grids by employing methods that, for example, increase the reliability of energy grids and reduce users’ cost. Within energy grids, Smart Home scenarios can be characterized by a unique combination of appliances and user preferences. To increase their impact, a scenario-specific selection of the best performing DR methods is necessary. As the user faces a multitude of heterogeneous DR methods to choose from, a complex decision problem is present. The primary goal of this study is to develop a decision support framework that can determine the best- performing DR methods. Building on literature analyses, expert workshops and expert interviews, we identify seven requirements, derive solution concepts addressing these requirements, and develop the framework by combining the concepts using a benchmarking process as a template. To demonstrate the framework’s applicability, we conduct a simulation study that uses artificial (simulated) data for seven types of households. Within this study, we employ four DR methods, assume changing appliances over time and cost minimization as primary objective. The study indicates, that by using the framework and thus by identifying and using the best DR method for each scenario, the users can achieve further cost benefits. The application of the framework allows practitioners to increase the efficiency of the DR method selection process and to further enhance DR-related benefits, such as cost minimization, load profile flattening, and peak load reduction. Researchers benefit from guidance for benchmarking and evaluating DR methods. Keywords: Demand side management, Demand response simulation, Benchmarking process, Home energy management system Energy Informatics Energy Informatics Behrens et al. Energy Informatics (2018) 1:53 https://doi.org/10.1186/s42162-018-0059-6 Demand response in the residential context Technological improvements, changing environmental conditions, altered consump- tion patterns, and rising pollution levels (e.g., carbon emissions) challenge traditional energy grids (Lawrence et al. 2017). Renewable, but unstable, energy sources like photovoltaic (PV), wind power, and hydro power and new appliances such as electric vehicles (EVs) result in more volatile energy generation and consumption that affect both the supply side and the demand side (Seidel et al. 2013). Critical peaks, Behrens et al. Energy Informatics (2018) 1:53 Page 2 of 28 Page 2 of 28 contingencies, volatile load profiles, unreliable market performance, and inefficient in- frastructure use (Siano 2014) can result in blackouts, brownouts, shortages, and a high spinning reserve and can endanger the reliability of energy grids. The major transfor- mations in the residential context, in particular through the increased use of EVs and decentralized energy generation and energy storage, contributes to this problem be- cause the energy consumed in this sector amounts to more than a third of total con- sumption (Hu and Li 2013; Jovanovic et al. 2016). One possible solution to the challenges in this context is the management of appliances in Smart Homes (Koolen et al. 2017). Appliance management can be carried out by Demand Response (DR). DR optimizes consumption patterns by, for example, using external signals (e.g., pricing signals, direct controlling signals). Optimization is then performed by shifting or managing loads based on incentive-based or price-based programs (Merkert et al. 2015), the latter are often realized through dynamic pricing (e.g., time-of-use pricing (TOUP), critical-peak pricing, real-time pricing (RTP) (Siano 2014; Steen et al. 2012)). DR can also “transform domestic customers from static consumers into active participants” ((Molderink et al. 2010), p., 109), which especially promotes the implementation of decentralized energy generation (e.g., PV, storages) in the grid. Nevertheless, since DR is a complex task and pricing signals may change during the day (cf., RTP), it can hardly be carried out manu- ally by the users without assistance. Especially the reaction on changing price signals would require significant user efforts regarding time and participation as consumption plans need to be adapted on multiple times throughout the day. Therefore, applying DR in the residential context requires supporting infrastructure, including, for instance, a Smart Meter and a Home Energy Management System (HEMS) (e.g. (Chaudhari et al. 2014)). A HEMS is a system that enables DR applications for residential users (Zhao et al. Demand response in the residential context 2013) by monitoring and automatically managing appliances based on a user-specific set of requirements (REQs) (e.g., (Hu and Li 2013; Zhao et al. 2013; Han et al. 2014; Kuzlu et al. 2012)). Thereby, a HEMS can be assigned to a single home or multiple homes, especially in the latter case forming a microgrid (e.g., (Jiang and Fei 2015)). REQs are composed of the user-specific scenarios, including living behaviors, appli- ances, available storage, and energy-generation infrastructures, and consider environ- mental factors like weather, stock prices, and price signals (Fig. 1). To address these REQs, various DR methods can be applied that, for instance, differ regarding their objectives, optimization methods, and communication models (Kosek et al. 2013). Overviews can be found in (Balijepalli et al. 2011; Al-Sumaiti et al. 2014; Gerwig et al. 2015). DR methods are here understood as algorithms that are developed to solve optimization problems in the DR context in light of constraints and desired goals. However, (Khadar et al. 2017) denotes, that no sophisticated comparison of DR method performance is done yet. Moreover, to the best of our knowledge, no approaches have been developed so far that support this comparison and a selection of best performing DR methods for a user-specific scenario. Consequently, the development of a solution is required, that assists with the com- parison of DR methods and with the selection of the best one for a user-specific sce- nario. As HEMSs have heterogeneous structures and scenarios can change as new appliances and infrastructure components are developed, the solution should be Page 3 of 28 Page 3 of 28 Behrens et al. Energy Informatics (2018) 1:53 Fig. 1 Smart Home context adaptable for all kinds of HEMSs. A framework is suitable for this task, since it can provide a reusable structure and it can be adapted to fit individual needs (Johnson and Foote 1988). In this study, we develop a decision support framework that assists in selecting the best DR method for a user-specific scenario. The application of the framework allows practitioners to increase the efficiency of the DR method selection process and to further enhance DR-related benefits, such as cost minimization, load profile flattening, and peak load reduction. Researchers can use the framework to compare the effects of existing DR methods in varying scenarios and thus inform the enhancement of their methods and underlying algorithms. Demand response in the residential context We proceed by describing related work in Section “Related Work” and the chosen re- search design in Section “Research Design”. In the next step, we gather seven REQs based on the literature analyses, expert workshops and expert interviews (Section “Gathering Requirements”), derive solution concepts (SOCs) (Section “Deriving Solu- tion Concepts”), and assemble them into a decision support framework (Section “As- sembling the Framework”). To demonstrate our framework’s applicability, we implement a software prototype and conduct a simulation study with illustrative exam- ples extracted from artificial data that build on real-world scenarios (Section “Demon- stration”). Finally, we discuss our findings and provide an outlook on future research (Section “Conclusion”). Related work Based on an extensive literature analysis (see Appendix 1), we identified a set of contri- butions to the domain of DR methods that include comparisons, benchmarks, and eval- uations of DR methods. Although some of these studies perform benchmarking, most provide evaluations, especially argumentative comparisons of types of methods (e.g., (Sianaki et al. 2010)), proofs that a particular method can find an optimum (e.g., (Lab 2018)), evaluations against the status quo (e.g., (Gerwig et al. 2015)), and descriptions of methods with a variation of different input factors (e.g., (Mohsenian-Rad et al. 2010)). Among these, proving a method’s ability to find an optimum is in many cases not possible since a complex optimization problem is solved with a heuristic or the Behrens et al. Energy Informatics (2018) 1:53 Page 4 of 28 solving process is too complex and time-consuming. Decision support systems are often used in the energy field (e.g., (Sellak et al. 2017)), but we found no suitable system or framework that could meet our research goal and no solution in the extant literature could be discovered. Moreover, most working groups and research projects that address this topic focus on the organizational levels and grid levels and do not compare or evaluate DR methods. For instance, “MOSAIK” (a flexible simulation framework for smart grids (MOSAIK 2018)), uses a co-simulation approach to simulate methods and scenarios for a large energy grid with multiple elements like different consumers, decentralized en- ergy generation and EVs (Schloegl et al. 2015). “DER-CAM” focuses on minimizing costs by optimizing on-site generation and heat and power systems (Lab 2018) in build- ings and microgrids, and bases on an optimization problem with multiple objectives. However, the authors state that the optimization problem must be solved by a heuristic, a greedy-based solving strategy. So even if the method’s ability to find the optimal solu- tion is proven, a solution is often found with a heuristic, and the results differ based on the chosen algorithm. Other projects can be found, such as Smart Grid Algorithm Engineering, Smart Nord, and D-Flex (Blank et al. 2015), have been addressed, but none realizes a way to select a DR method for individual scenarios. Besides the litera- ture search and the analysis of existing projects and research groups, the energy informatics field might offer helpful insights (Goebel et al. 2014). Related work Our research aims at increasing energy efficiency by supporting the user-specific se- lection of DR methods that are used on HEMS in residential buildings (here Smart Homes) (Goebel et al. 2014). Similar research activities fail to compare, evaluate, and benchmark DR methods, although they address, for example, distributed and renewable energy resources (e.g., (Berthold et al. 2017; Dyson et al. 2014)), islanded grids (e.g., (Vergara et al. 2018; Ma and Billanes 2016)) or grids in general (e.g., (Steinbrink et al. 2017)), the energy grid in smart cities (e.g., (Masera et al. 2018; Stoyanova et al. 2017)), security concerns (e.g., (Fulli et al. 2017)), EVs and batteries in general (e.g., (Park et al. 2016; Ma et al. 2010)), energy contracts (e.g., (Basmadjian et al. 2016)), or data and privacy concerns (e.g., (Cupelli et al. 2018)). Leaving the immediate field of DR methods, Ketter et al. (Ketter et al. 2016) present competitive benchmarking, an approach for the comparison of solutions to wicked problems. For demonstration, they select the topic of sustainable energy and financial market stability (Ketter et al. 2016) and the Trading-Agent-Competition (TAC) (Ketter et al. 2011). The TAC, which has several customer models that represent households, businesses, and so forth, takes a supplier perspective, while the demand perspective is “fixed”. However, by fixing the demand perspective, the stated research question is not sufficiently answered, as the focus lies on the Smart Home Context. In this context, the supplier is the black box and delivers information like a cost function or penalty costs for high peaks (O’Connell et al. 2014) Although benchmarking is frequently addressed in the DR field, to the best of our knowledge, the field currently offers no support for decision-making in our context. Focusing on benchmarking procedures in general, that aim at identification of best practices and performance improvement (e.g., (Andersen and Pettersen 1995; Camp 2006)), Lugauer et al. (Lugauer et al. 2012) analyze 35 benchmarking frameworks in terms of the four phases—planning, collecting, analyzing, and improving—based on the Behrens et al. Energy Informatics (2018) 1:53 Page 5 of 28 Page 5 of 28 “plan-do-check-act”-cycle from Deming (Deming 1982). The planning phase is about what to benchmark, and identifying who and which resources are involved. Afterwards, in the collecting phase, benchmarking objects need to be analyzed by defining needed data and measurements. Related work In the analyzing phase, the performance of the benchmarking objects is compared, and gaps between these performances are identified. During the improving phase, necessary actions need to be identified, to move, originally the busi- ness, forward and improve, for example business processes. Phase 1: Gathering Requirements In this phase, REQs are derived from the knowledge base inductively. We build on existing literature (literature analysis) and expert knowledge (expert interviews) and de- rive several REQs based on Deming’s general benchmarking approach (Deming 1982). Research design Our research began with the identification of a practical problem. An industry partner that plans to design and implement a HEMS, asked, what kind of services could be additionally offered to the customers, based on this HEMS. They state the delivery of an optimized energy consumption plan to energy-users to be useful, because users can realize cost benefits. As the HEMS should be used in diverse surroundings, an “opti- mal” DR method could not be chosen easily. Because of the high individuality of user scenarios and the variety of DR methods, a decision-making approach must be imple- mented on these HEMS that supports the ability to select a method that best fits each scenario. To develop the framework, we carried out a staged research design (Fig. 2) that consists of gathering REQs (Section “Gathering Requirements”), suggesting SOCs (Section “Deriving Solution Concepts”), assembling the decision support framework from these SOCs (Section “Assembling the Framework”), and demonstrating the applic- ability of the framework by simulating various scenarios (Section “Demonstration”). In the following, each of these phases is described in detail. Phase 3: Assembling the framework After gathering REQs and deriving SOCs, we used Deming’s (Deming 1982) bench- marking process as guidance and matched each SOC to it. We also analyzed informa- tion flows from both inside and outside the framework and added, for example, event triggers for changes in the scenario. Phase 2: Deriving Solution Concepts We suggested SOCs that address each REQ individually by searching for information in existing research and by interviewing experts. These SOCs are only addressing the REQs, but still must be combined to realize the decision support framework. Fig. 2 Staged research design Page 6 of 28 Behrens et al. Energy Informatics (2018) 1:53 Phase 4: Demonstration For the demonstration, we implemented a software prototype that features four DR methods and a database suitable for conducting simulations. The aim of this research is not to evaluate the exemplarily implemented methods but to show that our framework is feasible and applicable. As the framework should be reusable and adaptable, it is not HEMS-specific and so is (re-)useable on many kinds of systems. We used artificial data that have minor disadvantages but are of sufficient quality (Cao et al. 2013) to conduct meaningful DR simulations. As the simulation is an artificial setting, we impose several events to simulate changes in the scenario (e. g., additional appliances, defects of appli- ances, altered user behavior). Gathering requirements This section presents seven REQs for a decision support framework grounded in gen- eral benchmarking approaches (that follow a plan-collect-analyze-improve cycle (Lugauer et al. 2012)), a literature analysis, a review of several DR methods (e.g., found in (Balijepalli et al. 2011; Al-Sumaiti et al. 2014; Gerwig et al. 2015; Barbato and Capone 2014)), and expert interviews (Behrens et al. 2017). Inspired by the first benchmarking phase (planning phase) (Camp 2006), we define the benchmarking objects and benchmarking goals (Heib et al. 1997). Benchmarking objects reference to the entities to be compared, such as companies, divisions in a com- pany, or even products, processes, and functions (Heib et al. 1997). In our study, these benchmarking objects are DR methods. Benchmarking goals are the objectives that the benchmarking process seeks to achieve, such as improving the energy efficiency of Smart Homes by selecting the best DR method for a certain situation. However, “best” depends on how the term is defined. As it relates to DR (Gellings 1985), “best” can be the method that reduces peak loads and costs or the method that ensures a flattened load profile. These objectives may differ in different homes or over time according to the users’ preferences. Consequently: REQ1—Objective(s) Specification: Provide features that allow users to specify their individual objectives. The benchmarking approach requires collecting suitable data. In addition to the indi- vidual objective, these data comprise the scenario of the user, which can be very hetero- geneous (Hoogsteen et al. 2016) based on, for example, living behaviors, daily routines, existing infrastructure, and appliances (Pflugradt 2017). Various data sources, such as recorded data, simulated data, and predicted data, can be used (e. g., (Monacchi et al. 2014; Behrens et al. 2014; Behrens et al. 2016)). To identify the best method for an Behrens et al. Energy Informatics (2018) 1:53 Page 7 of 28 Page 7 of 28 individual scenario, this specific scenario needs to be represented first—regardless which data is chosen (e.g., forecasting data, past data, manual data from the user). DR can be divided into two phases: data acquisition (often broken down to forecasting) and optimization (Simmhan et al. 2011) (Fig. 3). User-specific data (e.g., deadlines for finishing appliances and times during which the user cannot run appliances) must be added in or after the first phase and used in the second. Gathering requirements We need an adjustment between these two phases, for example, to identify the pos- sible using intervals of appliances (e.g., (Behrens et al. 2017)). This can be done auto- matically (e.g., by predicting user’s behavior and needs) or manually by the user. Consequently: REQ2—Scenario Building: Provide features that allow users to build individual scenarios. REQ2—Scenario Building: Provide features that allow users to build individual scenarios. Another part of collecting data is to ensure its quality, which has been identified as a problem in DR research (e.g., (Cao et al. 2013; Barta et al. 2014)). Data on energy use is often recorded in heterogeneous ways, so the data structures are not standardized and information (e.g., whether an appliance is deferrable) may be missing (Behrens et al. 2016). Moreover, gaps in the data (Monacchi et al. 2014; Barta et al. 2014), such as missing recordings and zero values that are often caused by a malfunction of the Smart Meters, occur (Hoogsteen et al. 2016). Regardless of which data is chosen, it must be in a suitable format and of high quality (Cao et al. 2013). High quality thereby means, that needed information are given, the recording frequency is adequate, and missing values can either be predicted or filled without falsifying the data. Thus, data quality needs to be guaranteed through a suitable preprocessing (cf., (Barta et al. 2014)). Consequently: REQ3—Data Preprocessing: Provide features that allow the quality of the data to be ensured. The second DR phase deals with optimization by DR methods and therefore delivers data for the analysis (third benchmarking phase). However, not all DR methods have directly comparable characteristics and therefore additional demarcation is needed. Dif- ferent characteristics, regarding their functionality and the considered appliances, infra- structure and constraints (Kosek et al. 2013; Gerwig et al. 2015; Behrens et al. 2017) need to be addressed. Benchmarking must guarantee that the only methods that are compared are those that, for example, fulfill the same constraints (Behrens et al. 2017). The methods themselves and the additional information need to be stored to enable the HEMS accessing them. Consequently: Fig. 3 DR phases, data sources, and scenario data Fig. 3 DR phases, data sources, and scenario data Page 8 of 28 Page 8 of 28 Behrens et al. Energy Informatics (2018) 1:53 REQ4—Method Comparability: Provide features that ensure the comparability of DR methods. The calculation of assessment criteria is the last step of the data-collection phase. Assessment criteria like measures of energy consumption and savings must be calcu- lated and analyzed (Gerwig et al. 2015). However, many possible criteria are offered in the DR field (e.g., (Balijepalli et al. 2011; Al-Sumaiti et al. 2014)), so the appropriate choices must be made. Hence, these criteria must be identified in a first step. REQ5—Result Calculation: Provide features that allow DR methods to be assessed based on established criteria. REQ5—Result Calculation: Provide features that allow DR methods to be assessed based on established criteria. Analysis, the third benchmarking phase, identifies best practices and performance gaps to enable improvement (e.g., (Lugauer et al. 2012; American Productivity and Quality Center 1993)). In the DR context, this means we need a way to identify the best DR method to enable a decision making. “Best” in this case depends heavily on the user’s objectives (REQ1) and the user-specific scenario (REQ2). Based on this “objecti- ve-function” identification, the calculated assessment criteria (REQ6) are interpreted. As we have multiple criteria, we must add a suitable decision making process (e.g., (Ho et al. 2010; de Boer et al. 2001)). Multiple criteria are difficult to handle because their units of measure may differ, their levels of importance may differ, and so on. Conse- quently, the suitable decision making must be supported by identifying the best DR method, which can be selected by the user and then be deployed on the HEMS. Consequently: REQ6—Decision Support: Provide features that allow the best DR method for a specified scenario to be identified. REQ2—Scenario Building: Provide features that allow users to build individual scenarios. Based on these definitions and after the methods calculated their results for a certain scenario, the assessment criteria need to be calculated. For ensuring a uniform calculation of the criteria’s measurements and comparability, we need a centralized computation, as the methods might have a different calculation or might even miss the calculation of single measurements. The DR methods must transfer the results in a suitable format to our framework, which—afterwards—calculates all assessment criteria for all comparable methods. Consequently: Deriving solution concepts Deriving solution concepts We suggest possible SOCs to address the REQs, which are derived inductively from a literature review and interviews with experts. SOC1—Let the user select the objectives to be achieved The user should be presented a variety of options so he or she can choose which objec- tives are most important. Our literature search identified several DR objectives, shown in Table 1, each of which can be reached using DR methods and can be provided for decision-making. The user can select the objective(s) to be reached (Fig. 7 in Appendix 2). As the “welfare” objective is difficult to measure but important for users (cf., (Jovanovic et al. 2016)), we consider it a given. The user can state his or her preferences based on usage patterns during scenario-building (REQ2) or select an automated way to predict preferences, for example, via forecasting based on historical data. REQ6—Decision Support: Provide features that allow the best DR method for a specified scenario to be identified. Improving is the last step of the benchmarking approach that especially in the DR field is needed if changes occur that may impact the effectiveness of the DR method chosen. Changes can include the addition of a new appliance or a new operating status (“Appliance Event”) (cf., (Bui et al. 2017)) or changes in the objectives (“Objective Event”). These changes can occur because of unexpected malfunctions or use patterns or insufficient forecasts. A monitoring and a suitable reaction mechanism are needed to facilitate the ability to react to such events. Consequently, the selection needs to be repeated and is a continuous task, as also stated in the benchmarking phase of (Lugauer et al. 2012). We need a starting point for the “Initialization”, as no data is set before. The last requirement therefore faces the challenge of reacting on events to realize the most beneficial method selection. Consequently: Page 9 of 28 Page 9 of 28 Behrens et al. Energy Informatics (2018) 1:53 REQ7—Event Reaction: Provide features that allow users to react to events. REQ7—Event Reaction: Provide features that allow users to react to events. SOC2—Provide energy consumption data and let the users specify their energy consumption profile The framework should provide several options for the user with which to select their energy composition: a predefined dataset (e.g., the last day or similar households), where only single loads are adapted to the user’s habits (e.g., an EV is added or the use intervals are changed); an empty dataset to build a scenario and add all appliances, plus additional information “bottom up”; past data recorded from the HEMS; or forecasted data. This step might be done manually (by the user) or automatically when the user gives a starting point and the framework then uses forecasting data. As privacy and data security are important topics (for example due to the general data protection regu- lation), data must not leave the HEMS. Based on the data provider, the user-specific scenario can be built (e.g., (Hoogsteen et al. 2016)). Besides selecting the data, additional user-specific information regarding the user’s welfare is needed, but defining the user’s wel- fare is not easy, so a utility function is often used in the DR context (e.g., (Li et al. 2012; Table 1 DR objectives derived from the literature # DR objective Source (e.g.) Ob1 Cost minimization for a (single) participant (savings in electricity bills) or even market-wide (e.g., through more effi- cient use of the infrastructure). (Balijepalli et al. 2011; Albadi and El-Saadany 2008; Aghaei and Alizadeh 2013; Abdollahi et al. 2012; Gkatzikis et al. 2013) Ob2 Minimization of peak loads in the consumption profile, to reduce the need of high spinning reserves, etc. (Balijepalli et al. 2011; Conejo et al. 2010; Raza et al. 2013; Gyamfi et al. 2010) Ob3 Load profile flattening by shifting demand, thereby reducing the cost of producing electrical energy. (Conejo et al. 2010; Raza et al. 2013) Ob4 Ensuring reliability to reduce the risk of outages and ensure the ability to react to contingencies by holding a spinning reserve. (Abdollahi et al. 2012; Conejo et al. 2010; Raza et al. 2013; Zakariazadeh et al. 2014; Wang et al. 2013) Ob5 Maximization of market performance by allowing consumers to affect the market, thereby reducing price volatility in the spot market. (Albadi and El-Saadany 2008) Ob6 Maximization of users’ utility (often also called welfare), to maximize the users comfort and preferences. (Conejo et al. 2010) Ob7 Efficiency of infrastructure usage to save external kWh. (Balijepalli et al. 2011; Aghaei and Alizadeh 2013; Raza et al. SOC4—Add metadata to the methods and form groups Suitable metadata for DR methods must be identified in terms of its classification and characteristics. DR methods can be classified by two dimensions: Place of decision-making and used communication (Kosek et al. 2013). The loads addressed and constraints met must also be determined, as, for example, methods that do not con- sider the same constraints cannot be compared (c.f., (Behrens et al. 2017)). Every method must be comparable with other methods from the same “class” and suitable for the scenario. Therefore, we add additional data (metadata) based on the DR method’s classification (Kosek et al. 2013), the compliances addressed and the constraints consid- ered (Behrens et al. 2017). The methods themselves must be available in a repository, either locally on the HEMS or in the cloud. SOC2—Provide energy consumption data and let the users specify their energy consumption profile 2013) Table 1 DR objectives derived from the literature Ob7 Efficiency of infrastructure usage to save external kWh. Behrens et al. Energy Informatics (2018) 1:53 Page 10 of 28 Samadi et al. 2012; Mas-Colell et al. 1995)), for example known from the microeconomics context. A questionnaire can be used to derive a utility function (Jovanovic et al. 2016). However, no standard has been defined for measuring the user’s welfare in a real use-case yet, so we let the user define the times when an appliance may run (e.g., the EV must be charged in the morning or the washing machine must be finished by 6:00 p.m.) (e.g., (Jova- novic et al. 2016)). SOC3—Sample the data, fill gaps, and reject unreliable data To provide appropriate data, gaps and data-quality issues (e.g., time resolution) must be addressed. Most DR methods use a fifteen- or thirty-minute cycle to optimize load positioning (Abdulla et al. 2017), as cycles of less than 1 minute or continuous record- ings are not required (and are difficult to achieve). We suggest a down-sampling with a frequency of 1 minute (e.g., (Hoogsteen et al. 2016) or (Palensky and Dietrich 2011)) for 1440 recording points over 24 h, which is suitable for most DR methods. The gaps can be divided into two dimensions: the number of gaps for a certain day and the num- ber of (completely) missing days in the data. These gaps can be filled by writing in zero values or approximating the missing values (Cao et al. 2013). However, if too many values are missing to interpolate these values or by filling with zero values the data would be too much impure, the data is insufficient for the next steps and sorted out. SOC5—Establish measurements and provide needed data To provide the indicators, measurement information, and DR objective, assignments needed to rate the methods, we analyzed literature reviews on several DR methods (found in the literature reviews of (Balijepalli et al. 2011; Al-Sumaiti et al. 2014; Gerwig et al. 2015)). The criteria we derived with which to rate the methods consisted of one or more DR objective, an indicator, and a measurement unit (Table 2). Additional infor- mation, such as a cost function, is also needed. Energy consumption is needed and must be transferred in a suitable format to the framework to calculate the indicator, so an interface must be designed. Other information, such as PV or battery use, could also be required. Our literature analysis showed that one indicator can represent multiple objectives, so we enabled the user to state criteria for each stated objective and support a “default criteria” (underlined objective in Table 2). Page 11 of 28 Page 11 of 28 Behrens et al. Energy Informatics (2018) 1:53 Table 2 Assessment criteria derived from literature (underlined objective number = default) Table 2 Assessment criteria derived from literature (underlined objective number = default) Indicator and description Unit Source (e.g.) Objective Peak-to-Average-Ratio (PAR): Highest peak from the average (positive or negative) over the day. [kWh] (Soliman and Leon-Garcia 2014; Song et al. 2014; Fathi and Gholami 2012; Verschae et al. 2014) Ob2, Ob4, Ob5 Mean-Squared-Error (MSE): The arithmetic middle of the squared error during the day; often also indicted as Root- Mean-Squared-Error (RMSE). [kWh2] (Verschae et al. 2014; Javed et al. 2012) Ob3, Ob4, Ob5 Normalized MSE (NMSE): The normalized MSE regarding the status quo (no DR); often used to quantify consumption- flattening. [kWh] (Verschae et al. 2014) Ob3, Ob4, Ob5 Costs: How much has to be paid? Cost function or pricing scheme needed. [€],[$] (Atabay et al. 2013; Ali et al. 2012; Alam et al. 2013) Ob1 Savings: How much savings can be realized compared to no DR in either percentage or money? [%] or [€] (Maqbool et al. 2012; Bassamzadeh et al. 2014) Ob1 Infrastructure use: How good is the infrastructure used? That is, how much of the generated energy is used to reduce external kWh? [kWh] (Mohamed et al. 2012; Beaude et al. 2012; Bashash and Fathy 2013; Keerthisinghe et al. 2014) Ob7 Utility: Is the user’s utility increased by using DR? No unit or standardization was found here. [/] (Behrens et al. SOC6—Identify the best method(s) based on user objectives and scenarios To support decision-making, we rank the DR methods based on their results (SOC5) and the stated user objectives (SOC1). As we may have multiple criteria and objectives, To support decision making, we rank the DR methods based on their results (SOC5) and the stated user objectives (SOC1). As we may have multiple criteria and objectives, we match a multi-objective function with multiple criteria. Additional difficulties occur because individual objectives might be weighted differently when their importance to the users differs. Hence, three cases are derived: only a single objective is selected, mul- tiple objectives with different weights are selected, and multiple objectives are selected but no weights are given. To make the results comparable, a single number from the criteria (e.g., (Charnes et al. 1978; Cooper et al. 2006)) using one of three strategies is formed: If the user selects only a single objective, since we enable only one measure for each objective, we can directly compare the criteria with each other. If the user selects two or more objectives and weights for each objective, a combined value with the formula ∑Criteriai ∗Weighti for each DR method can be calculated. If the user selects two or more objectives but no weights, equal weights are supposed and calculate the comparative value as in the second strategy. SOC5—Establish measurements and provide needed data 2016; Alam et al. 2013; Yang et al. 2012) Ob6 Table 2 Assessment criteria derived from literature (underlined objective number = default) Indicator and description Unit Source (e.g.) Objectiv SOC7—React to events by repeating the decision support process Reacting to events—here, “Appliance Events” and “Objective Events”—requires two ac- tions: implementing a monitoring strategy and implementing a suitable reaction strat- egy. The monitoring determines whether changes, such as a new appliance being added or a change in the infrastructure (e.g., because of a malfunction) occurred. The frame- work repeats the selection process if the user changes the objectives (“Objective Event”) but the process can jump to data collection phase and rebuild the database if the event is an “Appliance Event.” When the process is run for the first time, an “Initialization” is con- ducted, meaning that the process starts at the beginning with the user stating his or her ob- jective(s). In the monitoring phase, while the DR method is used, the framework receives Behrens et al. Energy Informatics (2018) 1:53 Page 12 of 28 event triggers and additional information from the HEMS. After receiving such an event, a specific phase is called and the information (e.g., the load profile of the new appliance) is handed over. Assembling the framework The next step in developing the decision support framework is to assemble and embed the SOCs to the framework and into the HEMS and into the environment. Next, we describe how we assembled the framework and how the framework interacts with the HEMS and the environment. Assemble the Solution Concepts The REQs and SOCs are inspired by the benchmarking phases of (Lugauer et al. 2012), so the framework is divided into four phases (see Fig. 4 for a visualization of the process and Table 3 for an overview of the REQs and SOCs): The first phase, Planning, involves SOC1, the specification of the objective. The second phase, Demand Response, involves collecting data, including building the scenario (SOC2), preparing the data (SOC3), ensuring the comparability of methods (SOC4), and the calculating the mea- sures (SOC5). We divided this second phase into two sub-phases: data acquisition (SOC2 and SOC3) and optimization of the consumption plan (SOC4 and SOC5). The third phase, Decision and Deployment, identifies the best DR method based on the col- lected data, stated objectives, and so forth (SOC6), which is then deployed on the HEMS. In the last phase, Monitoring, the framework reacts to events that occur during the DR method’s use. If an event occurs, the framework guides either the HEMS (auto- mated) or the user (manual) back to the planning phase or the demand response phase (SOC7). When the decision process is first run, it begins with SOC1 (objective specification). Fig. 4 Decision support framework for DR methods with the SOCs addressed Page 13 of 28 Page 13 of 28 Behrens et al. Energy Informatics (2018) 1:53 Table 3 Derived REQs and SOCs (overview) #REQ Name #SOC Solution concept Framework phase Benchmarking phase REQ1 Objective(s) Specification SOC1 Let the user decide the objectives to be achieved. Planning Plan REQ2 Scenario-Building SOC2 Provide data and let the users select their energy composition. Demand Response Collect REQ3 Data Preprocessing SOC3 Sample the data, fill gaps, and reject unreliable data. Demand Response Collect REQ4 Method Comparability SOC4 Add metadata to the methods and form groups. Demand Response Collect REQ5 Result Calculation SOC5 Establish measurement and provide needed data. Demand Response Collect REQ6 Decision Support SOC6 Identify the best method(s), based on user objectives and scenarios. Decision and Deployment Analyze REQ7 Event Reaction SOC7 React to events by repeating the decision support process. Monitoring Improve Embed the Framework The framework requires inputs from the HEMS, the users, and the environment, so the framework must interact with those three entities. Both the inputs and the interactions are specified for each phase of the framework. In the Planning phase, users state their objectives, potentially on the HEMS’ (graphical) user interface (e.g., (Han et al. 2014; Khadar et al. 2017; Yener et al. 2017)), as this interface is a suitable way to enable the user to transport his or her objectives into the framework. This phase occurs either on the initial use of the DR method or when an “Objective Event” occurs. In the Demand Response phase, the framework provides suitable data to the user for the purpose of scenario-building, again, potentially using the HEMS’ interface, and the user specifies individual appliances, intervals, etc. The HEMS can transfer these data to the frame- work. Even if an automated process of scenario-building (e.g., through forecasting) is chosen, the user can monitor and alter the scenario. In the third phase, Decision and Deployment, the framework shows the ranked DR methods in the HEMS’ interface based on their results and the users’ stated preferences. Then either the user chooses his or her favored method, or the framework performs this choice automatically. The method is then deployed on the HEMS. Finally, in the Monitoring phase, the deployed method is used on the HEMS, and any change events that occur are reported to the framework, as well as additional information (e.g., new objective given, new appliance installed). If an “Objective Event” occurs, for example because the user changes the ob- jectives in the HEMS’ interface, these altered objective(s) need to be reported to the framework. If an “Appliance Event” occurs, the altered appliance(s) need to be re- ported, for example the consumption profile of the (new) appliance. The framework then continues with the decision process and delivers the new information. Demonstration case To build a suitable foundation for this test case, appropriate data must be selected and prepared (SOC2 and SOC3). At the beginning, we used recorded data from naturalistic homes (analyzed, for example, in (Monacchi et al. 2014; Behrens et al. 2016)), which cover existing scenarios of real users, so no assumptions needed to be made (cf. (Barta et al. 2014)). However, we had to add information about, for example, the time of use of certain appliances, so we added artificial data using the LoadProfileGenerator (LPG) (Pflu- gradt 2017) to ensure sufficient data quality (cf., (Cao et al. 2013; Hoogsteen et al. 2016)). LPG has several predefined load profiles and uses a behavior model to simulate the data. We used seven predefined households from the LPG as a demonstration case. House- holds’ inhabitants differ in terms of their ages, jobs, habits, and daily routines. Conse- quently, the consumption amounts and patterns in these households differ. Instantiation of framework We implemented the software prototype in Java so we could use common libraries to visualize the results or read out data. At the beginning of the decision process, the user must select the objectives to be achieved (SOC1). In our demonstration case, we use cost minimization, represented by savings, as measurement (see Fig. 7 in Appendix 2). As for the user-specific data for the selection process (SOC2), we use artificial data, for example, for use intervals. We use a TOUP, and as no storage or generation infrastructure is in the demon- stration scenario, these data are not added (see Fig. 8 in Appendix 2). The data itself must be in a suitable format (SOC3), so we choose a 15-minute interval to match the proposed DR methods. As the data are simulated, there are no gaps. Each DR method has a meta file that provides additional metadata (SOC4). Once optimized, the resulting consumption plan is sent to the framework. Next, the indicators are calculated and written into an output file for documentation, and the measurements are calculated with the information given (SOC5). With these criteria and based on the objectives from SOC1, a ranked list of methods is cre- ated (SOC6), and the results are displayed (see Fig. 9 in Appendix 2). The selected method is deployed on the HEMS (see Fig. 10 in Appendix 2). Afterwards, the framework switches to the monitoring phase (SOC7), where two events occur, and the analysis is repeated. Demonstration To demonstrate the framework’s feasibility and applicability, we conducted several sim- ulations. We instantiated the SOCs (Section “Instantiation of Framework”) and de- signed an illustrative scenario (Section “Demonstration Case”). The instantiation consists of the framework itself (visualized with a graphical user interface, simulating the HEMS interface) and four DR methods, each of which follows the same optimization problem and fulfills the same constraints (for the description of the Behrens et al. Energy Informatics (2018) 1:53 Page 14 of 28 optimization problem and the constraints, see Appendix 4). We exemplarily imple- mented two solving strategies with two variations per strategy: greedy-based (min and max) (Sianaki et al. 2010) and multi-agent-system-based (with and without communi- cation) (Mohsenian-Rad et al. 2010) (for additional information see Appendix 3). We have chosen an artificial setting for the demonstration scenario. Consequently, events are triggered manually and the users’ objective(s), appliances, and so on are simu- lated. To consider multiple homes and the effects of a method selection, we simulated a microgrid consisting of several homes. We included different home configurations (e.g., varying appliances, usage patterns, etc.). To simulate changes, we introduce two appli- ances events, namely the consideration of an EV with regular, domestic (AC) charging and furthermore, the installation of corresponding fast-charging infrastructure (DC). Basic scenario details We built the following scenario (SOC2) of seven homes: Page 15 of 28 Behrens et al. Energy Informatics (2018) 1:53 1. A couple (male, age 40; female, age 35), both of whom work outside the home, with three children (male, age 13; male, age 6; female, age 4), requires 4001.24 kWh throughout the year (min (day): 0.16 watts, max (day): 8914.97watts). 2. A working woman (age 30) with two children (males, ages 11 and 7) requires 3277.88 kWh throughout the year (min: 0.16 watts, max: 7364.95 watts). 3. A multigenerational home with a working couple (male, age 40; female, age 32), two children (female, age 15; male, age 4), and two seniors (male, age 70; female age 68) requires 8279.49 kWh throughout the year (min: 0.50 watts, max: 13,624.86 watts). 4. A working woman, age 25, requires 1778.51 kWh throughout the year (min: 0.16 watts, max: 8644.84 watts). 5. A working man, age 26, requires 1512.73 kWh throughout the year (min: 0.16 watts, max: 7208.11 watts). 6. A working couple (male, age 25; female, age 23) requires 2448.96 kWh throughout the year (min: 0.16 watts, max: 6370.82 watts). 7. A couple (working man, age 45; female homemaker, age 43) with two children (male, age 20; female, age 14) requires 4112.76 kWh throughout the year (min: 0.16 watts, max: 10,643.47 watts). Deferrable appliances within the homes are washing machines, dryers, and dishwashers. The duration, time, and quantity of appliances, which depend on the life styles and behavior of the residents, are provided. We added the following information, that are visualized in Fig. 5: The deferrable appliances may not run between 12 a.m. and 7 a.m. The working residents leave at 7 a.m. and return at 6 p.m., thus an EV cannot be charged in between. Our test case uses a TOUP. The price from 0:00–06:00 and 22:00–24:00 is €0.24 per kWh, and the price from 06:00–22:00 is €0.36 per kWh. Scenario variations Events can call for adjustments to the chosen DR method. For initialization, the user first states objectives to be achieved. In our scenario, we choose cost minimization as the only goal, which means that we have just one objective. Measuring this, the savings are considered as criteria—this means, how much cost (in percent) from not perform- ing DR can be saved. In the second step, we assemble the scenario details, which mean the user adds the appliances and usage patterns (e.g., intervals) to the scenario. In the demonstration case the predefined consumption plans are used. After the implemented DR methods calculated an optimized consumption plan, a ranked order is derived, and the users deploy the favored method on the HEMS. As a starting point, we assume that our users have no EVs. Later, they will buy an EV (we assume a Volkswagen e-Up) and charge it with 3.6 kW (AC) (Appliance Event 1). The alteration is monitored by the HEMS. If changes occur in a Smart Home or microgrid (here events, see for example (Bui et al. 2017)) the scenario needs to be re-analyzed, ensuring that still the best method is selected. Correspondingly, the decision process starts from the second phase and the scenario must be adjusted. Later on, a fast charging station is installed Page 16 of 28 Behrens et al. Energy Informatics (2018) 1:53 Fig. 5 Summarized consumption of all households, EV charging times, and TOUP borders Fig. 5 Summarized consumption of all households, EV charging times, and TOUP borders (Appliance Event 2). As a result, we have three variations of our scenario, caused by two Appliance Events (see Table 4 for a summarized scenario description). Table 4 Scenario Description # Description Basic scenario details Homes: (1) working couple, 3 children; (2) working woman, 2 children; (3) multigenerational family (working couple, 2 children, 2 seniors); (4) working woman under age 30; (5) working man under age 30; (6) working under age 30; (7) couple with 2 children, man at work. Appliances: washing machine (1, 2, 3, 4, 5, 6, 7); dryer (1, 7); dishwasher (1, 2, 3, 7). Cost function: We assume that we have a TOUP from the supplier. Infrastructure: No generation or storage data were added. Additional Information: No appliances may run between 24:00 and 07:00; an EV is added to each household and regarded as a deferrable load; the residents leave at 07:00 and return at 18:00, so the EV cannot be charged between these times. Variation 1 No EV Variation 2 (Event 1) EV (Volkswagen e-Up) with 3.6 kW charging (AC) Variation 3 (Event 2) EV with fast-charging infrastructure (DC) Results of the demonstration case After we built the demonstration case, the implemented DR methods calculate the new consumption plans and transfer them to the framework. The measurements are calculated, and the best method is identified based on the selected objective, in this case, cost savings (see Fig. 6). The results indicate that the performances of the particular DR method depends on the scenario. Without an EV, the best methods are the two greedy methods. This changes, if an EV is considered, as this shifts load to the morning hours which flattens the load profile. At this point, the greedy (max) is most beneficial, but after the households switch to DC fast char- ging, the greedy (min) generates the most savings. Each event (integrating EVs and changing the charging mode) requires adjustments in the grid; if the methods were not re-selected after the events occurred, we would have a loss of 3.1% (event 1) respectively 0.7% (event 2) in savings. The overall savings for performing DR at all are much higher (up to 34.2%). Therefore, we generated additional savings of about €1.00 each day just by Table 4 Scenario Description # Description Basic scenario details Homes: (1) working couple, 3 children; (2) working woman, 2 children; (3) multigenerational family (working couple, 2 children, 2 seniors); (4) working woman under age 30; (5) working man under age 30; (6) working under age 30; (7) couple with 2 children, man at work. Appliances: washing machine (1, 2, 3, 4, 5, 6, 7); dryer (1, 7); dishwasher (1, 2, 3, 7). Cost function: We assume that we have a TOUP from the supplier. Infrastructure: No generation or storage data were added. Additional Information: No appliances may run between 24:00 and 07:00; an EV is added to each household and regarded as a deferrable load; the residents leave at 07:00 and return at 18:00, so the EV cannot be charged between these times. Variation 1 No EV Variation 2 (Event 1) EV (Volkswagen e-Up) with 3.6 kW charging (AC) Variation 3 (Event 2) EV with fast-charging infrastructure (DC) Page 17 of 28 Behrens et al. Energy Informatics (2018) 1:53 selecting the best method over the second best. Comparing the best to the worst methods, we save 1.6% in scenario 1, 14.2% in scenario 2, and 0.8% in scenario 3. Results of the demonstration case While these savings may not sound significant, they are realized on top of the savings created by using the “wrong” DR method and just because we re-selected the method with our framework (see Appendix 5 for more detailed results). Conclusion This study focusses on the Smart Home context, which has several challenges. To address these challenges, we gathered a set of REQs for a decision support framework for DR methods in the residential context, derived SOCs to fulfill the Fig. 6 Results of method benchmarking. Without EV (top), EV charged with 3.6 kW (middle), and EV charged with DC (bottom). The best method for each is highlighted (in light gray). Values = Savings [%] of each method. (For more detailed results see Appendix 5) Fig. 6 Results of method benchmarking. Without EV (top), EV charged with 3.6 kW (middle), and EV charged with DC (bottom). The best method for each is highlighted (in light gray). Values = Savings [%] of each method. (For more detailed results see Appendix 5) Fig. 6 Results of method benchmarking. Without EV (top), EV charged with 3.6 kW (middle), and EV charged with DC (bottom). The best method for each is highlighted (in light gray). Values = Savings [%] of each method. (For more detailed results see Appendix 5) Behrens et al. Energy Informatics (2018) 1:53 Page 18 of 28 REQs, and assembled them into a framework. To demonstrate the framework’s feasibility and applicability of this framework, we implemented a software proto- type and conducted simulations with different scenarios. The results indicate that our framework supports the identification of the best DR method for a user-specific scenario and thus allows a decision making. The developed frame- work thereby aims at increasing the efficiency of DR method usage in Smart Homes and thus allows to realize DR related objectives such as improving grid reliability, minimizing energy costs, and reducing peak loads. To do so, the deci- sion process is structured to enable a systematic and scenario-specific selection. To support this decision process, the framework contains valuable advice for (technical) implementation(s) on different HEMS, derived from literature and experts. From a research perspective, our results can be used for supporting the manage- ment of complex energy structures in changing scenarios and for investigations of whether such a framework can increase user acceptance by encouraging more sus- tainable behavior and consumption. Moreover, researchers can compare DR methods in specific scenarios (e.g., to compare methods they develop to other methods). Making an evaluation with benchmarking datasets possible increases the comparability of methods. Conclusion For practice, the integration of the framework in a HEMS can support the management of increasing complexity in the Smart Home context and in urban infrastructures (e.g., Smart Cities). Consequently, new appliances and new infrastructure can be used more ef- ficiently; for example, a PV panel can be combined with energy storage when the best method can be selected more efficiently. Moreover, user acceptance may increase, as the user can see his or her advantages by performing DR as well as choosing the best per- forming DR method and that his or her preferences (welfare) are considered. The demonstration case aims at showing the frameworks feasibility and applicability in answering the research question by identifying the best DR method for a specific situation. It thereby covers only a limited choice of all possible scenario specifications. Other microgrid consumptions, cost functions, and infrastructures would deliver other DR method results. However, the decision process remains the same and will also sup- port the selection of the best performing DR method. Moreover, as intelligent energy management systems, especially HEMS, have not seen a wide implementation, a demonstration and evaluation with real-world data was not possible. Consequently, as the rarely available (real) data lacks in addition on informa- tion, we relied on artificial data and their corresponding limitations such as insufficient event integration, abrupt changes in user behavior, etc. The developed framework focusses on the residential context. Other contexts, discussed in the literature, are industrial and commercial contexts (Gellings and Chamberlin 1987). Future research can apply our framework to these contexts to investigate its applicability there. For example, available DR methods will differ, appliances might have other restrictions and available infrastructure is more di- verse. In general, we assume that our approach is capable of supporting a deci- sion support in other contexts as well. Most aspects of these contexts are similar to the residential context, such as objectives and criteria. However, changes to the REQs might be necessary. For example, other constraints or variations like Behrens et al. Energy Informatics (2018) 1:53 Page 19 of 28 Page 19 of 28 Page 19 of 28 deadlines (e.g., production deadlines) would have to be taken into account in a more detailed way (e.g., because the supply chain needs more precise planning or because machines can switch among multiple modes (Behrens et al. 2017)). Conclusion Moreover, pricing schemes might differ from the residential context, and flexibil- ity might alter, as machines react differently to changes compared to a private user. Real data lack on additional information, for example usage times, deadline of single appliances, etc. Since recording the additional information required from the user is difficult, we plan to design and implement a tool that enables users to transfer user-specific data, such as behavior patterns and preferences. To increase user acceptance and penetration, consumers who have no knowledge in the field should also use the framework, supported by an assistant, guiding the user. Our approach can be combined with the TAC, from (Ketter et al. 2011). Ket- ter et al. (Ketter et al. 2016) called the entire field a “wicked problem.” As the TAC is a “system within a system,” the users in the TAC might change their strategy when they have such benchmarking as decision support. Therefore, the users react to external factors like price signals as well as events. A challenge, then, would be to implement agents not only on the supplier side but also on the consumer side to reach the best result for both sides or to deal with decision-making on the supplier side. Agents on the supplier side would then benefit from our framework and the improved decision making (selecting the best performing DR method). DR objectives are manifold and differ on indicators and dimensions. The con- sideration of multiple indicators with various dimensions and no predefined weightings thereby can impede the decision process. One method that can be used in this case (multiple indicators, various dimensions, no predefined weights) is the Data-Envelopment Analysis (e.g., (Charnes et al. 1978; Vine et al. 1994; Suganthi and Samuel 2012)). So far, our framework can only consider a single ob- jective respectively indicator or multiple ones, if the user defines weights or there are equal weights for each objective. It seems possible to consider efficiency as well, so not only the quality of a method’s result can be considered but also the effort needed to achieve these results. Appendix 1 To identify current approaches for supporting the selection of DR methods we started with conducting a cross-database literature search with Google Scholar, IEEE Xplorer and AISeL, based on the methodology of (Vom Brocke et al. 2009). Afterwards, existing re- search projects and research groups are analyzed as well as the energy informatics field and nearby research contexts. Used search items for conducting the first literature search were: “Demand Side Management”, “Demand Response”, “Evaluation”, “Comparison”, “Benchmark” and “Selection”. Demand-Side-Management is included, because it is often used as synonym to DR (Barbato and Capone 2014), even it is the superordinate concept and thus includes DR. We further included evaluation, comparison and benchmark as search items because we discovered during the iterative search that these items are rele- vant to the DR method selection process. Page 20 of 28 Page 20 of 28 Behrens et al. Energy Informatics (2018) 1:53 Appendix 2 Fig. 7 Defining the objectives (schematic drawing) Appendix 3 Table 5 Description of the chosen methods Classification Method Procedure Direct Control Max Greedy Start Take maximum/minimum load 02 Search the cheapest place for the load 03 Calculate consumption – generation Direct Control Min Greedy 04 Find lowest result 05 Place the load at this place End Take the next load and start again Transactional Control MAS with comm. Start Select agent and hand over consumption plan 02 Agent places its loads (Max Knapsack) 03 Agent commits overall consumption plan End Select next agent Autonomous Control MAS without comm. All agents place their loads simultaneously Agents commit individual consumption plans All four methods use the same optimization model, with an objective function aiming at cost reduction, as this was selected by the user in our demonstration scenario. Both greedy based methods are classified as direct control. This means, the centralized HEMS is gathering the data from all seven households and shifting the loads afterwards by sending a controlling signal. However, the data is not leaving the HEMS and the utility company does not receive any user-specific data. MAS with communication means, that each household has an agent, which monitors and controls its own appli- ances. Each agent can be considered as a HEMS. Consolidation is then achieved by communicating the summarized load profiles to the other agents. As the agents are communicating with each other, this might result in less created peaks. Contrary to this approach, the MAS without communication do not communicate with the other agents. Therefore, artificial and new peaks might be derived, because all agents or HEMS shift their load to the same time. Appendix 2 Appendix 2 Fig. 8 Composition of a user-specific scenario (schematic drawing) Page 21 of 28 Page 21 of 28 Behrens et al. Energy Informatics (2018) 1:53 Fig. 9 Analysis of the results (schematic drawing) Fig. 10 Monitoring and event reaction (schematic drawing) Fig. 10 Monitoring and event reaction (schematic drawing) Page 22 of 28 Behrens et al. Energy Informatics (2018) 1:53 Appendix 4 Based on (Behrens et al. 2017; Behrens et al. 2014) we derived a mathematical model. Let N be all considered living units and An be all the appliances of living unit n ∈N and ω be the sample rate of the discrete model (number of time periods) over one day. Moreover, let xh ¼ P n∈N X a∈An hh n;a with h∈Z ¼ f0; 1; …; ωg be the sum of all appliances a ∈An of all living unit n ∈N in the timeslot h. a ∈An of all living unit n ∈N in the timeslot h. Let lk n;a be the load profile in a local time interval k∈T l ¼ f0; 1; ::; δn;ag; ln;a ¼ X Let lk n;a be the load profile in a local time interval k∈T l ¼ f0; 1; ::; δn;ag; ln;a ¼ X k∈T l lk n;a the load sum and δn, a the length of load a ∈An. In doing so, we can transform a given horizontal and inseparable load profile from its local time interval Tl to the global one T (C1) through shifting the whole Tl by an appropriate constant mn, a, i.e., hk = k + mn, a with 0 ≤mn, a ≤ω −δn, a. Behrens et al. Energy Informatics (2018) 1:53 Page 23 of 28 Furthermore, let γh; min n;a be the min and γh; max n;a be the max borders for a load xh n;a with h ∈T, a ∈An so we can specify, in which borders the intensity of load a can be shifted, i.e., γh; min n;a ≤xh n;a ≤γh; max n;a (C2). We note that the given load profiles have to satisfy the in- equality γh; min n;a ≤lhk n;a ≤γh; max n;a for all k ∈Tl to get a feasible solution. Let αn, a be the starting and βn, a be the ending time slot of a load for an appliance a, then we can restrict time interval T to [αn, a, βn, a] (C3). We note that the interval length between αn, a and βn, a has to be at least the length of the load profile δn, a. to get a feasible solution, i.e., βn, a −αn, a ≥δn, a. Appendix 4 To turn constraint i on and off the for each appliance a let ci a∈f0; 1g be a binary vari- able that shows if a constraint is turned on (ci a ¼ 1) or not (ci a ¼ 0). The objective function describes the total cost of the given load profiles, while the cost in a time slot h is a function depending on h and the total load xh i.e., ch = c(h, xh) ∗xh. Generally, c is a concave function with regard to the load xh, which means that the optimal load profile is smoothed. The resulting mathematical model that minimizes the energy costs and holds the constraints can be formulated as follows. minc xhn;a ¼ X w h¼0 ch ¼ X w h¼0 c h; xh xh ð1Þ minc xhn;a ¼ X w h¼0 ch ¼ X w h¼0 c h; xh xh ð1Þ X w h¼0 xh n;a ¼ ln;a ∀n∈N; a∈An ð2Þ xhk n;a−lk n;a c1 a ¼ 0 ∀k ¼ 0; …; δn;a; ∀n∈N; a∈An ðC1Þ xhk n;a−lk n;a c1 a ¼ 0 ∀k ¼ 0; …; δn;a; ∀n∈N; a∈An ðC2Þ ðC3Þ ð3Þ xh n;a ≥0; ci a∈0; 1 f g ∀h∈T; n∈N; a∈An; i ¼ 1; 2; 3 ð3 xh n;a ≥0; ci a∈0; 1 f g ∀h∈T; n∈N; a∈An; i ¼ 1; 2; 3 Page 24 of 28 Page 24 of 28 Behrens et al. Abbreviations API A li i API: Application Programming Interface; DR: Demand Response; DSR: Design Science Research; EV: Electric Vehicle; HEMS: Home Energy Management Systems; kWh: Kilowatt hour; LPG: LoadProfileGenerator; MAS: Multi-Agent-System; PV: Photovoltaic; REQ: Requirement; SOC: Solution Concept; TAC: Trading-Agent-Competition; TOUP: Time-of-Use Pricing Authors’ contributions DB conceived, designed and performed the experiments and implemented the prototype; DB and TS mainly wrote the paper and derived the framework. DB, TS and SB revised the manuscript. SB and RK contributed by overall suggestions through the research steps and contributing by co-authoring the motivation and outlook section. All au- thors read and approved the final manuscript. Availability of data and materials The data used is simulated by the LoadProfileGenerator from (Pflugradt 2017). Appendix 4 Energy Informatics (2018) 1:53 Appendix 5 Table 6 Results from the simulation Method Criteria No EV 3.6 kW DC No DSM Mean [kWh] 0.632 1.993 1.993 PAR 2.182 6.281 51.901 MSE [kWh2] 34.243 731.601 4607.545 RMSE [kWh] 5.852 27.048 67.879 Costs 20.034 56.780 70.997 savings 0.000 0.000 0.000 Greedy Min Mean [kWh] 0.632 1.993 1.993 PAR 1.942 3.774 14.251 MSE [kWh2] 26.718 291.121 1030.782 RMSE [kWh] 5.169 17.062 32.106 Costs 19.275 50.218 46.717 savings 0.038 0.116 0.342 Greedy Max Mean [kWh] 0.632 1.993 1.993 PAR 1.942 3.774 13.443 MSE [kWh2] 26.302 370.660 969.994 RMSE [kWh] 5.129 19.253 31.145 Costs 19.275 48.449 47.616 savings 0.038 0.147 0.329 MAS without communication Mean [kWh] 0.632 1.993 1.993 PAR 1.942 4.511 19.300 MSE [kWh2] 24.542 361.142 1206.356 RMSE [kWh] 4.954 19.004 34.733 Costs 19.446 56.473 46.888 savings 0.029 0.005 0.340 MAS with communication Mean [kWh] 0.632 1.993 1.993 PAR 1.942 3.725 11.876 MSE [kWh2] 25.154 308.974 884.948 RMSE [kWh] 5.015 17.578 29.748 Costs 19.716 48.693 47.158 savings 0.016 0.142 0.336 Authors’ information Dennis Behrens—Dennis Behrens is research assistant and PhD student at the department of Enterprise Modeling and Information Systems at the University of Hildesheim, Germany. His research interests focus on sustainability, demand side management, and green business modelling. Especially Demand Response is in his focus. Dennis Behrens—Dennis Behrens is research assistant and PhD student at the department of Enterprise Modeling and Information Systems at the University of Hildesheim, Germany. His research interests focus on sustainability, demand side management, and green business modelling. Especially Demand Response is in his focus. Thorsten Schoormann—Thorsten Schoormann is research assistant and PhD student at the department of Enterprise Modeling and Information Systems at the University of Hildesheim, Germany. His research interests focus on business Thorsten Schoormann—Thorsten Schoormann is research assistant and PhD student at the department of Enterprise Modeling and Information Systems at the University of Hildesheim, Germany. His research interests focus on business Page 25 of 28 Page 25 of 28 Behrens et al. Energy Informatics (2018) 1:53 modelling approaches as well as business process modelling languages and supporting tools (e.g., business model development tools) that contribute to the creation, representation, and assessment of sustainability in such models. Especially carsharing (e.g., with electric vehicles) is in the focus of his research. modelling approaches as well as business process modelling languages and supporting tools (e.g., business model development tools) that contribute to the creation, representation, and assessment of sustainability in such models. Especially carsharing (e.g., with electric vehicles) is in the focus of his research. Sebastian Bräuer—Sebastian Bräuer works as a postdoctoral research assistant at the department of Enterprise Modeling and Information Systems at the University of Hildesheim, Germany. His main research interests comprise innovative services, especially in the context of electric vehicles, including aspects of service engineering, value networks, and product-service systems. Previous to his work at the institute, he received his doctoral degree at the University of Münster, Germany and worked at the European Center for Information Systems (ERCIS). Ralf Knackstedt—Ralf Knackstedt is Full Professor of the Institute for Economics and Information Systems, department for Enterprise Modelling and Information Systems at the University of Hildesheim, Germany. His research areas include reference modelling, product-service systems, conceptual modelling, and business process management. Before his work at the institute, he received his doctoral degree and his habilitation at the University of Münster, Germany and worked at the European Center for Information Systems (ERCIS). Competing interests Competing interests The authors declare that they have no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 15 May 2018 Accepted: 17 September 2018 Received: 15 May 2018 Accepted: 17 September 2018 Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. References Abdollahi A, Moghaddam MP, Rashidinejad M, Sheikh-El-Eslami MK (2012) Investigation of Economic and Environmental- Driven Demand Response Measures Incorporating UC. IEEE Trans Smart Grid 3:12–25. https://doi.org/10.1109/TSG.2011 2172996 Abdollahi A, Moghaddam MP, Rashidinejad M, Sheikh-El-Eslami MK (2012) Investigation of Economic and Environmental- Driven Demand Response Measures Incorporating UC. 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Authors’ information His work has been published in leading academic journals and conferences such as Business Information Systems Engineering, IEEE Transactions on Engineering Manage- ment, Communications of the AIS, Scandinavian Journal of Information Systems and Enterprise Modelling, and Infor- mation Systems Architectures. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Not applicable. 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W4388919976.txt	https://journals.uco.es/cco/article/download/14512/12994	de		BRUNS, Peter und Thomas Kremer (eds.), Studia Syriaca. Beiträge des IX. Deutschen Syrologentages in Eichsstätt 2016	Collectanea christiana orientalia	2,020	cc-by	1,196	BRUNS, Peter und Thomas Kremer (eds.), Studia Syriaca. Beiträge des IX. Deutschen Syrologentages in Eichsstätt 2016. «Eichstätter Beiträge zum Christlichen Orient» 6 (Wiesbaden: Harrassowitz Verlag, 2018), VIII+229 pp. ISBN: 9783447110143 El libro se abre con un prólogo en el que sus autores dan cuenta del marco que sirvió para acoger los dieciséis trabajos que componen el presente volumen, concretamente el noveno congreso de los siriólogos alemanes, celebrado en la localidad de Eichsstätt, entre los días 1 y 3 de julio de 2016. Tras el prólogo (p. VII), la disposición de las dieciséis colaboraciones sigue el orden alfabético de los autores. La primera (“Zur Rolle syrisch-aramäischer Ärzte in Antike und Mittelalter”, pp. 1-11) se debe a Besim Akdemir y como su título índica está dedicado al papel desempeñado por los médicos de lengua siriaca durante la Antigüedad y la Edad Media, estructurando su trabajo en dos ámbitos: el geográfico (Edesa, Nísibe, Gundishapur, Bagdad) y el de autoría (Sergio de Reshayna, Actio de Amida, Yaḥyā ibn Māsawayḥ, Ḥunaynn b. Isḥāq y Barhebreo, entre otros). La segunda aportación (“Some Approaches to Jacob of Sarug’s Remarks on Pagan Cults”, pp. 13-24), de Simon Birol, ofrece un estudio del “sermón” (memrā) titulado “Sobre la caída de los ídolos” < ‘Al mafūltā d-ptakrē). Para ello, el autor analiza el contexto y la transmisión de este texto homilético, describe la práctica de culto pagano de la época y expone los recursos retóricos de que hace gala Jacobo de Serug en el texto, para acabar prestando atención al auditorio para el que fuera concebida la homilía. El tercer trabajo (“Der Arme and der Tür ein Sinnbild für den Erlöser? Zur Deutung der Lazarusperikope in der syrischen Literatur”, pp. 25-44), de Peter Bruns, uno de los dos editores de la obra, contiene el análisis de la simbología de la imagen del pobre y la puerta contenida en la perícopa de Lázaro, con el objeto de poder demostrar que se trata de un símbolo del Redentor en el seno de la literatura siriaca. A Winfried Büttner corresponde la cuarta aportación (“Einige Aspekte aus dem syrischen Schrifttum zum psychophysischen Problem”, pp. 45-52), en la que nos presenta la actitud de algunos autores siriacos (Job de Edesa, Moshē bar Kephā y Simeón de Ṭaybuleth) ante determinados problemas de naturaleza psico-física. Collectanea Christiana Orientalia 17 (2020), pp. 323-326; ISSN-e 2386–7442 324 Reseñas Armando Elkhoury (“Fisher- and Net-Imageries of the Church and Her Missionary Role in the Poetry of Jacob of Sarug”, pp. 53-60), en el quinto trabajo, estudia la imagen del pescador y la red en tanto que símbolo de la Iglesia y el papel misionero de ésta en la producción poética de Jacobo de Sarug. La sexta colaboración (“Educational Background of Mar Narsai: Between the “Tradition of the School” and Theodore of Mompsuestia’s Exegesis”, pp. 61-69), correspondiente a Sofia Fomicheva, indaga en los antecedentes formativos de Mār Narsay a partir de dos influencias educacionales experimentadas por el autor, la tradicional de la ‘Escuela’ y la de la exégesis aportada por Teodor de Mompsuestia. Para tal menester, la autora estudia la expresión syag namūsā (“la cerca de la Ley”) y bucea en la extensa producción homilética de Narsay, compuesta por un corpus de más de trescientas homilías. El séptimo trabajo (“The Assyrian Orphanage of the German Orient Mission in Urmia: from its Foundation to its End”, pp. 71-87), de Vincent Pascal Gucha, contiene una exposición de la llegada de los activistas, así como de las actividades humanitarias llevadas a cabo por estos en el Orfanato asirio de la ciudad iraní de Urmia, como parte de la ‘Mision Oriente’ germana desde el año 1900 hasta 1914, final de ésta como consecuencia del estallido de la Primera Guerra Mundial. La octava, de Martin Illert (“Die Erfahrung des Heiligen Geistes bei Pseudo-Makarios und in den frühen syrischen Liturgien”, pp. 89-93), ofrece un inventario de motivos y sus posibilidades combinatorias sobre de la experiencia del Espíritu Santo en el Pseudo Macario y en la primitiva liturgia siriaca. A Hubert Kaufhold, en el noveno trabajo (“Christliche Motive auf Briefmarken nahöstlicher Staaten”, pp. 95-125), debemos un interesante trabajo de corte sociológico (filatélico), gracias al repertorio de motivos cristianos que figuran en los sellos expedidos en países del Oriente Próximo, en concreto Jordania, Palestina, Israel, Líbano, Siria, Iraq, Egipto e Irán. La décima aportación (“Der Brief des Mārā bar Serapiyōn: Stoische Indifferenz und biblische Weisheit in der Welt des Aion”, pp. 127-137) se debe a Matthias Perkams y estudia las relaciones que se dan en la ‘Epístola de Mārā bar Serapiyōn’ entre dos motivos, filosófico uno (la Reseñas 325 indiferencia estoica) y otro sapiencial (la sabiduría bíblica). El autor analiza en la obra la relación entre mundo y filosofía, así como los términos Dios y tiempo con el objeto de mostrar la conexión (Verbindung) que exhibe la obra entre las tradiciones sapienciales judías orientales con material griego, desdeñando la influencia de elementos dogmáticos cristianos así como los metafísicos platónicos. Lukas Pieper en su aportación, la décima primera (“Paulos Mar Gregorios: (Dis-)Kontinuität syrischer Tradition im indischen Kontext”, pp. 139-145), analiza el doble proceso de continuidad/discontinuidad experimentado por la tradición siriaca en autores y obras generados en un contexto cultural hindú a partir de una contextualización histórica-cultural. El trabajo décimo segundo (“Griechisches Dogma in syrischer Gestalt. Die Christologie des Briefes des Sophronius von Jerusalem an Arkadius von Zypern”, pp. 147-154), obra de Catalin-Stefan Popa, está centrado en el tema de la cristología, temática griega vertida en molde siriaco a partir del material epistolar de dos autores: Sofronio de Jerusalén y Arcadio de Chipre. En décimo tercer lugar tenemos la aportación de Alexander M. Schilling (“Ein Koran-Florileium in syrischer Überlieferung. Alphonse Mingana und der „Disput gegen die Nation der Araber“ des Dionysios bar Ṣalībī”, pp. 155-180) en la que estudia la traducción e interpretación de textos coránicos, así como aspectos terminológicos, todos ellos de enorme relevancia para la historia de la polémica islamo-cristiana. Martin Tamcke, en la décimo cuarta colaboración (“„Reform der Kirche des ostens von innen?“ Programm und Lebenswerk des Yuhannon Pera”, pp. 181-192) explora la problemática de la “reforma” experimentada en el seno de la Iglesia de Oriente vista a partir de la trayectoria vivida por el eclesiástico Yuḥannon Pera. La penúltima aportación, la décimo quinta, es la de Jürgen Tubach (“Die Personennamen der Doctrina Addai”, pp. 193-211), que ofrece un interesante trabajo de corte filológico sobre el componente antroponímico de la obra siriaca conocida como “Doctrina del Apóstol Adday” (Malpanuṯā d-Adday Šlīḥā). El décimo sexto y último trabajo se debe a Matthias Westerhoff (“Afrems trinitarische Skizzen”, pp. 213-229), que como su propio 326 Reseñas título indica contiene una serie de bosquejos sobre formulaciones trinitarias en un contexto de polémica anti-arriana, a partir de una obra menor, memrā d-haymanūtā (‘Homilía sobre la fe’), de Efrén el Sirio. Con este volumen sexto, la colección EBCO refuerza su solidez académica, al tiempo que nos proporciona un nuevo producto resultante de la dinámica que mantienen sin descanso nuestros colegas siriólogos de la escuela alemana, con cuyos trabajos, y este es uno más en su ya larga y fecunda trayectoria, siguen alimentando incasablemente la llama de los estudios siriacos a nivel tanto nacional como internacional. JUAN PEDRO MONFERRER-SALA Universidad de Córdoba
https://openalex.org/W2331748108	https://www.arca.fiocruz.br/bitstream/icict/29305/2/Survival%20benefits%20of%20antiretroviral%20therapy%20in%20Brazil_Paula_Luz_etal_INI_Lapclin-AIDS_2016.pdf	English	null	Survival benefits of antiretroviral therapy in Brazil: a model‐based analysis	Journal of the International AIDS Society	2,016	cc-by	9,985	Abstract Abstract Objective: In Brazil, universal provision of antiretroviral therapy (ART) has been guaranteed free of charge to eligible HIV-positive patients since December 1996. We sought to quantify the survival benefits of ART attributable to this programme. Objective: In Brazil, universal provision of antiretroviral therapy (ART) has been guaranteed free of charge to eligible HIV-positive patients since December 1996. We sought to quantify the survival benefits of ART attributable to this programme. Methods: We used a previously published microsimulation model of HIV disease and treatment (CEPAC-International) and data f l l f f l d d d h d f Objective: In Brazil, universal provision of antiretroviral therapy (ART) has been guaranteed free of charge to eligible HIV-positive patients since December 1996. We sought to quantify the survival benefits of ART attributable to this programme. Methods: We used a previously published microsimulation model of HIV disease and treatment (CEPAC-International) and data from Brazil to estimate life expectancy increase for HIV-positive patients initiating ART in Brazil. We divided the period of 1997 to 2014 into six eras reflecting increased drug regimen efficacy, regimen availability and era-specific mean CD4 count at ART initiation. Patients were simulated first without ART and then with ART. The 2014-censored and lifetime survival benefits attributable to ART in each era were calculated as the product of the number of patients initiating ART in a given era and the patients since December 1996. We sought to quantify the survival benefits of ART attributable to this programme. Methods: We used a previously published microsimulation model of HIV disease and treatment (CEPAC-International) and data from Brazil to estimate life expectancy increase for HIV-positive patients initiating ART in Brazil. We divided the period of 1997 to 2014 into six eras reflecting increased drug regimen efficacy, regimen availability and era-specific mean CD4 count at ART initiation. Patients were simulated first without ART and then with ART. The 2014-censored and lifetime survival benefits attributable to ART in each era were calculated as the product of the number of patients initiating ART in a given era and the increase in life expectancy attributable to ART in that era. Results: In total, we estimated that 598,741 individuals initiated ART. Projected life expectancy increased from 2.7, 3.3, 4.1, 4.9, 5.5 and 7.1 years without ART to 11.0, 17.5, 20.7, 23.0, 25.3, and 27.0 years with ART in Eras 1 through 6, respectively. Abstract Of the total projected lifetime survival benefit of 9.3 million life-years, 16% (or 1.5 million life-years) has been realized as of December 2014. Conclusions: Provision of ART through a national programme has led to dramatic survival benefits in Brazil, the majority of which are still to be realized. Improvements in initial and subsequent ART regimens and higher CD4 counts at ART initiation have contributed to these increasing benefits. Keywords: HIV; Brazil; highly active antiretroviral therapy; survival; modelling; Latin America. Keywords: HIV; Brazil; highly active antiretroviral therapy; survival; modelling; Latin America. To access the supplementary material to this article please see Supplementary Files under Article Tools online. Received 5 August 2015; Revised 29 January 2016; Accepted 22 February 2016; Published 29 March 2016 Copyright: – 2016 Luz PM et al; licensee International AIDS Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 Unported (CC BY 3.0) License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To access the supplementary material to this article please see Supplementary Files under Article Tools online. Received 5 August 2015; Revised 29 January 2016; Accepted 22 February 2016; Published 29 March 2016 Copyright: – 2016 Luz PM et al; licensee International AIDS Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 Unported (CC BY 3.0) License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. and efavirenz [2]; other therapies have been adopted for second-line and salvage regimens. Brazil’s aggressive response to the HIV epidemic has been described as exemplary among low- and middle-income countries [3]. Survival benefits of antiretroviral therapy in Brazil: a model-based analysis §Corresponding author: Paula M Luz, Instituto Nacional de Infectologia Evandro Chagas, Fundac¸a˜o Oswaldo Cruz, Avenida Brasil 4365, Rio de Janeiro 21040-360, Brazil. Tel: 55 21 2270 7064, Fax: 55 21 2270 7064. (paula.luz@ipec.fiocruz.br) Results from this manuscript were presented both as a poster and in a themed discussion section at the 2015 Conference on Retroviruses and Opportunistic Infections (CROI) in Seattle, WA on February 23 to 26, 2015. Luz PM et al. Journal of the International AIDS Society 2016, 19:20623 Luz PM et al. Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.doi.org/10.7448/IAS.19.1.20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.doi.org/10.7448/IAS.19.1.20623 Research article Introduction In 1996, the Brazilian government instituted the universal provision of combination antiretroviral therapy (ART), with virologic and immunologic monitoring, free of charge to eligible HIV-positive patients [1]. Along with these provisions, the Department of Sexually Transmitted Diseases, AIDS and Viral Hepatitis of the Brazilian Ministry of Health designated an expert panel to provide country-specific guidelines for the treatment of HIV-positive individuals. These guidelines have kept pace with widely accepted international standards of care for persons with HIV. In 1996, first-line ART consisted of zidovudine, lamivudine and indinavir, and individuals initiated ART upon presentation with an opportunistic infection (OI) or with CD4 B200/mL. The most recent (2014) guidelines advocate treatment for all HIV-positive patients, irrespective of CD4 count, with a first-line regimen of tenofovir, lamivudine Even 20 years after its inception, the Brazilian universal ART treatment programme remains vulnerable. To secure contin- ued public support for the programme, evidence of both its past benefit and, more importantly, its likely lifesaving performance in the years ahead is essential. Additionally, evidence of the success of the Brazilian ART experience could be influential in motivating expansion of ART programmes in other Latin American countries whose HIV epidemics closely resemble that in Brazil. Few epidemiological studies have quantified the impact of ART on population survival in Brazil over time. Early studies characterized pre-ART survival only [4,5]. Studies from the late 1990s and early 2000s Luz PM et al. Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.doi.org/10.7448/IAS.19.1.20623 care, patients on ART receive CD4 and HIV RNA monitoring every six months [2]. After regimen failure defined as a WHO Stage 3 or 4 OI or a confirmed detectable viral load has been detected, patients switch to a subsequent line of ART, which is defined by regimen availability in Brazil at the time of failure for any given era. After failing the last available line, patients remain on their final ARTregimen until death. Patients are also subject to a monthly, adherence-dependent probability of becoming lost to follow-up. Those lost are subject to virologic rebound and HIV disease natural history; they also have a monthly probability of returningto care.Cohorts ofone million patients are simulated to achieve stable per-person estimates of survival and clinical events. A more complete description of the CEPAC-I model can be found in the Supplementary file and in previous publications [18,20,21] or in the CEPAC Model User’s Guide [22]. The CEPAC-I model Natural history was derived from a study sample that included adult patients (]18 years) who enrolled in the INI cohort from 1986 through 2010. These patients were followed for a minimum of 60 days post-enrolment. Cohort procedures include several means of checking patient’s vital status to minimize the risk of bias when reporting on mortality (see Supplementary file for details). Natural history parameters included incidence and mortality rates of OIs, both stratified by CD4 count (but not by age and gender); these parameters were assumed to remain constant for the time period evaluated and were converted into monthly probabilities for model input [18]. Brazil-specific non-AIDS mortality probabilities were age- and gender-stratified from the life tables of the United Nations Department of Economic and Social Affairs [26].To account for lower life expectancy for HIV-positive individuals attributable to risk factors associated with HIV infection (e.g. injection drug use), non-AIDS mortality rates were adjusted by standardized mortality ratios corresponding to various HIV risk groups [27]. Rates of off-ART CD4 decline were stratified by CD4 count and CEPAC-I is an individual patient-level microsimulation model of HIV natural history, disease and treatment. Simulated patients are randomly generated from user-specifiable cohort characteristic distributions and are subject to treatment regimens and HIV monitoring policies defined by the model user. HIV disease progression, defined as immune system deterioration, onset and relapse of OIs, and mortality, is influenced by CD4 count, HIV viral load and OI history. ART use decreases morbidity and mortality through viral suppres- sion and CD4 count increase. OI incidence, OI mortality and AIDS-related mortality are stratified by a patient’s CD4 count. Patients eligible for ART are assigned a level of treatment adherence drawn from a distribution of pharmacy withdrawal records at an HIV/AIDS reference centre in Sa˜o Paulo, Brazil [19]. Initial suppression on ART and subsequent virologic failure rates are dependent upon a patient’s level of adherence [20], with higher adherence corresponding to higher likelihood of virologic suppression. Following the Brazilian standard of CEPAC-I is an individual patient-level microsimulation model of HIV natural history, disease and treatment. Simulated patients are randomly generated from user-specifiable cohort characteristic distributions and are subject to treatment regimens and HIV monitoring policies defined by the model user. HIV disease progression, defined as immune system deterioration, onset and relapse of OIs, and mortality, is influenced by CD4 count, HIV viral load and OI history. Introduction characterized survival for subsets of patients [612] or with alternative AIDS definitions [13]. Although no analysis could portray the past counterfactual in all its detail nor portray the future course of the epidemic with precision, our objective was to offer a credible window on both. We used a model-based approach to quantify the 2014-censored and projected life- time survival benefits achieved with the incremental improve- ments of the Brazilian HIV treatment programme. Cohort characteristics Characteristics of the simulated annual cohorts were derived from the 2290 treatment-naı¨ve HIV-positive adults initiating ARTat INI between 2000 and 2014. Mean (SD) age was 37 (10) years, and 70% of patients were male (Table 1) with negligible variation by era. In contrast, other parameters varied by era, includingCD4countatARTinitiation,whichrangedfrom185/mL (Era 1) to 518/mL (Era 6), and the fraction of patients with an OI history (i.e. a diagnosis of AIDS) at ART initiation (see Supplementary Figures 1 and 2 for additional details). Input parameters Model parameters for cohort characteristics and natural history were derived from the HIV Clinical Cohort at the Instituto Nacional de Infectologia Evandro Chagas (INI), Fundac¸a˜o Oswaldo Cruz, and from published Brazilian govern- mental data. INI is a public research and healthcare institution situated in Rio de Janeiro, Brazil, and is one of Brazil’s largest reference centres for HIV research and treatment. It has provided care to over 5500 HIV-positive patients in the Rio de Janeiro metropolitan area since 1986; we had access to client- level data for model parameterization [2325]. Analytic overview y We used a previously published microsimulation model of HIV disease and treatment [1418], the Cost-Effectiveness of Preventing AIDS Complications-International (CEPAC-I) model, using data from Brazil [18] to project the survival benefits attributable to provision of ART by the Brazilian national HIV treatment programme for patients initiating ART from 1997 to 2014. We divided this time frame into six eras, reflecting improvements in drug regimen efficacy and avail- ability over time, as reported in the Brazilian HIV Treatment Guidelines [2]: Era 1 (1997 to 1999), Era 2 (2000 to 2003), Era 3 (2004 to 2007), Era 4 (2008 to 2012), Era 5 (2013) and Era 6 (2014). ARTeligibility criteria also changed over time, which we modelled by varying cohort characteristics, including CD4 count at ART initiation. Annual cohorts of patients with characteristics reflective of their respective eras were simulated from treatment initiation in that year under two scenarios: without ARTand with ART.To limit the analysis to the benefit of ART, both the treatment and no-treatment scenarios included the use of guideline-concordant OI prophylaxis. Life expec- tancy censored at 2014 for each cohort was recorded. Increase in life expectancy was calculated for each era and then multiplied by the projected number of patients initiating ART in that era to obtain survival benefits. Results were extended to uncensored life expectancy to determine the projected lifetime survival benefits of ARTand to estimate the proportion of benefits that has already been realized. Median and 20-year survival were estimated for each era for the two scenarios, without ART and with ART. The CEPAC-I model ART use decreases morbidity and mortality through viral suppres- sion and CD4 count increase. OI incidence, OI mortality and AIDS-related mortality are stratified by a patient’s CD4 count. Patients eligible for ART are assigned a level of treatment adherence drawn from a distribution of pharmacy withdrawal records at an HIV/AIDS reference centre in Sa˜o Paulo, Brazil [19]. Initial suppression on ART and subsequent virologic failure rates are dependent upon a patient’s level of adherence [20], with higher adherence corresponding to higher likelihood of virologic suppression. Following the Brazilian standard of 2 Luz PM et al. Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.do Luz PM et al. Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.doi.org/10.7448/IAS.19.1.20623 Luz PM et al. Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.doi.org/10.7448/IAS.19.1.20623 HIV RNA and obtained from an analysis of two multicentre cohorts of untreated HIV-positive patients [28,29]. We used regimen, including subsequent lines of therapy. The cohort adherence distribution was from a study of ART pharmacy Table 1. The CEPAC-I model Select model parameter inputs Model parameter Base case value Ranges examined Reference Cohort characteristics INI Age (SD), (years) 37 (10) 2747 Male sex, (%) 70 6080 Mean (SD) CD4 at ART initiation, (/mL) Era 1 185 (123) 92277 Era 2 231 (154) 116347 Era 3 284 (189) 142426 Era 4 343 (229) 172515 Era 5 383 (255) 192575 Era 6 518 (273) 259777 Proportion of patients with OI history at ART initiation, (%) Era 1 63.7 31.995.6 Era 2 54.0 27.081.0 Era 3 42.5 21.363.8 Era 4 30.7 15.346.0 Era 5 23.7 11.935.6 Era 6 13.0 6.519.5 HIV RNA level at ART initiation, all Eras 100,000 45.1 30,001100,000 25.7 10,00130,000 18.3 300110,000 10.9 53000 0.0 Disease natural history INI Monthly risk of chronic AIDS death, (%) Without history of OI 0.820.02 With history of OI 5.670.02 Antiretroviral therapy Adherence distribution [19] 95% 38.9 19.558.4 8095% 30.2 B80% 30.9 First-line virologic suppression at six months, (%) Era 1 60 4080 [30] Eras 24 80 7090 [38] Eras 56 90 8595 [38] Virologic failure rate, (/100PM) Currently prescribed regimens 0.16 0.080.24 [40,41] Formerly prescribed regimens 3.96 1.985.94 [42] Rate of loss to follow-up, (/1000PY) 10.1 5.115.2 [43] Rate of return to care, (/1000PY) 818 4091227 INI SD: standard deviation; INI: Instituto Nacional de Infectologia Evandro Chagas; OI: opportunistic infection; PM: person-months; PY: person-years. Risks are stratified by CD4 count, with higher risk associated with lower CD4 count. SD: standard deviation; INI: Instituto Nacional de Infectologia Evandro Chagas; OI: opportunistic infection; PM: person-months; Risks are stratified by CD4 count, with higher risk associated with lower CD4 count. regimen, including subsequent lines of therapy. The cohort adherence distribution was from a study of ART pharmacy withdrawals in Sa˜o Paulo, Brazil, and was assumed to be constant across all eras; mean adherence was 83.5%, mean- ing that the average patient was in possession of 83.5% of his or her prescribed medication in the previous 12 months [19]. ART regimen sequencing and availability varied by era; patients in earlier eras who survived to subsequent eras HIV RNA and obtained from an analysis of two multicentre cohorts of untreated HIV-positive patients [28,29]. We used these natural history parameters to simulate survival for patients without ART. ART efficacy and availability Per universal access in Brazil [1], the model assumes universal and era-dependent access to every available ART 3 3 Luz PM et al. Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.doi.org/10.7448/IAS.19.1.20623 months for all ART lines, proportion of patients initiating ART with a history of OI, virologic failure rate for suppressed patients, loss to follow-up rate, return to care rate for patients lost to follow-up. All parameters were varied from 50 to 150% of base case values. skipped obsolete regimens (Table 2) and switched to newer, more effective regimens upon virologic failure. Rates of virologic suppression and CD4 count increase for suppressed patients were derived from clinical trials [3039]. Virologic suppression for first-line ART at 24 weeks, which was dependent on adherence, ranged from an average of 60% (Era 1) to 90% (Eras 5 to 6). The rate of later virologic failure for those who achieved suppression, also dependent on adherence, averaged 0.16/100PM for regimens currently prescribed in Brazil [40,41] and 3.96/100PM for older regi- mens no longer in use [42]. months for all ART lines, proportion of patients initiating ART with a history of OI, virologic failure rate for suppressed patients, loss to follow-up rate, return to care rate for patients lost to follow-up. All parameters were varied from 50 to 150% of base case values. skipped obsolete regimens (Table 2) and switched to newer, more effective regimens upon virologic failure. Rates of virologic suppression and CD4 count increase for suppressed patients were derived from clinical trials [3039]. Virologic suppression for first-line ART at 24 weeks, which was dependent on adherence, ranged from an average of 60% (Era 1) to 90% (Eras 5 to 6). The rate of later virologic failure for those who achieved suppression, also dependent on adherence, averaged 0.16/100PM for regimens currently prescribed in Brazil [40,41] and 3.96/100PM for older regi- mens no longer in use [42]. In multivariate sensitivity analysis, we created best-case and worst-case scenarios, using the most and least optimistic parameter assumptions (50 or 150% of the base case values). In the worst-case scenario, we assumed decreased mean CD4 count at ART initiation, increased proportion of patients initiating ART with a history of OI, increased ART failure at six months, increased virologic failure rate for suppressed patients, increased loss to follow-up rate and decreased return to care rate for patients lost to follow-up. 2014-censored and lifetime survival benefits Per-person survival benefits for each era were calculated as the difference in 2014-censored life expectancy for patients on ART in a given era and that of patients without ART in that era. This means that patients in the 1997 cohort accrued a maximum of 18 years of survival through 2014, whereas those in the 2014 cohort accrued a maximum of only one year. To obtain total cohort benefits for a given era, we multiplied the per-person benefits by the number of patients initiating ART in that era. Survival benefits were summed across all eras to obtain total population estimates of survival benefits attribu- table to the Brazilian national ART programme. In addition to 2014-censored results, uncensored survival benefits results were calculated in the same way to estimate projected lifetime benefits of the Brazilian national ART programme for these patients and to estimate the proportion of survival benefits that has not yet been realized. For lifetime outcomes, Era 1 had a life expectancy of 2.7 years without ART compared to 11.0 years with ART, for a per-person increase of 8.3 years (Table 3, columns B?D?). Per-person survival benefits for subsequent eras were even greater, ranging from 14.2 years for Era 2 to 19.9 years for Era 6. Per-person life expectancy in each era is illustrated in Figure 1, as is the survival benefit attributable to ART availability in each era compared to no ART and compared to the era immediately before. Increased per-person survival yielded a total population anticipated lifetime survival benefit of 9.3 million life-years, 7.8 million (84%) of which have yet to be realized (Figure 1). Loss to follow-up and return to care The loss to follow-up rate of 10.1/1000 person-years (PY) was derived from the HIV-Brazil Cohort Study [43], a nationwide, multicentre study of over 5000 patients receiving ART from 2003 to 2010. The rate of return to care (818/1000PY) was from the INI clinical cohort. ART efficacy and availability The opposite was assumed for the best-case scenario. Adherence, since it affects ART failure at six months, virologic failure for suppressed patients and loss to follow-up, was not modified in the best- case and worst-case scenarios to avoid double counting effects. Results Base case The number of patients initiating ART in each of the six eras was 114,062, 115,363, 92,895, 189,741, 43,755, and 42,925, respectively, for a total of 598,741 patients initiating ART between 1997 and 2014 (Table 3). Results censored in December 2014 demonstrate that Era 1 yielded a life expectancy of 2.7 years without ART compared to 6.5 years with ART, a per-person increase of 3.8 years (Table 3, columns BD). Survival increased in those without ARTowing to higher CD4 counts at presentation as the eras progressed. To under- stand this, note that each era is characterized not only by improved treatment regimens but also by earlier presentation to care and treatment initiation. Consequently, patients have higher mean CD4 counts at simulation entry even those who do not receive ART. Survival benefits censored at 2014 for subsequent eras [26] decreased as the eras progressed due to the shorter time horizon for survival accrual relative to the earlier eras and due to longer survival without ART.The sum of products of the number of patients initiating ART and the 2014-censored per-person survival benefits yielded an esti- mated 1.5 million life-years saved as of December 2014; approximately 87% of this benefit was accrued by patients initiating ART in the first three eras. Estimates of number initiating ART The Brazilian Ministry of Health reports the annual number of patients receiving ART through the national ART programme from 1999 to 2012 [27]. We created two linear models by extrapolating backward two years (1997 and 1998) and forward two years (2013 and 2014) to obtain estimates for the entire time frame of interest, 1997 to 2014 (see Supplementary Figure 3). We assumed that all patients on ART in 1997 (60,316) initiated ART that year. Since the number of patients initiating ART in each year is not available, we calculated this as the reported number of patients on ART in that year minus the number of patients on ART from the previous year who survived and were not lost to follow-up according to CEPAC-I model survival output, a methodology used in a previous survival benefits analysis [44]. Sensitivity analyses We varied uncertain model parameters across a plausible range (Table 1) using the number of patients initiating ART in each era to evaluate the robustness of the survival benefit estimates. These parameters, varied in all eras, included the following: mean CD4 count at ART initiation, proportion of patients with greater than 95% adherence, ART failure at six Twenty-year survival, both without and with ART, increased in each subsequent era (Figure 2, Table 4). In the without-ART analyses (dashed), this resulted from increased CD4 counts at 4 Luz PM et al. Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.do Luz PM et al. Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.doi.org/10.7448/IAS.19.1.20623 Table 2. Sensitivity analyses ART regimen sequencing and efficacy by era Era Regimen Year regimen available Proportion of patients achieving virologic suppression at 24 weeksa Estimated CD4 increase at 12 months for suppressed patients (cells/mL) Reference ERA 1 (19971999) 1) AZT3TCIDV 1997 60 140 [30] 2) Alternative regimens after PI failure, w/o genotype 1997 22b 100 [31] 3) EFV2NRTIs (3TC/d4T or 3TC/ddI or d4T/ddI) 2000 62 140 [32] 4) LPV/r2NRTIs* 2000 62 100 [33] 5) ATV/rTDF1NRTI 2004 65 110 [33] 6) ENFoptimized background* 2006 30 140 [34] 7) RALDRV/r2NRTIs 2008 75 150 [35] 8) ETVPI/r2NRTIs 2013 70 150 [36] 9) CCR5PI/r2NRTIs 2013 60 140 [37] ERA 2 (20002003) 1) EFVAZT3TC 2000 80 180 [38] 2) LPV/R2NRTIs* 2000 62 100 [33] 3) Alternative regimens after PI failure, with genotype* 2000 34b 90 [31] 4) ATV/rTDF1NRTI 2004 65 110 [33] 5) ENFoptimized background* 2006 30 140 [34] 6) RALDRV/r2NRTIs 2008 75 150 [35] 7) ETVPI/r2NRTIs 2013 70 150 [36] 8) CCR5PI/r2NRTIs 2013 60 140 [37] ERA 3 (20042007) 1) EFVAZT3TC 2004 80 180 [38] 2) LPV/r2NRTIs 2004 62 100 [33] 3) ATV/rTDF1NRTI 2004 65 110 [33] 4) ENFoptimized background* 2006 30 140 [34] 5) RALDRV/r2NRTIs 2008 75 150 [35] 6) ETVPI/r2NRTIs 2013 70 150 [36] 7) CCR5PI/r2NRTIs 2013 60 140 [37] ERA 4 (20082012) 1) EFVAZT3TC 2008 80 180 [38] 2) LPV/r2NRTIs* 2008 62 100 [33] 3) ATV/rTDF1NRTI 2008 80 120 [39] 4) RALDRV/r2NRTIs 2008 75 150 [35] 5) ENFoptimized background* 2008 30 140 [34] 6) ETVPI/r2NRTIs 2013 70 150 [36] 7) CCR5PI/r2NRTIs 2013 60 140 [37] ERA 5 (2013) 1) TDFFTCEFV 2013 90 200 [38] 2) ATV/r2NRTIs 2013 80 130 [39] 3) RALDRV/r2NRTIs 2013 75 150 [35] 4) ETVPI/r2NRTIs 2013 70 150 [36] 5) CCR5PI/r2NRTIs 2013 60 140 [37] ERA 6 (2014) 1) TDFFTCEFV 2014 90 200 [38] 2) ATV/r2NRTIs 2014 80 130 [39] 3) RALDRV/r2NRTIs 2014 75 150 [35] 5 Table 2. ART regimen sequencing and efficacy by era Era Luz PM et al. Sensitivity analyses Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.doi.org/10.7448/IAS.19.1.20623 Table 2 (Continued ) Era Regimen Year regimen available Proportion of patients achieving virologic suppression at 24 weeksa Estimated CD4 increase at 12 months for suppressed patients (cells/mL) Reference 4) ETVPI/r2NRTIs 2014 70 150 [36] 5) CCR5PI/r2NRTIs 2014 60 140 [37] HVL: HIV viral load; AZT: zidovudine; 3TC: lamivudine; IDV: indinavir; PI: protease inhibitor; EFV: efavirenz; NRTI: nucleoside reverse transcriptase inhibitor, d4T: stavudine; ddI: didanosine; LPV: lopinavir; r: boosted ritonavir; ATV: atazanavir; TDF: tenofovir; FTC: emtricitabine; ENF: enfurvitide; RAL: raltegravir; DRV: darunavir; ETV: etravirine; CCR5: C-C chemokine receptor type 5. aVirologic suppression defined as HVL B400 or B500 copies/mL, depending on the source; bsuppression was assessed at 12 weeks because of reporting in the source. Indicates a regimen that will be skipped if a subsequent, more effective regimen is available. Bolded regimens are those available at the beginning of each era. Era HVL: HIV viral load; AZT: zidovudine; 3TC: lamivudine; IDV: indinavir; PI: protease inhibitor; EFV: efavirenz; NRTI: nucleoside reverse transcriptase inhibitor, d4T: stavudine; ddI: didanosine; LPV: lopinavir; r: boosted ritonavir; ATV: atazanavir; TDF: tenofovir; FTC: emtricitabine; ENF: enfurvitide; RAL: raltegravir; DRV: darunavir; ETV: etravirine; CCR5: C-C chemokine receptor type 5. aVirologic suppression defined as HVL B400 or B500 copies/mL, depending on the source; bsuppression was assessed at 12 weeks because of reporting in the source. Indicates a regimen that will be skipped if a subsequent, more effective regimen is available. Bolded regimens are those available at the beginning of each era. Indicates a regimen that will be skipped if a subsequent, more effective regimen is available. Bolded regimens are those available at the beginning of each era. of patients with 95% adherence (7.8 to 10.1 million life- years), ART failure at six months for all regimens (8.3 to 10.1 million life-years) and the percentage of patients starting ART with a history of OIs (8.8 to 9.7 million life-years). Rate of loss to follow-up and return to care did not substantially influ- ence lifetime survival benefits. In the worst-case scenario, lifetime survival benefits were estimated at 5.5 million life-years. The best-case scenario yielded projected lifetime survival benefits of 11.1 million life-years. ART initiation and the decreased proportion of patients presenting with OIs. Sensitivity analyses Twenty-year survival for patients in Era 1 increased from 0.2% without ART to 20.8% with ART; median survivalforthesepatientsincreasedfrom2.0yearswithoutART to3.3yearswith ART(seeFigure2andTable4).InEra6,20-year survival increased from 3.4% without ART to 60.6% with ART; median survival for these patients increased from 5.7 years without ART to 25.7 years with ART. Sensitivity analyses Projected lifetime survival benefits were generally stable across the ranges of parameters examined (Figure 3).The most influential parameter was mean CD4 count at ART initiation for all eras; decreasing or increasing this by a relative 50% gave lifetime survival benefit estimates of 7.3 and 9.9 million life-years. Other influential parameters included the proportion -zero but is reported as zero due to rounding. p y Value is non-zero but is reported as zero due to rounding. Discussion Based on reporting by the Brazilian Ministry of Health [27], we estimated that almost 600,000 individuals have recei- ved ART, free of charge, in Brazil between 1997 and 2014. Our results predict that the lifetime survival benefits Table 3. 2014-censored and lifetime survival benefits of the Brazilian national ART program for patients starting ART between 1997 and 2014 2014 censored results (LY) Lifetime results (LY) Era A: Persons initiating ARTa B: Per capita life expectancy, without ART C: Per capita life expectancy, with ARTb D: Per capita survival benefitc [CB] E: Survival benefit [AD] B’: Per capita life expectancy, without ARTd C’: Per capita life expectancy, with ART D’: Per capita survival benefit [C’-B’] E’: Survival benefit [AD’] Era 1 (19971999) 114,062 2.7 6.5 3.8 433,436 2.7 11.0 8.3 946,715 Era 2 (20002003) 115,363 3.2 8.1 4.9 565,279 3.3 17.5 14.2 1,638,155 Era 3 (20042007) 92,895 3.7 7.0 3.3 306,554 4.1 20.7 16.6 1,542,057 Era 4 (20082012) 189,741 3.3 4.3 1.0 189,741 4.9 23.0 18.1 3,434,312 Era 5 (2013) 43,755 1.8 1.9 0.1 4375 5.5 25.3 19.8 866,349 Era 6 (2014) 42,925 1.0 1.0 0.0* 385 7.1 27.0 19.9 854,208 Total 598,741 1,499,770 9,281,796 aCalculated as the number of patients on ART in the given era minus the patients still alive from previous eras; bcensored life expectancy for 1997 cohort is out of a possible 18 years whereas that for 2014 is out of a possible one year; ccalculated by subtracting life expectancies during 19972014 for the Without ART simulations (column B) from those for the ART simulations (column C). Survival gains in 1997 are thus measured over 18 years, whereas those in 2014 are measured over one year; dincrease in life expectancy without ART is due to an increase in CD4 count at presentation by era. *Value is non-zero but is reported as zero due to rounding. Table 3. 2014-censored and lifetime survival benefits of the Brazilian national ART program for patients starting ART between 1997 and 2014 ored and lifetime survival benefits of the Brazilian national ART program for patients starting ART between 1997 Table 3. 2014-censored and lifetime survival benefits of the Brazilian national ART program for patients starting AR and 2014 Table 3. Discussion Bar width corresponds to the number of patients in each era and total coloured area corresponds to lifetime survival benefits. Survival benefits realized as of December 2014 are shaded with diagonal lines. YLS: years of life saved; ART: antiretroviral therapy. Figure 1. Years of life saved per-person in each era produced by model simulations. Bar width corresponds to the number of patients in each era and total coloured area corresponds to lifetime survival benefits realized as of December 2014 are shaded with diagonal lines. YLS: years of life saved; ART: antiretroviral therapy. p p p y responds to the number of patients in each era and total coloured area corresponds to lifetime survival benefits. Survival benefits December 2014 are shaded with diagonal lines. YLS: years of life saved; ART: antiretroviral therapy. Peru and Venezuela have similarly concentrated epidemics, with an HIV prevalence in the general population below 1%. In these countries, the proportion of eligible HIV-positive individuals on ART is similar to that reported in Brazil (estimated from 60 to 86%, with a region-wide estimate of 75%; see Supplementary Table 2). This suggests that survival benefits for the recent eras reported in this paper might be generalizable to other countries in the region with similar ART guidelines. Nevertheless, care is needed when extra- polating this achievement, given that Brazil’s HIV treatment programme dates to 1996, whereas most other Latin American countries, such as Mexico, Peru and Chile, initiated national-level treatment programmes in 2001, 2004 and 2005, respectively [4547]. Further, specific enhancements such as treatment monitoring (incorporated in Brazil in conjunction with HIV treatment), genotype resistance testing after failure (started in Brazil in early 2000) and availability of third-line and salvage regimens should be acknowledged, as these are often not uniformly available and can influence the applicability of our results to other settings. achievable by these individuals will reach 9.3 million life- years. Of this total, we found that 16% of the benefits have been realized as of December 2014 (1.5 million life-years), whereas 7.8 million life-years have yet to be realized. Our results also predict that lifetime per-person survival benefits surpass a decade of life for Era 2 (spanning 2000 to 2004) and that these individual benefits for Era 3 and beyond are even greater, 16 additional years of life. Discussion 2014-censored and lifetime survival benefits of the Brazilian national ART program for patients starting ART be d 2014 Era aCalculated as the number of patients on ART in the given era minus the patients still alive from previous eras; bcensored life expectancy for 1997 cohort is out of a possible 18 years whereas that for 2014 is out of a possible one year; ccalculated by subtracting life expectancies during 19972014 for the Without ART simulations (column B) from those for the ART simulations (column C). Survival gains in 1997 are thus measured over 18 years, whereas those in 2014 are measured over one year; dincrease in life expectancy without ART is due to an increase in CD4 count at presentation by era aCalculated as the number of patients on ART in the given era minus the patients still alive from previous eras; bcensored life expectancy for 1997 cohort is out of a possible 18 years whereas that for 2014 is out of a possible one year; ccalculated by subtracting life expectancies during 19972014 for the Without ART simulations (column B) from those for the ART simulations (column C). Survival gains in 1997 are thus measured over 18 years, whereas those in 2014 are measured over one year; dincrease in life expectancy without ART is due to an increase in CD4 count at presentation by era. 6 Luz PM et al. Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.doi.org/10.7448/IAS.19.1.20623 0 5 10 15 20 25 30 Per-person Life Expectancy (years) Improvement from previous era Benefit from previous eras Benefit realized through Dec 2014 Life expectancy with pre-ART prophylaxis alone Era 6 2014 Era 5 2013 Era 4 2008–2012 Era 3 2004–2007 Era 2 2000–2003 Era 1 1997–1999 0.9 million YLS 114,062 patients 1.6 million YLS 115,363 patients 1.5 million YLS 92,895 patients 3.4 million YLS 189,741 patients 0.9 million YLS 43,755 patients 0.9 million YLS 42,925 patients Figure 1. Years of life saved per-person in each era produced by model simulations. Bar width corresponds to the number of patients in each era and total coloured area corresponds to lifetime survival benefits. Survival benefits realized as of December 2014 are shaded with diagonal lines. YLS: years of life saved; ART: antiretroviral therapy. Era 3 2004–2007 Era 2 2000–2003 Era 1 1997–1999 Figure 1. Years of life saved per-person in each era produced by model simulations. Discussion Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.doi.org/10.7448/IAS.19.1.20623 0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 Percentage alive Years from treatment initiation Era 6 - 2014 Era 5 - 2013 Era 4 - 2008 Era 3 - 2004 Era 2 - 2000 Era 1 - 1997 Era 6 - 2014 No ART Era 5 - 2013 No ART Era 4 - 2008 No ART Era 3 - 2004 No ART Era 2 - 2000 No ART Era 1 - 1997 No ART Figure 2. Survival curve over 20 years from treatment initiation for each era of ART in Brazil. Survival with ART is displayed in solid lines, and survival without ART is displayed in dotted lines. ART: antiretroviral therapy. Figure 2. Survival curve over 20 years from treatment initiation for each era of ART in Brazil. Survival with ART is displayed in solid lines, and survival without ART is displayed in dotted lines. ART: antiretroviral therapy. variation in mortality and other key model parameters that might more accurately reflect Brazil’s heterogeneity; thus, our analysis cannot speak to variation in survival on or off ART that may result from underlying life expectancy differences between regions. Additionally, though we did not explicitly model changes in Brazil’s ART initiation criteria, we modelled the evolution of the Brazilian HIV Treatment Guidelines as closely as possible with respect to drugs and regimens and conservatively assumed no improvements in treatment or care in our projected lifetime results. Thus, our results are likely consistent with ART initiation criteria since, in general, patients initiated ART later than recommended in Brazil in all eras [48]. Importantly, our results exclude the as a proxy for medication adherence country-wide. Because adherence in the Sa˜o Paulo study may not reflect the country as a whole and because pharmacy withdrawals may not correlate perfectly with actual adherence we employed sensitivity analysis to consider the impact of varying this parameter on outcomes; when adherence was 50 and 150% of the base case, survival benefits changed to 7.8 and 10.1 million life-years, respectively. Next, although we did not adjust the model for risk group, age or sex, we did compare similar cohort compositions both with and without ART to achieve survival benefit totals; as such, this limitation should not greatly influence the present analysis. Discussion These results, which corroborate the literature on the survival benefits of ART in Brazil [611,13], provide a comprehensive view of the health benefits accrued through the Brazilian HIV treatment programme. achievable by these individuals will reach 9.3 million life- years. Of this total, we found that 16% of the benefits have been realized as of December 2014 (1.5 million life-years), whereas 7.8 million life-years have yet to be realized. Our results also predict that lifetime per-person survival benefits surpass a decade of life for Era 2 (spanning 2000 to 2004) and that these individual benefits for Era 3 and beyond are even greater, 16 additional years of life. These results, which corroborate the literature on the survival benefits of ART in Brazil [611,13], provide a comprehensive view of the health benefits accrued through the Brazilian HIV treatment programme. We have structured this analysis to portray the regimens used in Brazil and incorporated country-specific values for the prevalence of AIDS-related infections, late presentation to care and suboptimal adherence, all of which conserva- tively reduce survival benefits. Additionally, to restrict the calculation of survival benefits to only those conferred by ART, we incorporated other important facets of the Brazilian response to the HIV epidemic, including prophylaxis and comprehensive OI care, when modelling the eras without ART. To the extent that the simulations without and with ART were similar in all aspects except for ART (i.e. treatment adherence, clinical care, among other parameters), we can infer that the benefits reported here derive solely from the use of ART. Moreover, per-person life expectancy increases in subsequent eras can be attributed to improved ART regimens as drugs with increased efficacy were incorporated. This analysis has several limitations. A single source was used for parameters such as mean CD4 count at ART initiation, proportion of patients initiating ART with a history of OI, medication adherence, loss to follow up rate and return-to-care rate for patients previously lost. We acknowl- edge that input parameters from a single site may not always be representative of the entire country and have used sensitivity analyses to demonstrate where variation would have a major impact on results. For example, we used pharmacy withdrawal information from a study in Sa˜o Paulo Currently, the HIV epidemic of other Latin American countries resembles that of Brazil. Argentina, Chile, Mexico, 7 Luz PM et al. Discussion Further, our analysis did not incorporate regional and other demographic Table 4. Predicted median survival and 20-year survival for all six eras, without and with ART Era Median survival, without ART (years) Median survival, with ART (years) Predicted proportion alive at 20 years, without ART (%) Predicted proportion alive at 20 years, with ART (%) Era 1 (19971999) 2.0 3.3 0.2 20.8 Era 2 (20002003) 2.4 9.5 0.4 36.9 Era 3 (20042007) 3.0 16.4 0.8 45.0 Era 4 (20082012) 3.8 20.4 1.4 50.6 Era 5 (2013) 4.3 23.7 1.9 56.4 Era 6 (2014) 5.7 25.7 3.4 60.6 Table 4. Predicted median survival and 20-year survival for all six eras, without and with ART 8 Luz PM et al. Journal of the International AIDS Society 2016, 19:20623 http://www.jiasociety.org/index.php/jias/article/view/20623 \| http://dx.doi.org/10.7448/IAS.19.1.20623 4 5 6 7 8 9 10 11 12 Return to care (50–150%) Loss to follow-up (150–50%) Rate of virologic failure (150–50%) % OI history at ART initiation (150–50%) ART failure at 6 months, all lines (50–150%) Adherence, % with adherence >95% (50–150%) CD4 at ART initiation (50–150%) Parameter extremes (worst case to best case) Base Case = 9.3 Million YLS Survival Benefits (Millions of YLS) 4 5 6 7 8 9 10 11 12 Return to care (50–150%) Loss to follow-up (150–50%) Rate of virologic failure (150–50%) % OI history at ART initiation (150–50%) ART failure at 6 months, all lines (50–150%) Adherence, % with adherence >95% (50–150%) CD4 at ART initiation (50–150%) Parameter extremes (worst case to best case) Base Case = 9.3 Million YLS Survival Benefits (Millions of YLS) Figure 3. Tornado diagram of one-way sensitivity analyses of lifetime survival benefits of ART in Brazil. Each horizontal bar represents the range of survival benefits produced by varying a given model parameter across the ranges in parentheses. The vertical line represents the base case. Base Case = 9.3 Million YLS Figure 3. Tornado diagram of one-way sensitivity analyses of lifetime survival benefits of ART in Brazil. Each horizontal bar represents the range of survival benefits produced by varying a given model parameter across the ranges in parentheses. The vertical line represents the base case. Figure 3. Tornado diagram of one-way sensitivity analyses of lifetime survival benefits of ART in Brazil. Each horizontal bar represents the range of survival benefits produced by varying a given model parameter across the ranges in parentheses. Authors’ affiliations 1 1The Instituto Nacional de Infectologia Evandro Chagas, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brasil; 2Medical Practice Evaluation Center, Massachusetts General Hospital Boston, MA, USA; 3Division of General Internal Medicine, Massachusetts General Hospital Boston, MA, USA; 4Division of Infectious Disease, Massachusetts General Hospital Boston, MA, USA; 5HarvardUniversity CenterforAIDSResearch,HarvardMedicalSchoolBoston,MA,USA; 6Department of Epidemiology, Boston University School of Public Health Boston, MA, USA; 7Department of Health Policy and Management, Harvard School of Public Health Boston, MA, USA; 8Department of Orthopedic Surgery, Brigham and Women’s Hospital Boston, MA, USA; 9Department of Biostatistics, Boston Authors’ contributions PML, MPG, BG, KAF, VGV, EL, RAP, ADP and RPW conceived and designed the study. PML and CJS performed the statistical analysis. MPG and RLM performed the model analysis and created the tables and figures. PML, MPG, BG, KAF, VGV, EL, RLM, RAP, ADP and RPW analyzed and interpreted the data. PML and MPG drafted the article. All authors critically revised the article for important intellectual content. Funding This work was supported by the National Institute of Allergy and Infectious Diseases (R01 AI058736). Discussion The i l li h b University School of Public Health Boston, MA, USA; 10Biostatistics Center, MassachusettsGeneral HospitalBoston,MA, USA; 11Department of Health Policy and Management,Yale Schoolof Public Health, New Haven,CT, USA; 12Division of Infectious Disease, Brigham and Women’s Hospital Boston, MA, USA benefits attributable to the prevention of mother-to-child transmission, the treatment of children and adolescents and the reduction of horizontal HIV transmission due to sup- pressed viral load. Our results also exclude the economic returns of ART treatment [49]. Furthermore, we did not explicitly model expansion of HIV prevention, testing and treatment infrastructure. Finally, the current version of the CEPAC-I model does not include long-term co-morbidities related to ART exposure. Including these conditions may reduce the projected survival benefits of ART. benefits attributable to the prevention of mother-to-child transmission, the treatment of children and adolescents and the reduction of horizontal HIV transmission due to sup- pressed viral load. Our results also exclude the economic returns of ART treatment [49]. Furthermore, we did not explicitly model expansion of HIV prevention, testing and treatment infrastructure. Finally, the current version of the CEPAC-I model does not include long-term co-morbidities related to ART exposure. Including these conditions may reduce the projected survival benefits of ART. Acknowledgements PML, BG and CJS acknowledge funding from the National Council of Technological and Scientific Development and the Research Funding Agency of the State of Rio de Janeiro. RPW also acknowledges the support of the Massachusetts General Hospital Research Scholar Award. Competing interests The authors declare no competing interests. The authors declare no competing interests. Disclaimer: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Conclusions Over the past 20 years, Brazil’s national programme of free ART access to patients has led to dramatic survival benefits, the vast majority of which are still to be realized. Higher CD4 counts at treatment initiation and improvements in initial and subsequent ART regimens have all contributed substantially to these benefits. Increased HIV testing, with even earlier ART initiation and improved rates of linkage to care, as well as interventions to improve ART adherence, could lead to additional survival benefits in the future. 1The Instituto Nacional de Infectologia Evandro Chagas, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brasil; 2Medical Practice Evaluation Center, Massachusetts General Hospital Boston, MA, USA; 3Division of General Internal Medicine, Massachusetts General Hospital Boston, MA, USA; 4Division of Infectious Disease, Massachusetts General Hospital Boston, MA, USA; 5HarvardUniversity CenterforAIDSResearch,HarvardMedicalSchoolBoston,MA,USA; 6Department of Epidemiology, Boston University School of Public Health Boston, MA, USA; 7Department of Health Policy and Management, Harvard School of Public Health Boston, MA, USA; 8Department of Orthopedic Surgery, Brigham and Women’s Hospital Boston, MA, USA; 9Department of Biostatistics, Boston Luz PM et al. 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https://openalex.org/W4320449128	https://zenodo.org/records/7635837/files/SISTEMA%20P%C3%9ABLICO%20DE%20ESCRITURA%C3%87%C3%83O%20DIGITAL%20%E2%80%93%20SPED_%20PONTOS%20CR%C3%8DTICOS%20EM%20SUA%20IMPLANTA%C3%87%C3%83O%20NO%20COM%C3%89RCIO%20ATACADISTA%20DE%20ALIMENTOS%20DO%20MUNIC%C3%8DPIO%20DE%20BEL%C3%89M.%20%E2%80%93%20ISSN%201678-0817%20Qualis%20B2.pdf	Portuguese	null	SISTEMA PÚBLICO DE ESCRITURAÇÃO DIGITAL – SPED: PONTOS CRÍTICOS EM SUA IMPLANTAÇÃO NO COMÉRCIO ATACADISTA DE ALIMENTOS DO MUNICÍPIO DE BELÉM.	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	5,286	SISTEMA PÚBLICO DE ESCRITURAÇÃO DIGITAL – SPED: PONTOS CRÍTICOS EM SUA IMPLANTAÇÃO NO COMÉRCIO ATACADISTA DE ALIMENTOS DO MUNICÍPIO DE BELÉM. Edição 119 FEV/23 / 13/02/2023 REGISTRO DOI: 10.5281/zenodo.7635837 Rogério Moura Rego1 SISTEMA PÚBLICO DE ESCRITURAÇÃO DIGITAL – SPED: PONTOS CRÍTICOS EM SUA IMPLANTAÇÃO NO COMÉRCIO ATACADISTA DE ALIMENTOS DO MUNICÍPIO DE BELÉM. Edição 119 FEV/23 / 13/02/2023 REGISTRO DOI: 10.5281/zenodo.7635837 ISSN 1678-0817 Qualis B2 Revista Científica de Alto Impacto. SISTEMA PÚBLICO DE ESCRITURAÇÃO DIGITAL – SPED: PONTOS CRÍTICOS EM SUA IMPLANTAÇÃO NO COMÉRCIO ATACADISTA DE ALIMENTOS DO MUNICÍPIO DE BELÉM. RESUMO As organizações passam por um grande desafio no que tange as novas tendências de controle fiscal das três esferas do governo, a tecnologia da informação trouxe uma nova forma digital de assegurar o controle efetivo das transações fiscais. A Escrituração Fiscal Digital – EFD-ICMS-IPI, tem se tornado um entrave para as empresas, devido sua complexidade, tornando-se uma ferramenta de planejamento tributário no cenário atacadista de alimentos. Diante deste contexto o presente estudo visa analisar identificar e avaliar os pontos críticos encontrados na implantação da EFD-ICMS-IPI bem como sua adequação. A abordagem se torna essencial para o devido discernimento realizado em uma organização do segmento atacadista de alimentos, sendo que o estudo foi elaborado nos departamentos de contabilidade, tecnologia da informação, compras, estoque e vendas, de forma qualitativa descritiva e exploratória. Após o estudo conclui-se que a empresa apresentou 4 (quatro) pontos críticos por grau de relevância na implantação da escrituração fiscal digital, tais como: Investimento, qualificação, tecnologia e cultura organizacional. pontos críticos por grau de relevância na implantação da escrituração fiscal digital, tais como: Investimento, qualificação, tecnologia e cultura organizacional. Palavras-chave: Empresa, Tecnologia, Contabilidade, EFD-ICMS-IPI. Palavras-chave: Empresa, Tecnologia, Contabilidade, EFD-ICMS-IPI. Palavras-chave: Empresa, Tecnologia, Contabilidade, EFD-ICMS-IPI. ABSTRACT Organizations undergo a major challenge regarding the new trends of fiscal control of the three branches of government; information technology has brought a new digital way to ensure effective control of fiscal transactions. Digital Tax Bookkeeping – EFD-ICMS-IPI, has become an obstacle for companies, due to their complexity, making it a tax-planning tool in the wholesale food scene. Given this context, the present study aimed at identifying and assessing the critical issues encountered in the implementation of EFD-ICMS-IPI and its suitability. The approach is essential for proper discernment done in an organization of the wholesale segment of food, and the study was prepared in the accounting department, information technology, purchasing, inventory and sales, descriptive qualitative and exploratory. After the study is concluded that the company submitted four (4) critical points for degree of relevance in the implementation of digital fiscal bookkeeping, such as investment, training, technology and organizational culture. Keywords: Company, Technology, Accounting, EFD-ICMS-IPI. 1 INTRODUÇÃO O Brasil passa por transformações consideradas no que se refere ao avanço da tecnologia e o crescimento do consumismo, causando um aclive de empresas comerciais em ênfase ao segmento atacadista de alimentos, isso seria suficiente para o crescimento do País, entretanto o nível elevado da carga tributária, representando a obrigação principal das empresas, bem como as declarações prestadas ao fisco pelo contribuinte, demonstrando as obrigações acessórias, causa um entrave nas organizações. O surgimento do “Governo Eletrônico” trouxe um novo conceito de auditoria fiscal e tributária com advento da Emenda Constitucional no 42, a qual cria à integração as três esferas dos governos Federal, Estadual e Municipal, desenvolvendo uma nova gestão fiscalizadora e controle integrado de informações cadastrais e fiscais, os órgãos fiscalizadores tem a missão de criar mecanismos integrados de controle fiscal para garantir o cumprimento da legislação, originando desta forma o Sistema Público de Escrituração Digital. O projeto SPED (Sistema Público de Escrituração Digital), tem um potencial significativo no controle da sonegação fiscal e forte imposição de adequação para as organizações, esta nova ferramenta é subdivida em diversas obrigações acessórias, na qual focaremos a EFD-ICM-IPI, (Escrituração Fiscal digital), O projeto SPED (Sistema Público de Escrituração Digital), tem um potencial significativo no controle da sonegação fiscal e forte imposição de adequação para as organizações, esta nova ferramenta é subdivida em diversas obrigações acessórias, na qual focaremos a EFD-ICM-IPI, (Escrituração Fiscal digital), Mediante isso, os profissionais de contabilidade funcionam como instrumentos de mediação na relação Contribuinte x Fisco, por meio de analistas tributários para gerar informações consultivas às entidades particulares. Com isso, os contadores e as empresas se veem, cada vez mais, pressionados a promover os relatórios fiscais com maior presteza, uma vez observado, que qualquer dado falso ou omisso transmitido, pode resultar em uma notificação a caminho. Neste sentido foi necessário analisar os pontos críticos na implantação da EFD-ICMS- IPI, do comercio atacadista de alimentos de Belém, bem como a Identificação do processo de implantação, especificando os departamentos envolvidos e avaliando o nível de capacitação organizacional, caracterizando um estudo de caso na empresa Portugal Comercio e Atacado de Alimentos Ltda. O estudo é totalmente justificado pela analise, identificação e avaliação dos pontos críticos no processo de implantação do EFD-ICMS-IPI, através do levantamento das informações no cenário vivido entre os usuários primários e secundários. 2.1 Empresa Atacadista de Alimentos O histórico do comercio atacadista no Brasil evidenciado nos meados da década de 50, por portugueses imigrantes que aqui chegaram; Inicialmente, os imigrantes exerciam suas vendas em caixas de madeira (caixeiro-viajante) vendendo de casa em casa, ou apenas em pequeno varejo, posteriormente evoluindo para a modalidade atacado de balcão (estabelecimentos com equipes de vendedores internos preparados para atender os clientes). As primeiras grandes empresas do setor surgiram nas principais capitais brasileiras que, apesar de atuarem basicamente na forma de balcão, já possuíam vendedores cobrindo o interior do país, realizando negócios e enviando as mercadorias aos seus clientes por meio de transporte ferroviário. (MACHADO, 2000) O setor atacadista se compõe de empresas que percorrem o país e abastecem a população. Seu trabalho é de constantes desafios e dificuldades, tais como: as precárias estradas, riscos de assaltos e roubo de cargas, manutenção de frotas, perdas, altos custos operacionais, créditos restritos e o eterno peso da carga tributária entre outros. As novas tecnologias de gestão fiscalizadora trazem um olhar estratégico frente ao setor atacadista com a devida adequação ao atendimento de obrigações acessórias, fazendo com que este desafio seja uma característica de diferencial frente à concorrência. 1 INTRODUÇÃO Que são os empresários, colaboradores e contadores na empresa estudada, os quais podem ter dificuldade para implantação do SPED- Fiscal. Contribuindo para o discernimento dos discentes, docentes e da organização visto que ao identificar e analisar os pontos críticos reduz de forma significativa o risco de multas e auto de infração por não atender as obrigações acessórias impostas pela legislação tributária, aplicando de forma pratica o princípio da continuidade organizacional, com consequente sobrevivência da empresa no segmento atacadista de alimentos e permanência de seus colaboradores. 2.2 Perfil profissional contábil frente à tecnologia da informação Em razão do avanço da Informática, principalmente após a Segunda Guerra Mundial, tem-se hoje, recursos de alta eficiência e adaptabilidade aos mais variados ramos de organizações e, ainda mostram-se como tecnologias capazes de oferecer velocidade e precisão progressivas, identificando-se com o ambiente organizacional em uma sociedade competitiva e, por isso, a cada dia mais exigente em termos de qualidade em produtos, serviços e na satisfação nos relacionamentos. As empresas não buscam os profissionais contábeis apenas para contabilizar seus atos e fatos, mais sim para serem parceiros no atingimento das metas estabelecidas. Neste cenário é que está empregado o contador do futuro que tenta de maneira desafiadora conciliar sua profissão em concordância aos Princípios da contabilidade os quais são embasamentos ao exercício da profissão contábil, praticar cada princípio diferencia cada profissional na execução de suas tarefas sejam elas de natureza fiscal, contábil ou legal. O mercado atual é bastante amplo e requer modernidade, criatividade, novas tecnologias, novos conhecimentos e mudanças urgentes na visão através dos paradigmas, para a busca continua da competição, (SILVA, 2000). Como toda pessoa jurídica, necessita de, no mínimo, um contador, a área de consultoria contábil e fiscal, tornam-se pontos fortes entre as opções de emprego que estão em alta nesta área. As organizações têm procurado novos mercados e investido em modernização e treinamento pessoal, com o objetivo de conquistar segmentos em expansão como a consultoria. 2.3 Contabilidade Fiscal e Tributária Conceitua-se contabilidade tributária como um dos seguimentos da contabilidade que tem por objetivo aplicar na prática conceitos, princípios e normas básicas da contabilidade e da legislação tributária, de forma continua e adequada. (FABRETTI 2000). A importância da contabilidade passou a ser evidenciada com o aclive do desenvolvimento do capitalismo e o fortalecimento do mercado de ações, a partir deste momento a história a contabilidade passou a ser muito importante para a sociedade. A contabilidade deve ser feita segundo as Normas Brasileiras de Contabilidade (NBC) respeitando os chamados Princípios Fundamentais de Contabilidade e em observância da lei das sociedades por ações, o que por si só já está gerando muita discordância entre os contadores quanto a real utilidade dessas demonstrações contábeis para a tomada de decisões gerenciais. Neste contexto a empresa e o contador tem um papel vital diante da sociedade, otimizar recurso, cumprir a legislação fiscal e tributária de forma a garantir a sobrevivência da organização, delineando desta forma o planejamento tributário, com a devida adequação a escrituração fiscal digital. sobrevivência da organização, delineando desta forma o planejamento tributário, com a devida adequação a escrituração fiscal digital. 2.4 Sistema Público de Escrituração Digital (EFD – ICMS-IPI) A origem do sistema público nasce de maneira implícita através da Emenda Constitucional no 42 de 19 de dezembro de 2003, Art. 37, inciso XXII, onde descreve que, as administrações tributárias da União, dos Estados, do Distrito Federal e dos Municípios, atividades essenciais ao funcionamento do Estado, exercidas por servidores de carreiras específicas, terão recursos prioritários para a realização de suas atividades e atuarão de forma integrada, inclusive com o compartilhamento de cadastros e de informações fiscais, na forma da lei ou convênio. O SPED é instrumento que unifica as atividades de recepção, validação, armazenamento e autenticação de livros e documentos que integram a escrituração comercial e fiscal dos empresários e das sociedades empresárias, mediante fluxo único, computadorizado, de informações. (BARROSO; 2015). Legalmente o SPED foi instituído pelo Decreto no. 6.022 de 22 de janeiro de 2007 e pela instrução normativa da Receita Federal do Brasil RFB no. 787, de 19 de novembro de 2007. Neste projeto, o governo se uniu às empresas com um objetivo comum: mudar a forma de emissão e armazenamento de documentos fiscais, além de alterar a escrituração fiscal e contábil, colocando todos definitivamente na era do governo eletrônico (MARIANO; AZEVEDO, 2009). Sendo assim um novo avanço tecnológico que formaliza a sistemática de implementação ou usuário em arquivos virtuais a escrituração contábil-fiscal e auxiliará no armazenamento de documentos (MARASTONI, 2009). Neste sentido O projeto SPED pretende alterar a forma de cumprimento das obrigações acessórias realizadas pelos contribuintes, substituindo a emissão de livros e documentos contábeis e fiscais em papel, por documentos eletrônicos, cuja auditoria, integridade e validade jurídica são reconhecidas pelo uso do certificado digital (AZEVEDO; 2009). O foco deste estudo, esta em um dos subprojetos do sistema público de escrituração digital denominado Escrituração Fiscal Digital sob a sigla EFD- ICMS-IPI, o conjunto contábil onde são registradas as operações da empresa, ou seja, a escrita fiscal compreende os registros de entrada e saída, compra e revenda de notas fiscais eletrônicas efetuadas pelas empresas contribuintes dos Impostos ICMS e IPI. A EFD representa toda a movimentação de entrada, saída e inventário de mercadorias e serviços, além da apuração do ICMS e IPI. (DUARTE; 2008). A forma de se fazer o lançamento contábil fiscal não foi alterada, apenas os procedimentos de declará-lo e arquivá-lo (MAHLE, 2009). 2.4 Sistema Público de Escrituração Digital (EFD – ICMS-IPI) O contador deverá, por meio do PVA, importar o arquivo que contém a escrituração fiscal da empresa, que deve estar de acordo com o Ato Cotepe/ICMS no 09/2008. Assinado digitalmente e transmitindo para a Secretaria da Fazenda de Jurisdição do estabelecimento. 3 PROCEDIMENTOS METODOLÓGICOS Com intuito de aperfeiçoar e tornar compreensivo as ideias concernentes à identificação dos pontos críticos na implantação da Escrituração Fiscal Digital no comercio atacadista de alimentos do município de Belém, a pesquisa foi objetivada de forma exploratória e descritiva. A pesquisa exploratória tem como direcionamento principal traçar informações inerentes a um objeto, indicando assim um foco no todo, explorando as condições de especificações desse objeto. Neste contexto ela é uma inicialização para pesquisa explicativa. (SEVERINO 2007). Em se tratando dos procedimentos técnicos a pesquisa foi realizada através de um estudo de caso de uma empresa do ramo atacadista de alimentos nos departamentos de contabilidade, tecnologia da informação, compras, estoques e faturamento. O instrumento para a coleta de dados é a consolidação de todo trabalho de planejamento e execução das etapas iniciais do processo de pesquisa. É nesse documento onde serão apresentadas as perguntas aos pesquisadores, servindo também para anotações ou observações, relações de tópicos a serem seguidos durante uma entrevista, entre outros. (MATTAR 1996) O instrumento para a coleta de dados é a consolidação de todo trabalho de planejamento e execução das etapas iniciais do processo de pesquisa. É nesse documento onde serão apresentadas as perguntas aos pesquisadores, servindo também para anotações ou observações, relações de tópicos a serem seguidos durante uma entrevista, entre outros. (MATTAR 1996) O questionário é um instrumento de coleta de dados constituído por uma série ordenada de perguntas, que devem ser respondidas por escrito e sem a presença do entrevistador. Junto com o questionário deve-se enviar uma nota ou carta explicando a natureza da pesquisa, sua importância e necessidade de obter respostas, tentando despertar o interesse no recebedor para que ele preencha e devolva o questionário dentro de um prazo razoável (LAKATOS; MARCONI; 2002). A análise dos dados desse estudo será feita pelo próprio pesquisador, que utilizara métodos descritivos. Na definição analítica dos resultados, será feito também cruzamentos entre as variáveis, possibilitando estabelecer ralações entre as mesmas. Neste estudo foi empregado o método quantitativo e abordagem qualitativa que se caracterizou mais adequado ao objetivo proposto. Contudo foi possível também descobrir, a partir de opiniões e dados através da coleta de informações. 4.1 Histórico do estudo A empresa pesquisada foi constituída em 20 de agosto de 1982, devido à impossibilidade da divulgação de sua denominação optou-se pela generalização do termo comercio atacadista de alimentos no município de Belém, com uma filial no município de Ananindeua, seu objeto social principal é o comercio atacadista de alimentos, segmento bastante concorrido visto a grande concorrência de empresas do mesmo ramo. Diante do avanço da tecnologia da informação e o emprego do governo eletrônico nas organizações, passou por um momento de adequação as novas tendências do fisco Estadual Federal e Municipal, em ênfase por uma transição na implantação para Escrituração Fiscal Digital, esteve diante de uma nova ferramenta estratégica garantindo assim diferencial competitivo no segmento em relação às empresas não adaptáveis as novas tecnologias de fiscalização. Composta de 305 colaboradores sendo que o objeto de estudo foi elaborado nos departamentos de contabilidade, tecnologia da informação, compras, estoque e vendas, tendo como amostra 40 colaboradores selecionados aleatoriamente nos departamentos pesquisados através da entrevista e questionário. Os quais foram realizados no dia 20, 21 e 22 de outubro de 2015 em horário comercial nas dependências da empresa. 5 ANÁLISE DOS DADOS Neste artigo, a coleta de dados foi elaborada através de questionário com perguntas fechadas e abertas, a serem respondidas, foi desenvolvido um questionário buscando conhecer quais foram as dificuldades enfrentas pelos gestores dos departamentos de compra, estoque, revenda e contabilidade, no decorrer da implantação do projeto. As perguntas buscam de maneira sucinta responder os principais empecilhos enfrentados por estes gestores no início do processo de implantação até o termino, em seguida foi feito uma entrevista com os mesmos colaboradores para identificar a qualidade da informação evidenciando suas opiniões sobre a implantação do SPED Fiscal e colaborar desta forma com o desenvolvimento da pesquisa obtendo um retorno de 100% o que corresponde ao total de 40 colaboradores. 5.1 Qualificação especifica para implantação do EFD-ICMS-IPI aos Usuários de cada departamento 5.1 Qualificação especifica para implantação do EFD-ICMS-IPI aos Usuários de cada departamento A abordagem em destaque verificou-se o número de usuários que receberam treinamento especifico do EFD-ICMS-IPI, durante o processo da implantação sendo que 30% da área fiscal obtiveram treinamento, na área de compras não houve treinamento assim como no faturamento, por conseguinte a área de tecnologia 20% do departamento recebeu o treinamento, nesta análise verificou- se a falta de qualificação especifica dos usuários primários e secundários referente à implantação do EFD-ICMS-IPI, como demonstra o gráfico 01 a seguir: Treinamento Recebido para Implantação do EFD-ICMS-IPI Gráfico 01 – Treinamento recebido por usuários de cada departamento. Treinamento Recebido para Implantação do EFD-ICMS-IPI Treinamento Recebido para Implantação do EFD-ICMS-IPI Gráfico 01 – Treinamento recebido por usuários de cada departamento. 5.2 Dificuldade com relação à tecnologia em cada departamento envolvido no processo. 5.2 Dificuldade com relação à tecnologia em cada departamento envolvido no processo. De acordo com a análise o número de usuários que tiveram dificuldade com os novos conceitos tecnológicos tem um grau de 10% de dificuldade em relação ao departamento fiscal, fazendo uma analogia com o gráfico 01, podemos perceber que estes são os que mais receberam treinamento para a implantação, nos remetendo a entender a importância da qualificação no processo, já na área de compras a dificuldade foi de 80%, observa-se que os mesmos não obtiveram treinamento, assim como o departamento de faturamento na proporção de 80% de dificuldade, por conseguinte a área de tecnologia com 40% dificuldade, isso preceitua que para aplicar a tecnologia é necessário o conhecimento do EFD- ICMS-IPI, uma vez que este departamento obteve 20% de qualificação. Nesta vertente identifica-se que a tecnologia está atrelada ao sped fiscal, como demonstra o gráfico 02 a seguir: Dificuldade com relação à tecnologia por departamento Dificuldade com relação à tecnologia por departamento Gráfico 02. Dificuldade com relação à tecnologia por departamento Gráfico 02. Dificuldade com relação à tecnologia por departamento Gráfico 02. Dificuldade com relação à tecnologia por departamento 5.3 Investimento por departamento no processo da implantação do EFD- ICMS A análise neste quesito, refrete ao investimento alocado a cada departamento envolvido no processo, a demanda previu 30% de investimento no setor fiscal, levando em consideração aumento de salário, tecnologia e qualificação, para o departamento de compras não houve investimento, assim como o faturamento, já o de tecnologia houve um investimento de 40%, distribuídos na compra de software e treinamento, bem como alteração no salário, como demonstra o gráfica 03 a seguir: Investimento ao processo de implantação por departamento Gráfico 03 – Investimentos na implantação do EFD-ICMS. Investimento ao processo de implantação por departamento Gráfico 03 – Investimentos na implantação do EFD-ICMS. Investimento ao processo de implantação por departamento Investimento ao processo de implantação por departamento Gráfico 03 – Investimentos na implantação do EFD-ICMS. 5.4 A influência da cultura organizacional no processo de implantação do EFD-ICMS-IPI. 5.4 A influência da cultura organizacional no processo de implantação do EFD-ICMS-IPI. Nesta etapa foi possível perceber que a cultura organizacional da empresa influenciou diretamente nos processos da implantação, sendo que 30% dos entrevistados do departamento fiscal alegaram a dificuldade em empregar uma nova forma de trabalhar no processo visto que a empresa não aceitava as novas regras do fisco, por consequência 60% dos entrevistados do departamento de compras alegam a falta de readequação nos de Resistencia ao processo, seguido pelo departamento de tecnologia onde 25% disseram não poder melhorar o processo devido à falta de comprometimento dos próprios gestores e a mudança da forma de trabalhar como informa a proporção do gráfico 04 a seguir: Influência da cultura organizacional na implantação do EFD-ICMS-IPI. Gráfico 04 – Influência da cultura organizacional na implantação do EFD-ICMS- IPI. Influência da cultura organizacional na implantação do EFD-ICMS-IPI. uência da cultura organizacional na implantação do EFD-ICMS-IP Gráfico 04 – Influência da cultura organizacional na implantação do EFD-ICMS- IPI. 5.5 Principais dificuldades encontradas na implantação do sistema SPED- Fiscal de forma geral. 5.5 Principais dificuldades encontradas na implantação do sistema SPED- Fiscal de forma geral. Ao obter um norte anteriormente pesquisado nos gráficos 01 a 04, foram perceptível quatro pontos críticos, evidenciados os quais foram empregados em uma entrevista de maneira a diagnosticar o grau de relevância representado na empresa para cada ponto crítico, nestes moldes verifica-se que 40% dos entrevistados consideram que a falta de qualificação prejudica os resultados, 30% afirmam que a ausência da tecnologia eficaz é ponto chave no processo, 20% opinam pela falta de investimento da empresa para o devido resultado satisfatório, e 10% manifestam pela dificuldade da adaptação da cultura organizacional. Pontos Críticos relevantes na Implantação do EFD-ICMS-IPI Gráfico 05 – Pontos Críticos por relevância na implantação do EFD-ICMS-IPI. Pontos Críticos relevantes na Implantação do EFD-ICMS-IPI Pontos Críticos relevantes na Implantação do EFD-ICMS-IPI Gráfico 05 – Pontos Críticos por relevância na implantação do EFD-ICMS-IPI. 6 CONSIDERAÇÕES FINAIS Ao analisarmos os dados acima conclui-se que existem 4 (quarto) pontos crítico na implantação do EFD-ICMS-IPI no comercio atacadista do município de Belém, encontrados pela empresa ora pesquisada, evidenciados com grau de relevância, o primeiro versa sobre a falta de qualificação dos colaboradores que estão envolvidos no processo, ou seja, a necessidade de conhecimento teórico e prático sobre a escrituração, o segundo recai para a necessidade de um sistema de informação que atenda a legislação fiscal e tributária e gerencial da empresa, o terceiro desenvolve a partir da falta de investimento no processo de implantação por parte dos gestores da empresa, e por fim o quarto evidencia que a cultura organizacional da empresa interfere na adequação do Sped-Fiscal, pois existe uma resistência às novas tendências do governo eletrônico. No contexto da problemática para o estudo de caso da empresa comercio atacadista de alimentos especificou-se o seguinte problema: Quais os pontos críticos na implantação da EFD-ICMS-IPI, do comercio atacadista de alimentos no município de Belém. O confronto da referência teórica utilizada e os dados coletados no estudo de caso mediante questionários e entrevista, solucionou-se o problema levantado, uma vez que foi evidenciado nos preceitos do conhecimento em relação ao tratamento que se deve ter na empresa atacadista, bem como o atrelamento dos profissionais envolvidos, com um vasto conhecimento da tecnologia da informação, a contabilidade tributaria e o próprio EFD-ICMS-IPI. É perceptível que não houve uma junção da teoria referenciada com a prática enfeudada na implantação do sped-fiscal, visto que a empresa não trabalhou a forma amenizar os fatores fundamentais para o sucesso na adequação. Em detrimento aos objetivos da pesquisa de maneira geral, preconizava-se analisar os pontos críticos na implantação da EFD-ICMS-IPI, do comercio atacadista de alimentos de Belém. Este foi almejado mediante pesquisa bibliográfica aplicada ao estudo de caso, onde sacramentou que existem pontos críticos os a quais devem ser intensamente trabalhados no momento da implantação, mas uma, podendo ser empregado como possível solução às demais empresas do seguimento atacadista no município de Belém. Através da análise dos pontos críticos, é possível estabelecer um plano de ação para reduzir ao máximo as dificuldades enfrentadas pelos usuários primários e secundários. No tocante ao que refere aos objetivos específicos aplicados na organização estudada. 6 CONSIDERAÇÕES FINAIS Pretendia-se, Identificar os pontos críticos no processo de implantação da EFD-ICMS-IPI, do comercio atacadista de alimentos de Belém, concretizado através do estudo por ordem de relevância, sendo eles a qualificação dos envolvidos, tecnologia da informação, o investimento ao processo e a cultura organizacional. Ao fato de especificar os departamentos envolvidos, obtivemos os departamentos de contabilidade área fiscal, compras, faturamento, tecnologia da informação e, por conseguinte avaliar o nível de capacitação e cultura organizacional dos envolvidos, sendo demostrado a falta de qualificação nos departamentos de compras e faturamento, a conclusão foi determinada pelo questionamento e entrevista aos envolvidos que se mostraram descomprometidos com as questões pertinentes a organização e sua interação com o meio natural. Diante das hipóteses levantadas ao estudo, foi pressuposto que as organizações não dispõem de profissionais qualificados para a implantação do EFD-ICMS-IPI, no comercio atacadista de alimentos Belém, o que foi comprovado, visto que os departamentos não obtiveram qualificação necessária para o desenvolvimento da implantação, conseguinte, foi levantada de que os empresários não investem na implantação, comprovado de acordo com os dados coletados, acreditou-se que a cultura organizacional da empresa influenciaria na implantação da EFD- ICMS-IPI, no comercio atacadista de alimentos Belém, foi comprovado que a cultura organizacional como um dos pontos críticos no processo de implantação do EFD-ICMS-IPI. As hipóteses foram confirmadas pelo referencial teórico e ao estudo de caso. O referencial teórico conceituou e discerniu de maneira a garantir a solução do problema, bem como o devido alcance dos objetivos e, por conseguinte a confirmação das hipóteses levantadas O preceito é percebível pelos Artigos, Periódicos, Livros e a própria legislação do Sped-Fiscal. Deste modo para um embasamento, foram utilizadas as obras de autores inerentes ao objeto de estudo. 6 CONSIDERAÇÕES FINAIS As discussões principais neste estudo referem-se à interação entre empresa atacadista de alimentos e os colaboradores das empresas bem como os fiscos estaduais federais e municipais, levando em consideração a interação dos pensamentos a respeito de um tema tão importante na área organizacional, diante dos autores mais representativos ao tema destacam-se, sobre a evolução do comercio atacadista de alimentos temos o autor Machado (2000), em seguida destaca-se na abordagem da tecnologia da informação SILVA, (2000), com frente à contabilidade fiscal e tributária, FABRETTI (2000), para o estudo do Sped, temos a colaboração do autor Barroso (2015) com a mesma linha de pensamento para Mariano e Azevedo (2009) e MARASTONI (2009), para o estudo do EFD-ICMS-IPI, temos como autor destaque, DUARTE (2008). Quanto a os principais autores da linha de pesquisa dos pontos críticos na implantação do EFD-ICMS-IPI do comercio atacadista de Belém, temos Severino (2007) desenvolvendo a conceituação da pesquisa exploratória, seguido de Lakatos Marconi 2002, contribuíram no tocante ao discernimento da análise de dados. Quanto a os principais autores da linha de pesquisa dos pontos críticos na implantação do EFD-ICMS-IPI do comercio atacadista de Belém, temos Severino (2007) desenvolvendo a conceituação da pesquisa exploratória, seguido de Lakatos Marconi 2002, contribuíram no tocante ao discernimento da análise de dados. 7 RECOMENDAÇÕES PARA ESTUDOS FUTUROS Depois de concluso o estudo, podemos indicar com uma visão mais ampla dos acontecimentos, uma pesquisa voltada na aplicabilidade da qualidade da informação do EFD-ICMS – IPI. Neste entendimento podemos também demonstrar e recomendar um plano de ação para as empresas atacadista de alimentos do município de Belém, contribuindo para excelência na implantação como se segue: 1a AÇÃO: ADQUIRIR SISTEMA DE INFORMAÇÃO. O sistema a ser implantado deve atender as necessidades da empresa e do fisco, não há como organizar a empresa sem a aquisição de um sistema que seja coerente com a situação financeira da organização bem como sua facilidade de adaptação e o atendimento as obrigações acessórias do fisco. Faremos o orçamento dos sistemas presentes no mercado para assim verificar o mais adequado à organização. 4a AÇÃO: GERAR AS OBRIGAÇÕES PRINCIPAIS E ACESSORIAS. Após a devida inserção das informações corretas no sistema de informação, temos que gerar as obrigações acessórias, onde o principal, são o EFD-ICMS-IPI e o EFD-CONTRIBUIÇÕES, analisando essas informações no validador do EFD, verificando a quantidade de erros com suas respectivas crítica do validador e fazendo as devidas correções no sistema de informação, após este feito geraremos novamente essas informações para a transmissão ao fisco cumprindo o que rege a legislação em vigor. 5a AÇÃO DESENVOLVER UMA POLITICA DE MANUTENÇÃO DOS PROCESSOS. Todas expostas são de caráter decisivo e de vital importância para organização entrar no caminho correto e reduzir drasticamente o risco de um auto de infração ocasionado por informações incompletas transmitidas ao fisco. É necessária a manutenção da política da nova classificação tributaria, sendo a permanência de pessoas qualificadas para a inserção de novos produtos. 2a AÇÃO: QUALIFICAR OS PROFISSIONAIS. Selecionar pessoas que estejam dispostas a aprender e ajudar a organizar a empresa, treinando as mesmas no que se refere ao manuseio do sistema de informação adquirido pela empresa, dando discernimento ao que refere da classificação tributaria, utilizando assim o RICMS-PA, como base fundamental para o aprendizado, 3a AÇÃO. REALIZAR A CLASSIFICAÇÃO TRIBUTÁRIA. Após a devida qualificação dos profissionais envolvidos no que se refere ao discernimento do devido preenchimento da classificação tributaria, temos a enfrentar uma nova etapa que é a inserção destas informações na parametrização do sistema de informação. Juntaremos todas as notas fiscais eletrônicas de aquisição faremos devida classificação tributária de entrada e saída. REFERÊNCIAS BIBLIOGRÁFICAS AZEVEDO, Osmar Reis; MARIANO, Paulo Antônio. SPED – Sistema Público de Escrituração Digital, 1° edição. Editora IOB 2009. BECKMAN, T. The current state of knowledge management. In: LIEBOWITZ, J. (Ed.)Knowledge management handbook. New York: CRC Press, 1999. BECKMAN, T. The current state of knowledge management. In: LIEBOWITZ, J. (Ed.)Knowledge management handbook. New York: CRC Press, 1999. DAVENPORT, H. Thomas; PRUSAK, Laurence, Conhecimento empresarial: como as organizações gerenciam o seu capital intelectual. Rio de Janeiro: Campus, 1998. sponível em: BARROSO, Eveline. SPED, PIS/COFINS, Teoria e Prática. Disponível em: BARROSO, Eveline. SPED, PIS/COFINS, Teoria e Prática. http://www.crc-ce.org.br/crcnovo/files/Sped_Pis-Cofins-Maracanau.pdf. Acessado em: 24.09.2015. http://www.crc-ce.org.br/crcnovo/files/Sped_Pis-Cofins-Maracanau.pdf. Acessado em: 24.09.2015. Disponível em: RECEITA FEDERAL DO BRASIL. SPED – Escrituração Fiscal Digital. Disponível em: <http://www1.receita.fazenda.gov.br/sobre-o- projeto/parceiros.htm>. Acessado em: 24.09.2015. DUARTE, Roberto Dias. Big Brother Fiscal: na era do conhecimento. 2. ed. Belo Horizonte: Quanta 2008. LAKATOS, E. M.; MARCONI, M. de A. Técnicas de pesquisa. Planejamento e execução de pesquisas, amostragens e técnicas de pesquisa, elaboração, análise e interpretação de dados. 5. ed. São Paulo: Atlas, 2002. MAHLE, Marciane Maria; SANTANA, Alex Fabiano Bertollo. Sistema público de escrituração digital – SPED: um estudo nos escritórios de contabilidade no município de Pinhalzinho/SC. Revista Catarinense da Ciência Contábil (CRC-SC), Florianópolis, v.8, no 23, p. 73-92, abr./jul. 2009. MARASTONI, Carlos Eduardo. SPED – Sistema Público de Escrituração Digital NFE– Nota Fiscal Eletrônica. Unisescon – Universidade Corporativa, 2009. MARASTONI, Carlos Eduardo. SPED – Sistema Público de Escrituração Digital NFE– Nota Fiscal Eletrônica. Unisescon – Universidade Corporativa, 2009. MARIANO, Paulo Antônio; AZEVEDO, Osmar Reis. SPED – Sistema Público de Escrituração Digital. São Paulo: Iob, 2009. MARIANO, Paulo Antônio; AZEVEDO, Osmar Reis. SPED – Sistema Público de Escrituração Digital. São Paulo: Iob, 2009. PEREIRA, H. J. Proposição de um modelo organizacional baseado no conhecimento: um estudo de caso em empresa pública. In: MANAGEMENT IN IBEROAMERICAN COUNTRIES: CURRENT TRENDS AND FUTURE PROSPECTS, 3. 2003, São Paulo. Proceedings…São Paulo: Iberoamerican Academy of Management, Brazil, 1 CD ROM. PEREIRA, H. J. Proposição de um modelo organizacional baseado no conhecimento: um estudo de caso em empresa pública. In: MANAGEMENT IN IBEROAMERICAN COUNTRIES: CURRENT TRENDS AND FUTURE PROSPECTS, 3. 2003, São Paulo. Proceedings…São Paulo: Iberoamerican Academy of Management, Brazil, 1 CD ROM. SEVERINO, Antônio Joaquim. Teoria e Prática Científica. In: ______. Metodologia do Trabalho Científico. 23. ed. rev. e atualizada. 4. reimpressão. São Paulo: Cortez, 2007. SILVA, Tania Moura. Currículo Flexível: Evolução e Competência. Artigo publicado na Revista Brasileira de Contabilidade do CFC, edição Ano XXIX – No. 121 – Janeiro/Fevereiro 2000. Graduado em Ciências Contábeis pela Universidade da Amazônia – UNAMA, Bacharel em Administração pela Universidade da Amazônia – UNAMA. 1 Graduado em Ciências Contábeis pela Universidade da Amazônia – UNAMA, Bacharel em Administração pela Universidade da Amazônia – UNAMA. 1 RevistaFT A RevistaFT é uma Revista Científica Eletrônica Multidisciplinar Indexada de Alto Impacto e Qualis “B2” em 2023. Periodicidade mensal e de acesso livre. Leia gratuitamente todos os artigos e publique o seu também clicando aqui. Contato Queremos te ouvir. WhatsApp: 11 98597-3405 Queremos te ouvir. WhatsApp: 11 98597-3405 Queremos te ouvir. WhatsApp: 11 98597-3405 Queremos te ouvir. 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https://openalex.org/W2902358926	https://repository.arizona.edu/bitstream/10150/632363/1/fpsyt-09-00636.pdf	English	null	A Subset of Patients With Autism Spectrum Disorders Show a Distinctive Metabolic Profile by Dried Blood Spot Analyses	Frontiers in psychiatry	2,018	cc-by	9,766	A Subset of Patients With Autism Spectrum Disorders Show a Distinctive Metabolic Profile by Dried Blood Spot Analyses Item Type Article Authors Barone, Rita; Alaimo, Salvatore; Messina, Marianna; Pulvirenti, Alfredo; Bastin, Jean; Ferro, Alfredo; Frye, Richard E.; Rizzo, Renata Citation Barone R, Alaimo S, Messina M, Pulvirenti A, Bastin J, MIMIC- Autism Group, Ferro A, Frye RE and Rizzo R (2018) A Subset of Patients With Autism Spectrum Disorders Show a Distinctive Metabolic Profile by Dried Blood Spot Analyses. Front. Psychiatry 9:636. doi: 10.3389/fpsyt.2018.00636 DOI 10.3389/fpsyt.2018.00636 Publisher FRONTIERS MEDIA SA Journal FRONTIERS IN PSYCHIATRY Rights © 2018 Barone, Alaimo, Messina, Pulvirenti, Bastin, MIMIC- Autism Group, Ferro, Frye and Rizzo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Download date 24/10/2024 05:52:31 Item License https://creativecommons.org/licenses/by/4.0/ Version Final published version Link to Item http://hdl.handle.net/10150/632363 ORIGINAL RESEARCH published: 07 December 2018 doi: 10.3389/fpsyt.2018.00636 A Subset of Patients With Autism Spectrum Disorders Show a Distinctive Metabolic Proﬁle by Dried Blood Spot Analyses Rita Barone 1,2†, Salvatore Alaimo 3†, Marianna Messina 2, Alfredo Pulvirenti 3, Jean Bastin 4,5, MIMIC-Autism Group, Alfredo Ferro 3, Richard E. Frye 6,7 and Renata Rizzo 1 1 Child Neurology and Psychiatry, Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy, 2 Referral Centre for Inherited Metabolic Disorders, Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy, 3 Bioinformatics Unit, Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy, 4 Sorbonne Paris Cité, Faculté des Sciences Fondamentales et Biomédicales, Université Paris Descartes, Paris, France, 5 INSERM, UMR-S 1124, Toxicologie, Pharmacologie et Signalisation Cellulaire, Paris, France, 6 University of Arizona College of Medicine, Phoenix, AZ, United States, 7 Phoenix Children’s Hospital, Phoenix, AZ, United States Keywords: autism spectrum disorders, dried blood spots, ESI-MS/MS, mitochondrial fatty acid β-oxidation, machine learning Correspondence: Rita Barone rbarone@unict.it †These authors have contributed equally to this work Specialty section: This article was submitted to Molecular Psychiatry, a section of the journal Frontiers in Psychiatry Received: 06 July 2018 Accepted: 08 November 2018 Published: 07 December 2018 Autism spectrum disorder (ASD) is currently diagnosed according to behavioral criteria. Biomarkers that identify children with ASD could lead to more accurate and early diagnosis. ASD is a complex disorder with multifactorial and heterogeneous etiology supporting recognition of biomarkers that identify patient subsets. We investigated an easily testable blood metabolic proﬁle associated with ASD diagnosis using high throughput analyses of samples extracted from dried blood spots (DBS). A targeted panel of 45 ASD analytes including acyl-carnitines and amino acids extracted from DBS was examined in 83 children with ASD (60 males; age 6.06 ± 3.58, range: 2–10 years) and 79 matched, neurotypical (NT) control children (57 males; age 6.8 ± 4.11 years, range 2.5–11 years). Based on their chronological ages, participants were divided in two groups: younger or older than 5 years. Two-sided T-tests were used to identify signiﬁcant differences in measured metabolite levels between groups. Näive Bayes algorithm trained on the identiﬁed metabolites was used to proﬁle children with ASD vs. NT controls. Of the 45 analyzed metabolites, nine (20%) were signiﬁcantly increased in ASD patients including the amino acid citrulline and acyl-carnitines C2, C4DC/C5OH, C10, C12, C14:2, C16, C16:1, C18:1 (P: < 0.001). Näive Bayes algorithm using acyl- carnitine metabolites which were identiﬁed as signiﬁcantly abnormal showed the highest performances for classifying ASD in children younger than 5 years (n: 42; mean age 3.26 ± 0.89) with 72.3% sensitivity (95% CI: 71.3;73.9), 72.1% speciﬁcity (95% CI: 71.2;72.9) and a diagnostic odds ratio 11.25 (95% CI: 9.47;17.7). Re-test analyses as a measure of validity showed an accuracy of 73% in children with ASD aged ≤5 years. This easily testable, non-invasive proﬁle in DBS may support recognition of metabolic ASD individuals aged ≤5 years and represents a potential complementary tool to improve diagnosis at earlier stages of ASD development. Edited by: Chad A. Bousman, University of Calgary, Canada Reviewed by: Ahmad Abu-Akel, Université de Lausanne, Switzerland Frank Middleton, Upstate Medical University, United States Correspondence: Rita Barone rbarone@unict.it †These authors have contributed equally to this work Correspondence: Rita Barone rbarone@unict.it Correspondence: Rita Barone rbarone@unict.it INTRODUCTION increased oxidation damage marker, dityrosine, in plasma proteins, capable to classify the disease status (19). Autism spectrum disorder (ASD) is a neurodevelopmental disorder that aﬀects approximately 16.8 per 1,000 (one in 59) children aged 8 years in the US and with a male/female ratio of 4:1 (1). In Italy, a recent study reported an overall prevalence rate of one in 100 children at age 7–8 years (2). ASD is characterized by signiﬁcant defects of social communication and interaction and by restricted and repetitive patterns of interests and activities with onset in early childhood (3). The etiology remains poorly understood. Increasing evidence has converged on possible interactions among pleiotropic genetic background conferring vulnerabilities to environmental inputs leading to multiple systemic co-morbidities including metabolic disarrangement (4). Classic inborn errors of metabolism (IEM) aﬀect a subgroup of ASD patients accounting for 1–3% of patients (5). Acquired symptoms featuring autism or childhood disintegrative disorder may occur in the neuronopathic lysosomal storage disorders (6, 7). Among IEM, primary mitochondrial diseases aﬀect nearly 5% of patients with ASD, however the occurrence of abnormal biomarkers indicating mitochondrial dysfunction is higher in patients with ASD than in the general population (8). On a clinical ground, children with ASD may exhibit features of a mitochondrial disease such as hypotonia and delayed motor development as well as gastrointestinal disturbances and regression following fever or other environmental triggers (9). Since the 1980s, electrospray ionization (ESI) and tandem MS/MS technology endorsed high throughput analyses of samples extracted from dried blood spots (DBS) for newborn screening of IEM as health care standard (20). Thus, we hypothesized that ESI-MS/MS analyses of diﬀerent metabolites in DBS might represent a high throughput method for metabolic proﬁling of individuals with ASD by a single injection, in a rapid, low-cost, and suitable procedure. To test this hypothesis, we used a standardized ESI-MS/MS analyses in DBS to systematically examine the levels of a large panel of highly selective biochemical analytes in patients with ASD and healthy TD, matched-control subjects. The targeted metabolites include acyl-carnitines and amino acids representing a set of ASD candidate metabolic markers. We propose a novel approach applying machine learning methods to assess diﬀerences in the metabolic proﬁle between ASD and age-matched healthy TD controls. Citation: Barone R, Alaimo S, Messina M, Pulvirenti A, Bastin J, MIMIC-Autism Group, Ferro A, Frye RE and Rizzo R (2018) A Subset of Patients With Autism Spectrum Disorders Show a Distinctive Metabolic Proﬁle by Dried Blood Spot Analyses. Front. Psychiatry 9:636. doi: 10.3389/fpsyt.2018.00636 December 2018 \| Volume 9 \| Article 636 Frontiers in Psychiatry \| www.frontiersin.org 1 Metabolic Risk Proﬁle in ASD Barone et al. INTRODUCTION This represents a promising novelty in the ﬁeld given that previous analyses of multiple analytes in ASD often resort to a one-at- a-time approach that does not consider the data as a whole. Using univariate and multivariate data modeling, we outlined a metabolic risk proﬁle capable to classify a subset of ASD patients from TD children. The study supports identiﬁcation of metabolic ASD subtype whose distinguishing features suggest a reduced ﬂux through the mitochondrial fatty acid β-oxidation (FAO) pathway. Clinical diagnosis of ASD relies on behavioral tests. Early recognition and specialized intervention improve the outcome and are most eﬀective if initiated early in life (10). Thus, the development of multiple laboratory markers that can assist in the early and accurate diagnosis of ASD is envisaged. Urinary metabolomic studies (11–16) and a few studies performed on blood samples (17–19) collectively showed modiﬁcation of amino acid, purine and fatty acid metabolic pathways, increased oxidative stress, gut dysbiosis and altered gut permeability in individuals with ASD. Multiplatform analytical methodology and multivariate analysis may provide the best models discriminating between ASD and typically developing (TD) children. Through this approach, a rigorous analysis for the discovery of ASD biomarkers combined several mass spectrometry (MS)-based analyses of blood. This combined analysis resulted in 40 features could diﬀerential ASD and TD samples with an accuracy of 70% (17). More recently, a study in 38 children with ASD reported increased advanced glycation endproducts, Nε-carboxymethyllysine and Nω-carboxymethylarginine, and Abbreviations: ASD, autism spectrum disorders; IEM, inborn errors of metabolism; ESI-MS/MS, electrospray ionization-tandem mass spectrometry. DBS, dried blood spots; TD, typically developing; FAO, fatty acid β-oxidation; ADI-R, Autism Diagnostic Interview-Revised; ADOS, Autism Diagnostic Observation Schedule; CSS, Calibrated Severity Score; DD, developmental delay; ID, intellectual disability; PPV, Positive Predictive Value; NPV, Negative Predictive Value; DOR, Diagnostic Odds Ratio.; RF, Random Forest, SVM, Support Vector Machine; LM, Linear Regression; PART, Recursive Partition Tree; SCAD, short chain acyl-CoA dehydrogenase; MCAD, medium chain acyl-CoA dehydrogenase, VLCAD, very long chain acyl-CoA dehydrogenase; LCHAD, long chain 3- hydroxyacyl-CoA dehydrogenase; PA, propionic academia; PPA, propionic acid; NSC, neural stem cells; CPT, carnitine palmitoyl transferase. METHODS A total of 162 Caucasian subjects with age ranging from 30 months to 11 years were included in a case-control study during an 18-month period (January 1, 2016–June 30, 2017) at the Child Neurology and Psychiatry Unit of the University Children Hospital Catania, Italy. Participants comprised 83 children with the ASD diagnosis (60 males, 23 females; age 6.06 ± 3.6; range: 2–10 years) and 79 healthy TD controls, with a similar age and gender distribution as the patients (57 males, 22 females; age 6.8 ± 4.1; range 2.5–11 years) (Table 1). The Institutional Review Board at University Hospital of Catania approved the study that was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments (Helsinki Declaration 1975, revision 2013). Written informed consent was obtained from all ASD participants’ parent or legal guardian in order to enter clinical and laboratory data from the clinical ﬁles into the present study. Diagnosis of ASD was obtained according to strict criteria using standardized diagnostic tests including the Autism Diagnostic Interview-Revised (ADI-R) (21) and Autism Diagnostic Observation Schedule (ADOS) (22). The Calibrated Severity Score (CSS) from 4 to 10 was used as a measure of autism severity (23). Developmental quotient (DQ) and/or Intellectual quotient (IQ) were measured in all participants by a comprehensive, standardized neuropsychological assessment battery administered according to age. Among ASD individuals, exclusion criteria were the presence of December 2018 \| Volume 9 \| Article 636 Frontiers in Psychiatry \| www.frontiersin.org 2 Metabolic Risk Proﬁle in ASD Barone et al. TABLE 1 \| Demographic and clinical characteristics of ASD patients and TD controls divided by age. ASD TD Participant characteristics Total sample (n = 83) Age ≤5 (n = 42) Age > 5 (n = 41) P-value Total sample (n = 79) Age ≤5 (n = 35) Age > 5 (n = 44) P-value* Age (years) 6.06 ± 3.58 3.26 ± 0.89 8.9 ± 2.98 n.a. 6.8 ± 4.11 3.06 ± 1.5 9.7 ± 2.86 n.a. Boys (%) 60 (72.3) 29 (69.04) 31 (75.6) 0.653 57 (72.1%) 24 (68.5%) 33(75%) 0.692 DQ/IQ 63.2 ± 20.8 56.8 ± 17.1 69.6 ± 22.4 0.022 93.9 ± 14.2 89.6 ± 11.2 95.3 ± 10.5 0.752 DD/ID (%) 54 (65.1) 32 (76.2) 22 (53.6) 0.035 n.a n.a. n.a. n.a. Regression (%) 29 (35) 19 (45.2) 10 (24.4) 0.065 n.a n.a. n.a. n.a. Biospecimen Collection, Processing and MS/MS Analysis ( ) TD children were recruited among subjects that underwent morning fasting blood analyses screening for sideropenic anemia that was deﬁnitely ruled out in all included TD participants. Full informed consent was signed from parents to participate in the study. TD participants’ exclusion criteria included positive history for inherited metabolic diseases, intellectual disability or other developmental, neurological, or behavioral problems and inﬂammatory bowel diseases/celiac disease. The Social Communication Questionnaire (24) was used to screen and exclude autism in TD children. Since artifacts in plasma acylcarnitine levels are possible due to diet enriched with fatty acids (MCT-oil, ketogenic diet) (25), we ensured that no participants underwent fatty acids enriched diet, such as ketogenic diet or MCT-oil, at least 6 months before sample collection. To avoid systematic diﬀerences related to the time of sample collection, blood spots on ﬁlter paper card (Whatman card Specimen 903) were collected from each participant in the ASD and TD groups in the morning between 8.00 and 8.30 a.m. after nocturnal fasting. Samples from the NT children were prospectively collected in the same period, along with ASD children samples. Once dried, blood spots were stored at 4◦C in a unique refrigerator with controlled humidity rate and processed within 2 weeks after sampling. A 3.2 mm diameter blood dot of each individual was used for the analyses. Underivatized specimens were analyzed using electrospray ionization (ESI)-Tandem MS/MS system. Forty-ﬁve metabolites including amino acids, free carnitine and acyl-carnitines (saturated, unsaturated, hydroxylated, and dicarboxylated) were simultaneously measured in DBS. The analyte concentration was quantiﬁed by comparison with known concentration of corresponding stable-isotope internal standards. Results of targeted 45 metabolites in ASD participants were considered in comparison with age-matched reference ranges obtained from studied TD healthy subjects. A 3.2 mm diameter blood dot of each individual was used for the analyses. Underivatized specimens were analyzed using electrospray ionization (ESI)-Tandem MS/MS system. Forty-ﬁve metabolites including amino acids, free carnitine and acyl-carnitines (saturated, unsaturated, hydroxylated, and dicarboxylated) were simultaneously measured in DBS. The analyte concentration was quantiﬁed by comparison with known concentration of corresponding stable-isotope internal standards. Results of targeted 45 metabolites in ASD participants were considered in comparison with age-matched reference ranges obtained from studied TD healthy subjects. Metabolic Work-Up in ASD Subjects ASD patients underwent blood and urine collection in the morning between 8.00 and 8.30 a.m. after nocturnal fasting. METHODS Autism severity (ADOS CSS)◦ 6.7 ± 1.8 6.6 ± 1.8 6.8 ± 1.8 0.845 n.a. n.a n.a. n.a. Fisher’s Exact Test was performed for discrete variables gender, DD/ID and regression. T-test was performed for continuous variables DQ/IQ and ADOS-CSS. ◦The Social Communication Questionnaire was used to screen and exclude autism in TD children. DQ, developmental quotient; IQ, intelligence quotient; DD, developmental disability; ID, intellectual disability; ADOS, Autism Diagnostic Observation Schedule. CSS, Calibrated Severity Score. n.a., not applicable. TABLE 1 \| Demographic and clinical characteristics of ASD patients and TD controls divided by age. Fisher’s Exact Test was performed for discrete variables gender, DD/ID and regression. T-test was performed for continuous variables DQ/IQ and ADOS-CSS. ◦The Social Communication Questionnaire was used to screen and exclude autism in TD children. DQ, developmental quotient; IQ, intelligence quotient; DD, developmental disability; ID, intellectual disability; ADOS, Autism Diagnostic Observation Schedule. CSS, Calibrated Severity Score. n.a., not applicable. dehydrogenase deﬁciency (MCAD). Molecular analyses was not signiﬁcant for any mutations associated to MCAD in these patients. an associated monogenic disease (i.e., Fragile-X syndrome, Tuberous Sclerosis), positive chromosomal microarray analysis, positive history for mitochondrial disease or known medical conditions including autoimmune disease and inﬂammatory bowel diseases (IBD)/celiac disease. Biospecimen Collection, Processing and MS/MS Analysis Routine blood analyses including glucose, transaminases, cholesterol, triglycerides, creatine kinase, electrolytes and thyroid hormones were normal. Morning fasting lactate and ammonia blood levels were increased in 12.5 and 22.2% of patients, respectively in line with previous reported rates of increased markers of mitochondrial dysfunction in ASD (8). Twenty-ﬁve out of 40 studied subjects (62.5%) had signiﬁcantly decreased blood Vitamin D3 levels with normal Ca/P ratio. Urinary organic acids by using Gas Chromatography/MS detected increased excretion of ketone bodies in ﬁve patients. One patient showed increased urinary 3-hydroxy-isovaleric acid with normal plasma biotinidase activity. In two sibs with ASD, the acylcarnitine proﬁle showed increase of C8, C10, C10:1 carnitine levels suggesting medium-chain acyl-CoA Frontiers in Psychiatry \| www.frontiersin.org Metabolic Proﬁle of Target Analytes by ESI-MS/MS of Blood Spots in Patients With ASD and Healthy Control Subjects y j Over 45 analyzed metabolites in DBS, nine (20%) were signiﬁcantly increased in ASD patients with respect to healthy, age-matched subjects (Table 2). The increased metabolites in ASD patients included eight acyl-carnitines such as short-chain (2-5 carbon length) C2 and C4DC\C5OH, medium-chain (6– 12 carbon length) C10 and C12, and long-chain acyl-carnitines (13-18 carbon length) C14:2, C16, C16:1, C18:1. Among eleven studied amino acids, citrulline levels were increased in ASD patients (Table 2). Volcano plot showing the distribution of log-fold-changes vs. statistical signiﬁcance (p-value) of the metabolites and individual swarm plots for the subject data, split by diagnosis, for the nine changed metabolites are reported in supplementary materials (Figures S1–S4). We estimated an eﬀect size of 0.6 as the absolute diﬀerence between the mean of the most discriminant metabolites (short chain C2 and C4DC/C5OH acylcarnitines and long chain C10, C12, C14:2, C16, C16:1, C18:1 acylcarnitines, citrulline) within each class divided by the pooled variance observed between the two classes. This yielded a power of 0.93 at signiﬁcance level of 0.05 suggesting a high practical signiﬁcance. The power was also estimated within the two age groups at 0.79 for patient younger than 5 y. o. and 0.78 for older patients. The workﬂow of the study is depicted in Figure 1. Our dataset, comprising 83 ASD patients and 79 healthy controls, was randomly partitioned into a training set of 124 samples (67 ASD patients and 57 healthy controls) for identiﬁcation of the classiﬁcation modeling, and 38-sample holdout set (16 ASD patients and 22 healthy controls). Due to the small cohort size, keeping a large part of the samples in the training set is needed to proper identify the classiﬁcation model (28). For this reason, we kept two third of the samples in the training set and the remainder was used as a holdout validation set. Samples were properly randomized using diagnosis, age and gender to establish a similar proportion of factors on both training and holdout sets. Such holdout strategy was repeated 1,000 times to estimate average performances together with a 95% CI of the classiﬁcation model. In addition, a validation test was performed in an independent set of 29 ASD participants randomly recruited for re-test analyses. Neurotypical controls were not included in this analysis because further blood sampling was not achieved in the TD group. Metabolic Proﬁle of Target Analytes by ESI-MS/MS of Blood Spots in Patients With ASD and Healthy Control Subjects Finally, classiﬁcation performance was evaluated by permutation testing in order to establish a distribution of chance estimates. For this purpose, we trained the classiﬁer with the 124-sample training set with randomized group labels (ASD vs. TD) many times (≈1,000). This allowed establishing a chance distribution that could be used for comparison. Spearman correlations showed that in the ASD sample metabolite levels did not correlate with age, developmental or intellectual quotient and autism severity score (CSS) (Table 3). Statistical Analyses Blood levels of forty-ﬁve targeted analytes (µmol/L) obtained from 162 subjects, 83 ASD patients and 79 TD healthy controls, were evaluated. Metabolites, with statistically signiﬁcant diﬀerent blood levels between ASD and healthy TD control children were identiﬁed by using the R package limma (26). Since data supports equal population variances together with normal distributions, a T-test was applied. The p-value produced by the two-sided T-test, employed by limma, was corrected using the Benjamini & Hochberg method in order to estimate the False Discovery Rate (27). All diﬀerences were considered to be statistically December 2018 \| Volume 9 \| Article 636 Frontiers in Psychiatry \| www.frontiersin.org 3 Metabolic Risk Proﬁle in ASD Barone et al. signiﬁcant at a 5% probability level. Possible associations between the identiﬁed metabolites and clinical features of ASD patients were veriﬁed by Spearman correlations analyses. respectively). ASD patients did not fulﬁll diagnostic criteria for probable or deﬁnite mitochondrial disorder according to Morava mitochondrial disease criteria system (29). Less than 5% of studied ASD children had hypotonia and/or epileptic seizures. None presented with ataxia, peripheral neuropathy, sensorineural deafness, cardiomyopathy or endocrinological problems which are common features of mitochondrial diseases. Training and Testing Set Model Performance Next, we trained a classiﬁer based on the Naïve Bayes algorithm making use of the training set and adopting as predictor variables only the nine metabolites diﬀering signiﬁcantly between ASD and TD subjects (P: < 0.001) (Table 2). The results were veriﬁed on the holdout set with the purpose of checking the robustness of the procedure. Classiﬁcation Modeling Model development was performed with the aim to detect metabolic features useful to proﬁle ASD patients vs. healthy NT controls. For this purpose we trained an algorithm on the discriminant metabolites identiﬁed as described. The methodology was evaluated in terms of Sensitivity = TP/(TP + FN), Speciﬁcity = TN/(TN + FP), where TP is the number of true positives, i.e., the number of patients correctly classiﬁed; TN is the number of true negatives, i.e., number of controls correctly classiﬁed; FP is the number of false positives, i.e., number of controls classiﬁed as patients; and FN is the number of false negatives, i.e., number of patients classiﬁed as controls. Diagnostic odds ratios and 95% conﬁdence intervals were evaluated. Frontiers in Psychiatry \| www.frontiersin.org Participant Characteristics Participant Characteristics Based on their chronological ages, participants were divided in two groups: younger (ASD n.42; TD n.35) or older than 5 years (ASD n.41; TD n.44). Demographic data and clinical features of all participants in the two age groups, such as presence of developmental delay (DD), intellectual disability (ID) (IQ < 70) and symptoms of regressive autism are presented in Table 1. The rate and extent of DD/ID were higher in children with ASD younger than 5 years (P: 0.035 and P: 0.0228, respectively). No signiﬁcant diﬀerences were found in the rate of patients with regressive autism and in the degree of autism severity (CSS) between the two age groups (P: 0.065 and P: 0.845, p To assess the model and the predictive power of the selected metabolites, we compared the Naïve Bayes algorithm, with other classiﬁcation techniques such as C-tree, Random Forest (RF), Support Vector Machine (SVM), Linear Regression (LM), and Recursive Partition Tree (PART) (online methods). Our ﬁnal choice fell on the Naïve Bayes algorithm due to its robustness and stability. The training procedure led to the selection of acyl- carnitines C2 and C4DC\C5OH, C10, C12, C14:2, C16, C16:1, C18:1 as the most promising classiﬁcation variables. Naïve Bayes algorithm, using a 8 feature set, reaches an overall classiﬁcation Frontiers in Psychiatry \| www.frontiersin.org December 2018 \| Volume 9 \| Article 636 4 Metabolic Risk Proﬁle in ASD Barone et al. FIGURE 1 \| Workﬂow of the study. Blood acylcarnitines (m/z) C2 (204.3); C4DC\C5OH (262.2); C10 (316.2); C12 (344.3); C14:2 (368.3); C16 (400.3); C16:1 (398.3); C18:1 (286.2) (red asterisks) measured in DBS are increased in patients with ASD. Näive Bayes algorithm trained on the identiﬁed metabolites was used to proﬁle children with ASD vs. healthy controls. Blue asterisks indicate correspondent stable-isotope internal standards. FIGURE 1 \| Workﬂow of the study. Blood acylcarnitines (m/z) C2 (204.3); C4DC\C5OH (262.2); C10 (316.2); C12 (344.3); C14:2 (368.3); C16 (400.3); C16:1 (398.3); C18:1 (286.2) (red asterisks) measured in DBS are increased in patients with ASD. Näive Bayes algorithm trained on the identiﬁed metabolites was used to proﬁle children with ASD vs. healthy controls. Blue asterisks indicate correspondent stable-isotope internal standards. performance with 73.3% sensitivity (95% CI 72.6–73.9), 63.4% speciﬁcity (95% CI 62.8–64), 6.78 DOR (95% CI, 6.39–7.16). individuals older than 5 years (n = 18 samples, 67% accuracy). Participant Characteristics It should be noted that the re-test validation set only includes participants with an ASD diagnosis. Therefore, the validation can only test for true positive and false negative ASD classiﬁcations. Permutation Testing Taking into account that ASD are neurodevelopmental disorders we considered closely possible interactions of measured metabolites with participant ages. For this purpose, we divided the sample into two groups according to age (≤5 years and >5 years) and we applied the classiﬁer to discriminate among ASD and TD control subjects in each age group. Table 4 presents the predictive performances of the metabolic proﬁle (measures and 95% CI), using Naïve Bayes and other compared classiﬁers, for all participants; participants aged ≤5 years and >5 years. Classiﬁer performance was evaluated using permutation testing. Permutation testing can be used to evaluate the probability of getting speciﬁcity and sensitivity values higher than the ones obtained during the cross-validation procedure by chance. In order to establish a distribution of chance estimates, we trained the classiﬁer with the 124-sample training set each time randomly assigning patient and control labels to each sample many times (≈1,000) and repeated the cross-validation procedure. The results show that the quality of the classiﬁcation in such a case is even lower than expected values in the case of a random classiﬁer model with 43.9% accuracy (95% CI: 43.3;44.5) (Table S1). We deﬁnitely demonstrated that the accuracy of the classiﬁer (73%) is signiﬁcantly better than expected by chance alone as the classiﬁcation algorithms are actually able to extract molecular patterns that distinguish patients, with respect to a chance distribution. We found an increased competitiveness of the framework for classifying ASD in toddlers (n: 42 subjects mean age 3.26 ± 0.89) 72.3% sensitivity (95% CI: 71.3;73.9), 72.1% speciﬁcity (95% CI: 71.2;72.9), and diagnostic odds ratio (DOR) 11.25 (95% CI: 9.47;17.74). Furthermore, by applying our classiﬁcation framework to subjects older than 5 years of age, we found a reduction in performance compared to younger subjects: Sensitivity 67.5% (95% CI: 66.6; 68.4) Speciﬁcity 56.9% (56.1;57.7), DOR 4.29% (95% CI: 4.09;4.56). Frontiers in Psychiatry \| www.frontiersin.org Validation Test TABLE 2 \| Statistical signiﬁcant metabolites in ASD participants with respect to TD participants. Logarithm of the fold-change (Log-FC) between the classes, average concentration in each age subgroup, t-test statistic with its p-value and Benjamini & Hochberg adjusted p-value. Isomers or isobars metabolites. ASD, Autism Spectrum Disorders; TD, Typical Development; y, year. TABLE 3 \| Spearman correlations computed for the metabolites listed in Table 2 in relation to the quantitative clinical variables Age, DQ/IQ, and CSS. Age DQ/IQ CSS Metabolite p-Value p-Value p-Value C5OH\C4DC 0.0771 0.4939 0.1176 0.2959 −0.1122 0.3188 C2 −0.1497 0.1821 −0.0634 0.5741 −0.0083 0.9414 C12 0.1103 0.3271 −0.0415 0.7129 −0.1488 0.1850 C18:1 0.0093 0.9341 −0.0986 0.3814 0.0155 0.8906 C16 −0.0130 0.9084 −0.0380 0.7362 −0.1340 0.2328 CIT −0.1751 0.1180 −0.0685 0.5434 −0.1230 0.2741 C16:1 0.1286 0.2526 −0.0818 0.4677 −0.0795 0.4803 C14:2 −0.0410 0.7163 0.0060 0.9578 −0.0842 0.4551 C10 0.0843 0.4545 −0.0649 0.5647 −0.0607 0.5906 For each metabolite we report the computed correlation together with a p-value, which indicates whether the observed correlation is statistically signiﬁcant, under the null hypothesis that values are uncorrelated. Isomers or isobars metabolites. DQ, developmental quotient; IQ, intelligence quotient; CSS, Calibrated Severity Score. For each metabolite we report the computed correlation together with a p-value, which indicates whether the observed correlation is statistically signiﬁcant, under the null hypothesis that values are uncorrelated. Isomers or isobars metabolites. DQ, developmental quotient; IQ, intelligence quotient; CSS, Calibrated Severity Score. such as Crohn’s disease (33). Patients with classic citrullinemia (type I) (argininosuccinate synthetase 1 gene mutation) present with elevated citrulline levels along with hyperammonemia and variable neurological symptoms in the neonatal period or later on. Interestingly, it was demonstrated that cumulative exposure to ammonia and citrulline are the most reliable indicators of poorer cognitive functioning in patients with classic citrullinemia (34). a signiﬁcant increase of blood short-chain, long-chain acyl- carnitines and, to a lesser extent, medium-chain acyl-carnitines. Our ﬁndings in a Sicilian ASD population (Mediterranean area) conﬁrm the same, unique pattern of acyl-carnitine proﬁle, which has been ﬁrst systematically detected in ASD individuals from US, (32) deﬁning a broadest coverage of ethnic and regional groups. It is worth noting that distinct metabolite diﬀerences could be related to co-morbid undiagnosed medical conditions such as gastrointestinal disturbances that are frequently observed in ASD. In the present study we found signiﬁcantly increased citrulline levels in children with ASD. Citrulline is an intermediate metabolic amino acid produced primarily by enterocytes. Frontiers in Psychiatry \| www.frontiersin.org Validation Test ASD is a polygenic multifactorial disorder with variable underlying mechanisms including energy metabolism disarrangement among others (30). Recognition of speciﬁc classes of ASD patients by biological markers has been considering eﬀective for better understanding molecular mechanisms and to guide tailored therapeutic strategies in patient subset (31). In the current study we pursued to set up an easily testable blood metabolic proﬁle in DBS to support early recognition of metabolic subtype patients at risk for ASD diagnosis. We found in an ASD population without clinical relevant features secondary to primary mitochondrial disease, Results were conﬁrmed on independent validation test. For validation analyses, a set of 29 ASD participants was randomly recruited for re-test analyses. For this purpose, blood spot collection for metabolite analyses was repeated at the same conditions a second time after a mean time interval of 6.86 ± 3.8 months. Data from re-test were used as validation set. The initial values from total 132 subjects were used as training set. The results show an overall accuracy of 69% (20 patients correctly classiﬁed as ASD, and 9 misclassiﬁed as healthy). Splitting the re- test set by age we found a greater accuracy in younger subjects (≤5 years of age) (n = 11 samples, 73% accuracy) compared to December 2018 \| Volume 9 \| Article 636 Frontiers in Psychiatry \| www.frontiersin.org 5 Metabolic Risk Proﬁle in ASD Barone et al. TABLE 2 \| Statistical signiﬁcant metabolites in ASD participants with respect to TD participants. Metabolite Abbreviation Log-FC Average concentration t p-Value (ASD vs. TD) Adjusted p-Value ASD TD ≤5 y >5 y ≤5 y >5 y Citrulline CIT 0.3601 4.7594 4.5606 4.3556 4.2129 3.7337 0.0003 0.0020 Acetylcarnitine C2 0.3318 3.5333 3.5503 3.1886 3.2330 4.2770 0.0000 0.0007 Methylmalonyl/3-OH- isovalerylcarnitine* C4DC\C5OH 0.0762 0.4734 0.5062 0.4058 0.4224 4.4927 0.0000 0.0006 Decanoylcarnitine C10 0.0395 0.1468 0.1643 0.1035 0.1352 2.6288 0.0004 0.0470 Dodecanoylcarnitine C12 0.0246 0.0714 0.0762 0.0414 0.0556 4.1838 0.0000 0.0007 Tetradecadienoylcarnitine C14:2 0.0109 0.0398 0.0377 0.0211 0.0322 3.5912 0.0004 0.0025 Hexadecanoylcarnitine C16 0.1409 1.0594 1.0934 0.9499 0.9180 3.7904 0.0002 0.0019 Hexadecenoylcarnitine C16:1 0.0145 0.0758 0.0805 0.0634 0.0637 3.6726 0.0003 0.0021 Octadecenoylcarnitine C18:1 0.1302 1.0725 1.1304 0.9295 0.9945 3.8526 0.0002 0.0019 Logarithm of the fold-change (Log-FC) between the classes, average concentration in each age subgroup, t-test statistic with its p-value and Benjamini & Hochberg adjusted p-value. *Isomers or isobars metabolites. ASD, Autism Spectrum Disorders; TD, Typical Development; y, year. Validation Test Blood citrulline level is considered a biomarker of gastrointestinal mucosal surface and enterocyte integrity. Previous studies showed that citrulline levels are inversely correlated with severity of intestinal malabsorption disease (i.e., coeliac disease) and inﬂammatory bowel disease (IBD) Moreover, the existence of distinct metabolite diﬀerences could relate to concurrent vitamin D deﬁciency that was observed in a large proportion of patients with ASD (62%) in this study. Vitamin D has a pivotal role in neurodevelopment through several mechanisms including gene regulation and anti-inﬂammation/immunological modulation. Lower Vitamin D levels were consistently reported in subsets of patients with ASD compared to healthy controls (35). Carnitine is mainly provided in the diet, but is synthesized at extremely low rates from trimethyl-lysine residues generated during protein December 2018 \| Volume 9 \| Article 636 6 Metabolic Risk Proﬁle in ASD Barone et al. TABLE 4 \| Classiﬁers performances for all participants (A); participants aged ≤5 years (B) and >5 years (C). Classiﬁer Sensitivity Speciﬁcity DOR (A) All participants Naïve Bayes 0.7332 [0.7267; 0.7397] 0.6345 [0.6287; 0.6404] 6.7823 [6.3956; 7.1690] C-tree 0.6715 [0.6590; 0.6841] 0.4942 [0.4824; 0.5060] 2.7437 [2.6171; 2.8703] RF 0.7296 [0.7229; 0.7364] 0.5670 [0.5608; 0.5731] 4.8079 [4.5668; 5.0490] SVM 0.7319 [0.7250; 0.7388] 0.5939 [0.5875; 0.6003] 5.5428 [5.2650; 5.8207] LM 0.6324 [0.6250; 0.6397] 0.6585 [0.6523; 0.6646] 4.4804 [4.2328; 4.7280] PART 0.6281 [0.6196; 0.6365] 0.5657 [0.5576; 0.5738] 2.9980 [2.8474; 3.1486] (B) ≤age 5 years Naïve Bayes 0.7237 [0.7137; 0.7337] 0.7209 [0.7125; 0.7293] 10.1235 [9.4725; 10.7746] C-tree 0.7590 [0.7441; 0.7739] 0.3574 [0.3399; 0.3749] 2.8163 [2.6411; 2.9914] RF 0.8270 [0.8187; 0.8353] 0.5464 [0.5358; 0.5569] 7.1375 [6.5862; 7.6888] SVM 0.8017 [0.7925; 0.8109] 0.6202 [0.6105; 0.6300] 8.1844 [7.6207; 8.7481] LM 0.6567 [0.6450; 0.6684] 0.6871 [0.6771; 0.6972] 7.1923 [6.6648; 7.7198] PART 0.7031 [0.6914; 0.7149] 0.4532 [0.4407; 0.4658] 2.9037 [2.6924; 3.1150] (C) >age 5 years Naïve Bayes 0.6757 [0.6665; 0.6849] 0.5692 [0.5610; 0.5775] 4.2905 [4.0193; 4.5616] C-tree 0.5071 [0.4867; 0.5275] 0.5468 [0.5290; 0.5645] 1.6693 [1.5826; 1.7559] RF 0.5881 [0.5780; 0.5982] 0.5248 [0.5157; 0.5339] 2.3622 [2.2069; 2.5175] SVM 0.5878 [0.5780; 0.5976] 0.6295 [0.6208; 0.6382] 3.9369 [3.6500; 4.2238] LM 0.6039 [0.5939; 0.6139] 0.6022 [0.5933; 0.6112] 3.7105 [3.4456; 3.9755] PART 0.5264 [0.5143; 0.5386] 0.5367 [0.5260; 0.5474] 2.0030 [1.8714; 2.1347] Compared classiﬁcation algorithms: Naïve Bayes, C-tree, Random Forest (RF), Support Vector Machine (SVM), Linear Regression Model (LM), and Recursive Partition Tree (PART). For each classiﬁer sensitivity, speciﬁcity, of the model and diagnostic odds ratio (DOR) are shown. Validation Test All the measures are reported together with the bounds of the 95% CI. TABLE 4 \| Classiﬁers performances for all participants (A); participants aged ≤5 years (B) and >5 years (C). Classiﬁer Sensitivity Speciﬁcity d classiﬁcation algorithms: Naïve Bayes, C-tree, Random Forest (RF), Support Vector Machine (SVM), Linear Regression Model (LM), and Rec siﬁer sensitivity, speciﬁcity, of the model and diagnostic odds ratio (DOR) are shown. All the measures are reported together with the bounds Bayes, C-tree, Random Forest (RF), Support Vector Machine (SVM), Linear Regression Model (LM), and Recursive Partition Tree (PART). For model and diagnostic odds ratio (DOR) are shown. All the measures are reported together with the bounds of the 95% CI. that the validation set only includes participants with an ASD diagnosis and so the validation can test for true positive and false negative ASD classiﬁcations. catabolism and is excreted in the urine. In patients with nutritional rickets (vitamin D deﬁciency), an increased urinary excretion of carnitine may occur that is reversed by vitamin D supplementation (36). It may be argued that carnitine metabolism may be involved in patients with nutritional rickets. Possible links between vitamin D deﬁciency and carnitine deﬁciency should be further investigated also in view of the higher prevalence of both these conditions in patients with ASD. The present study conﬁrms that patients with ASD may show a distinct metabolic proﬁle, demonstrating that this can be used to identify a subset of ASD patients with respect to TD at younger ages. We veriﬁed that in each age group, clinical variables such as cognitive levels (DQ/IQ) and autism severity (CSS) did not correlate with the discriminant metabolite levels. This implies that both clinical features were irrelevant to clinically discriminate the identiﬁed patient subset. It would be interesting to further investigate if individual component of behavioral scores instead of global scores and/or additional neurological features might be more helpful at the clinical level using larger samples that allow patient stratiﬁcation (31). As ASD is developmental in nature, we considered possible interactions of measured metabolites with participant ages. Proﬁles of carnitine and acyl-carnitines change signiﬁcantly during the ﬁrst year of life, but kept at the same level between 2 and 15 years (37). We split the sample in two age categories (< 5 y.o. and ≥5 y.o.) to understand possible predictive metabolic signatures capable of distinguishing ASD and TD individuals at early stages of ASD development. Frontiers in Psychiatry \| www.frontiersin.org Validation Test As a whole, experimental ﬁndings show a pivotal role for mitochondrial fatty acid β-oxidation in controlling NSC-to-IPC transition in mammalian embryonic and adult brain, and propose NSC self-renewal as a cellular Validation Test This threshold is consistent with reliable ASD diagnosis and eﬀectiveness of early intervention. Indeed, the deﬁnite diagnosis of ASD is generally made between 3 and 5 years (38). Moreover, increasing evidences support the eﬀectiveness of early interventions (behavioral, developmental and educational approaches) in pre-schoolers (aged 24–71 months) with ASD (39). The predictive metabolic proﬁle identiﬁed in the present study is strongly supported by signiﬁcant biological and experimental data associated with ASD: (1) the present ﬁndings collectively suggest a reduced ﬂux through the mitochondrial β-oxidation pathway in a subset of patients with ASD. The acyl-carnitine pattern found in ASD patients is not consistent with any known genetic disorders of fatty acid oxidation and organic acid metabolism, electron transport chain or urea cycle dysfunction, or other inherited metabolic diseases. Genetic defects of mitochondrial β-oxidation are a group of IEM caused by failure of a single mitochondrial enzyme of β-oxidation such as short chain acyl-CoA dehydrogenase The results show higher classiﬁcation performance (sensitivity 72.3%, speciﬁcity 72.1%) at younger ages and potential application to improve diagnosis at earlier stages of ASD development. Re-test analyses as a measure of validity in independent samples showed an accuracy (proportion of observations that were correctly classiﬁed into patient or control group) of 73% in children aged ≤5 years. It has to be noted December 2018 \| Volume 9 \| Article 636 7 Metabolic Risk Proﬁle in ASD Barone et al. mechanism underlying the association between disturbances of mitochondrial fatty acid oxidation and autism (56, 57). (SCAD), medium chain acyl-CoA dehydrogenase (MCAD), very long chain acyl-CoA dehydrogenase (VLCAD) or long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD). Mitochondrial β-oxidation defects may be secondary to dysfunction of dependent processes, such as deﬁciencies of the carnitine fatty acid transporter system, or mitochondrial electron transfer ﬂavoprotein system (multiple acyl- CoA dehydrogenase deﬁciency) (40). The occurrence of developmental delay, autistic-like behavior or ASD in genetic defects of mitochondrial β-oxidation (41) particularly VLCAD (42) and LCHAD (43) suggests that impaired mitochondrial β-oxidation may contribute to dysfunctional energetic metabolism in subsets of patients with ASD. Deletion of the TMLHE gene, which is the ﬁrst step in carnitine synthesis pathway and located on the X chromosome, is found more often in males with non- dysmorphic autism suggesting that TMLHE deﬁciency is a risk factor for autism, albeit with low penetrance (estimated at 2–4%) (44). Validation Test (2) (2) ASD features and ASD have been reported in patients with propionic acidemia (PA), a severe organic acidemia caused by propionic acid (PPA) accumulation due to propionyl- CoA carboxylase enzyme deﬁciency (49). Endogenous PPA derives from the catabolism of branched-chain amino acids and from odd-chain fatty acid catabolism. PPA is a fermentation product of many autism associated gut bacteria, and also a common food preservative (50). Intracerebral PPA injections in rodents induce behavioral, electrographic and biochemical changes consistent with rodent ASD model (PPA model) (51). Brain lipid analyses of PPA model show increase of short- and long-chain acyl- carnitines but not medium- chain acyl-carnitines (52). The acyl-carnitine proﬁle of PPA model overlaps with those found in patients with ASD (32), also in the current study. (3) (3) Dysregulated cortical layer formation and layer-speciﬁc neuronal diﬀerentiation demonstrated in the neocortex of children with ASD, suggest possible defects in cell- cycle processes as well as in cell fate speciﬁcation (53). The carnitine palmitoyl transferase (CPT) system, which mediates the entry of long-chain fatty acids into the mitochondria for ß-oxidation, operates in astrocytes (54, 55) and in embryonic and adult neural stem cells (NSC) (56, 57). Recent evidences show that fatty acids might represent an important oxidative fuel during embryonic and early postnatal development and a reduced ﬂux through the mitochondrial fatty acid β-oxidation impairs NSC self- renewal in the mammalian embryonic brain and potentiates their transition to lineage-restricted cells (IPCs) (54–56). As a whole, experimental ﬁndings show a pivotal role for mitochondrial fatty acid β-oxidation in controlling NSC-to-IPC transition in mammalian embryonic and adult brain, and propose NSC self-renewal as a cellular (3) Dysregulated cortical layer formation and layer-speciﬁc neuronal diﬀerentiation demonstrated in the neocortex of children with ASD, suggest possible defects in cell- cycle processes as well as in cell fate speciﬁcation (53). The carnitine palmitoyl transferase (CPT) system, which mediates the entry of long-chain fatty acids into the mitochondria for ß-oxidation, operates in astrocytes (54, 55) and in embryonic and adult neural stem cells (NSC) (56, 57). Recent evidences show that fatty acids might represent an important oxidative fuel during embryonic and early postnatal development and a reduced ﬂux through the mitochondrial fatty acid β-oxidation impairs NSC self- renewal in the mammalian embryonic brain and potentiates their transition to lineage-restricted cells (IPCs) (54–56). Validation Test Children with ASD, as a group, are deﬁcient in Carnitine (45) with this deﬁciency potentially related to gastrointestinal symptom (46). Additionally, supplementing with Carnitine has been shown to improve core symptoms of ASD in two double-blind placebo controlled studies (47, 48). We found a combined acyl-carnitine pattern in patients with ASD indicative of impaired mitochondrial fatty acid β-oxidation. The identiﬁed acyl-carnitine proﬁle is characterized by a pattern of more elevated acyl-carnitine species in comparison with age- matched reference ranges. The presence of short-, medium-, and more elevated long-chain acyl-carnitine species, might reﬂect a mild generalized defect in FAO capacities, such as in FAO electron shuttle protein ETF (electron transferring factor), which is involved in the transfer of electrons coming from the short-chain, medium-chain and long-chain acyl-CoA dehydrogenases isoforms to the respiratory chain. Electrons from ETF feed the respiratory chain at the level of ETFDH (ETF dehydrogenase), a respiratory chain enzyme which transfers these electrons to coenzyme Q. Both inborn ETF and inborn ETFDH deﬁciency have been described in human, associated to a variety of phenotypes (58). The mechanisms responsible for expression of abnormal acyl-carnitine pattern in this subset of ASD patients cannot be inferred from the present study. Further studies are necessary to clarify if genetic variation of fatty acid oxidation and interaction with environmental factors including diet might account for acyl-carnitine accumulation. In view of the wide clinical features related to ASD we consecutively recruited patients with ASD diagnosis representing an heterogeneous ASD population: further studies are required to understand possible genetic and behavioral correlates of metabolic subtypes of ASD. One limitation of the present study is the lack of inclusion of a neurodevelopmental delay group to understand the performances of the algorithms for ASD vs. other developmental disabilities. Moreover, our study has been developed in a clinical sample. Similarly, classiﬁers have been applied to identify biomarkers of neurological and psychiatric diseases in clinical cohorts (28, 59). However, it has been recently highlighted that machine learning models should be adjusted to the epidemiological prevalence in the general population (60). Larger-scale studies or population analyses are therefore needed to assess performances in real life cohorts considering the actual prevalence rate. This will require resources for large-scale collaborative eﬀorts worldwide (61). REFERENCES 10. Dawson G, Rogers S, Munson J, Smith M, Winter J, Greenson J, et al. Randomized, controlled trial of an intervention for toddlers with autism: the Early Start Denver Model. Pediatrics (2010) 125:e17–23. doi: 10.1542/peds.2009-0958 1. Baio J, Wiggins L, Christensen DL, Maenner MJ, Daniels J, Warren Z, et al. Prevalence of autism spectrum disorder among children aged 8 years - autism and developmental disabilities monitoring network, 11 sites, United States, 2014. MMWR Surveill Summ. (2018) 67:1–23. doi: 10.15585/mmwr.ss6706a1 1. Baio J, Wiggins L, Christensen DL, Maenner MJ, Daniels J, Warren Z, et al. Prevalence of autism spectrum disorder among children aged 8 years - autism and developmental disabilities monitoring network, 11 sites, United States, 2014. MMWR Surveill Summ. (2018) 67:1–23. doi: 10.15585/mmwr.ss6706a1 11. Yap IK, Angley M, Veselkov KA, Holmes E, Lindon JC, Nicholson JK. Urinary metabolic phenotyping diﬀerentiates children with autism from their unaﬀected siblings and age-matched controls. J Proteome Res. (2010) 9:2996– 3004. doi: 10.1021/pr901188e 2. Narzisi A, Posada M, Barbieri F, Chericoni N, Ciuﬀolini D, Pinzino M, et al. Prevalence of autism spectrum disorder in a large italian catchment area: a school-based population study within the ASDEU project. Epidemiol Psychiatr Sci. (2018) 6:1–10. doi: 10.1017/S2045796018000483 12. Ming X, Stein TP, Barnes V, Rhodes N, Guo L. Metabolic perturbance in autism spectrum disorders: a metabolomics study. J Proteome Res. (2012) 11:5856–62. doi: 10.1021/pr300910n 3. American-Psychiatric-Association. Diagnostic and Statistical Manual of Mental Disorders. Washington, DC: American Psychiatric Publishing (2013). 3. American-Psychiatric-Association. Diagnostic and Statistical Manual of Mental Disorders. Washington, DC: American Psychiatric Publishing (2013). 13. Emond P, Mavel S, Aidoud N, Nadal-Desbarats L, Montigny F, Bonnet-Brilhault F, et al. GC-MS-based urine metabolic proﬁling of autism spectrum disorders. Anal Bioanal Chem. (2013) 405:5291–300. doi: 10.1007/s00216-013-6934-x 4. Constantino JN, Charman T. Diagnosis of autism spectrum disorder: reconciling the syndrome, its diverse origins, and variation in expression. Lancet Neurol. (2016) 15:279–91. doi: 10.1016/S1474-4422(15)00151-9 4. Constantino JN, Charman T. Diagnosis of autism spectrum disorder: reconciling the syndrome, its diverse origins, and variation in expression. Lancet Neurol. (2016) 15:279–91. doi: 10.1016/S1474-4422(15)00151-9 5. Kiykim E, Zeybek CA, Zubarioglu T, Cansever S, Yalcinkaya C, Soyucen E, et al. Inherited metabolic disorders in Turkish patients with autism spectrum disorders. Autism Res. (2016) 9: 217–23. doi: 10.1002/aur.1507 14. Noto A, Fanos V, Barberini L, Grapov D, Fattuoni C, Zaﬀanello M, et al. The urinary metabolomics proﬁle of an Italian autistic children population and their unaﬀected siblings. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualiﬁed researcher. SUPPLEMENTARY MATERIAL List of coauthors included in MIMIC (Metabolism-Immunity- Metals Integrated Concepts), Autism project: a multilevel approach to understand and cure Autism Spectrum Disorders: The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyt. 2018.00636/full#supplementary-material ACKNOWLEDGMENTS RB and SA conceived the project with contributions by MM, AP, and AF. MM and ADP analyzed the clinical samples. MM, ADP and GT acquired the data and performed data analyses. RB, MG, FM, AGF, GR carried out participants’ recruitment and clinical data analysis. SA, AP, AF performed statistical data analysis and computational analyses. RB and SA wrote the paper. RR, JB, RF performed critical revision of the manuscript for intellectual contents. All Authors read and approved the ﬁnal manuscript. We would like to thank the children and families who participated in this study. Partial ﬁnancial support from the University of Catania FIR-2014 ED99F1 MIMIC (Metabolism- Immunity-Metals Integrated Concepts) - Autism: a multilevel approach to understand and cure Autism Spectrum Disorders and from MIUR Fondo per le attività di base di ricerca to RB is gratefully acknowledged. CONCLUSION The present study supports early recognition of a distinctive metabolic proﬁle in DBS whose distinguishing features suggest a reduced ﬂux through the mitochondrial fatty acid β-oxidation pathway and provides insight into concealed molecular mechanisms determining ASD. The results show higher classiﬁcation performances in children with ASD younger than 5 years old suggesting a potential complementary and supportive ability to improve diagnosis at earlier stages of ASD development. The applied non-invasive methodology on DBS traditionally used for newborn screening is appropriate to evaluate metabolic proﬁle changes across development. The present ﬁndings yield the evidence that metabolic biomarkers that identify subset of patients with ASD are helpful. Considering the heterogeneity December 2018 \| Volume 9 \| Article 636 Frontiers in Psychiatry \| www.frontiersin.org 8 Metabolic Risk Proﬁle in ASD Barone et al. of ASD, metabolic proﬁling may support the identiﬁcation of phenotypes enabling individualized therapeutic approaches in children at risk of developing the disease. Prof. Agata Fiumara, Dr. Concetta Meli, Referral Centre for Inherited Metabolic Disorders,Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy; Dr. Mariangela Gulisano, Dr. Federica Maugeri, Dr. Adriana Prato, Child Neurology and Psychiatry, Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy; Prof. Giovanna Russo, Pediatric Hematology, Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy; Dr. Giovanni Tabbì, Institute of Biostructures and Bioimaging (IBB), National Research Council (CNR), Catania, Italy. REFERENCES (2005) 51:745–52. doi: 10.1373/clinchem.2004.043646 concentrations measured by tandem mass spectrometry. Clin Chem. (2005) 51:745–52. doi: 10.1373/clinchem.2004.043646 38. Mandell DS, Novak MM, Zubritsky CD. Factors associated with age of diagnosis among children with autism spectrum disorders. Pediatrics (2005) 116:1480–6. doi: 10.1542/peds.2005-0185 19. 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The enzymology of mitochondrial fatty acid beta-oxidation and its application to follow-up analysis of positive neonatal screening results. J Inherit Metab Dis. (2010) 33:479–94. doi: 10.1007/s10545-010-9104-8 Copyright © 2018 Barone, Alaimo, Messina, Pulvirenti, Bastin, MIMIC-Autism Group, Ferro, Frye and Rizzo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 59. Orrù G, Pettersson-Yeo W, Marquand AF, Sartori G, Mechelli A. Using support vector Machine to identify imaging biomarkers of neurological and psychiatric disease: a critical review. Neurosci Biobehav Rev. 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https://openalex.org/W3217137347	https://www.redalyc.org/journal/3842/384272295028/384272295028.pdf	Portuguese	null	Comentário a “Reminiscência e alma remêmora no Fédon de Platão”	Trans/Form/Ação	2,021	cc-by	1,618	Trans/Form/Ação ISSN: 0101-3173 ISSN: 1980-539X Trans/Form/Ação ISSN: 0101-3173 ISSN: 1980-539X Sistema de Informação Científica Redalyc Rede de Revistas Científicas da América Latina e do Caribe, Espanha e Portugal Sem fins lucrativos acadêmica projeto, desenvolvido no âmbito da iniciativa acesso aberto tigo do artigo redalyc.org Sistema de Informação Científica Redalyc Rede de Revistas Científicas da América Latina e do Caribe, Espanha e Portugal Sem fins lucrativos acadêmica projeto, desenvolvido no âmbito da iniciativa acesso aberto Como citar este artigo Número completo Mais informações do artigo Site da revista em redalyc.org Bocayuva, Izabela Aquino Comentário a “Reminiscência e alma remêmora no Fédon de Platão” Trans/Form/Ação, vol. 44, núm. 4, 2021, Outubro-Dezembro, pp. 353-356 Universidade Estadual Paulista, Departamento de Filosofia DOI: https://doi.org/10.1590/0101-3173.2021.v44n4.28.p353 Disponível em: https://www.redalyc.org/articulo.oa?id=384272295028 Como citar este artigo Número completo Mais informações do artigo Site da revista em redalyc.org Comentário Artigos / Articles 1 Professora Titular em Filosofia da Universidade do Estado do Rio de Janeiro (UERJ), Rio de Janeiro, RJ – Brasil. https://orcid.org/0000-0003-1571-1860. E-mail: izabelabocayuva@gmail.com. This is an open-access article distributed under the terms of the Creative Commons Attribution License. Comentário a “Reminiscência e alma remêmora no Fédon de Platão” Izabela Aquino Bocayuva 1 Izabela Aquino Bocayuva 1 https://doi.org/10.1590/0101-3173.2021.v44n4.28.p353 pensamento, a reflexão filosófica e a abordagem cultual. [...] A interpretação do Fédon permite aferir o nexo entre experiência cultual e análise racional pensamento, a reflexão filosófica e a abordagem cultual. [...] A interpretação do Fédon permite aferir o nexo entre experiência cultual e análise racional Na verdade, não é apenas a ética que se articula com o sagrado e a mitopoética tipicamente platônica, mas também sua epistemologia, sua política e sua metafísica das Formas. Platão constrói a imagem do filósofo como um homem de exceção, bem aos moldes do poeta do Poema de Parmênides que é separado da “senda dos mortais” para vir a ter a experiência numinosa da verdade em contato direto com uma deusa. O filósofo não se apresenta apenas como o homem moralmente excelente, incorruptível. Em Platão, o que o filósofo – ou sua alma – vê, sabe ou, como diz o texto de Rachid, o que ele lembra, rememora, pertence sempre ao âmbito imortal, verdadeiro, perene – o âmbito do ser e da verdade. Isso justifica que ele e só ele seja o rei que tem plena autoridade sábia, para até mesmo prescindir das leis escritas. No Fédon, o filósofo é o que exercita a morte, no sentido de praticar a separação do que é perecível, mutável, para, por fim, alçar-se, purificado, à imortalidade na ilha dos bem-aventurados ou terra dos heróis, uma vez que, em tese, a alma é imortal. Ainda no Fédon, mas também no Ménon, a questão da imortalidade da alma é inseparável da questão da reminiscência. Aprender é rememorar as Formas em si (puras, separadas) já contempladas antes do nascimento, tal como fica exposto no Fedro. O sensível pode ajudar nesse processo de rememoração, por semelhança ou dissemelhança, mas necessariamente mantendo distância ontológica em relação à Forma rememorada. O método dialético articulado com elementos empíricos também deve ajudar o filósofo a dar o salto súbito da recordação do ser. Tudo, na imagem dos filósofos, aponta para um para além, e Platão se vale explicitamente do mito para figurar essa dimensão, seja no Ménon, seja no Fédon, no Fedro – bem como na República, no Górgias etc. Pelo uso explícito do mito na argumentação racional, tanto ética quanto epistemológica e ontológica, a qual Platão leva a cabo, e pela relação estreita que observamos entre todos esses âmbitos discursivos, estamos de acordo com Rachid de que são indeslindáveis. Entretanto, vemos necessidade em questionar a abrangência da mitopoiesis platônica. Referência do artigo comentado: RACHID, R. J. R. Reminiscência e alma remêmora no Fédon de Platão. Trans/form/ação: revista de filosofia da Unesp, v. 44, n. 4, p. 327-348, 2021. Referência do artigo comentado: RACHID, R. J. R. Reminiscência e alma remêmora no Fédon de Platão. Trans/form/ação: revista de filosofia da Unesp, v. 44, n. 4, p. 327-348, 2021. Rodolfo José Rocha Rachid, em seu artigo “Reminiscência e alma remêmora no Fédon de Platão”, através de um vocabulário bastante rebuscado, contribui muito para o retorno à discussão em torno da imbricação entre mito e racionalidade, em Platão. Seu texto provoca esse retorno, à medida que acentua a necessidade de a argumentação racional platônica fundar-se em elementos míticos, como são o caso da reminiscência e da imortalidade da alma. A alma filosófica ou alma remêmora recupera, em sua nova vida, num exercício continuado de afastamento do sensível, sua condição sábia e sagrada de contato direto com o ser e a verdade, recuperação essa concomitante à purificação de seu ethos, decaído desde o nascimento, por ter sido obrigada, ainda no Hades, a beber da água do rio Lethe, o rio do esquecimento. Cito Rachid (2021, p. 346): A reflexão deontológica, preceituada por Sócrates, não prescinde da experiência do sagrado, de modo que coexistem no corpus platônico dois âmbitos considerados irreconciliáveis por parte das categorias modernas de https://doi.org/10.1590/0101-3173.2021.v44n4.28.p353 353 Trans/Form/Ação, Marília, v. 44, n. 4, p. 353-356, Out./Dez., 2021 BOCAYUVA, I. A. pensamento, a reflexão filosófica e a abordagem cultual. [...] A interpretação do Fédon permite aferir o nexo entre experiência cultual e análise racional Parece-nos que, mesmo nos diálogos finais, onde Platão revê sua metafísica canônica de dois mundos e não alude mais à imortalidade da alma e reminiscência, está operando na base de seu pensamento aquele para além que constitui fundamentalmente a imagem do filósofo e que, em diálogos 354 Trans/Form/Ação, Marília, v. 44, n. 4, p. 353-356, Out./Dez., 2021 Artigos / Articles Comentário anteriores, tinha nexo direto com os mitos por ele elaborados. No Parmênides, por exemplo, ele abandona a discussão dos dois mundos, para permanecer apenas na dimensão para além, o inteligível, sem qualquer referência ao sensível. No Sofista, outro exemplo, dá-se o mesmo: o não-ser passa à dimensão do inteligível, enquanto gênero da alteridade, sendo o gênero outro nome para o que anteriormente era denominado ideia. No Político, mais um exemplo: o rei ou político (ou filósofo – o que tem a ciência) é o único que sabe o Bem e, por isso, deve governar. Poderíamos entender que ele é o único que rememora o ser de todas as coisas. Assim, a dimensão celeste, sagrada, ideal, sem mais apelar para mitos, perdura. Parece que a mitopoiesis explícita inaugural no pensamento platônico havia já cumprido seu papel. A realidade de um “para além” havia se consolidado, entretanto, a dimensão mítica com a qual Platão contou para construir sua narrativa racional continua sendo imprescindível, ainda que oculta. Podemos dizer que o autor-filósofo Platão reúne nele dois traços necessariamente: o do político e o do educador. Digo isso, sabendo que há diversas exegeses do pensador grego que procuram eliminar o traço político, em seu pensamento, a começar com os neoplatônicos e terminando com os que ainda hoje querem apagar a influência nefasta da interpretação poperiana do pensamento platônico. No entanto, não vejo que seja possível pular a própria sombra. Toda obra platônica tem motivação política. E, como ele mesmo deixa muito claro, no Político, a primeira função do político (o rei- filósofo) é educar, é instruir minuciosamente, através de princípios claros, os que irão educar diretamente os homens. O autor Platão, que decide fazer a política perfeita, de exceção, não misturada nos meandros da política vil de todo dia, uma política que se afasta da multiplicidade e da variação, usa a sua obra como instrumento de educação dirigindo tudo e todos, passo a passo, para o seu princípio supremo: o um. Assim, sua metafísica inicial sempre apontou para a unidade da ideia. RACHID, R. J. R. Reminiscência e alma remêmora no Fédon de Platão. Trans/form/ ação: revista de filosofia da Unesp, v. 44, n. 4, p. 327-348, 2021. pensamento, a reflexão filosófica e a abordagem cultual. [...] A interpretação do Fédon permite aferir o nexo entre experiência cultual e análise racional Sua metafísica final mostra o mesmo, através da dialética, encaminhando tudo para o um, o conceito suficiente e necessário. Sua ética mostra ainda o mesmo, à medida que encaminha para a base fundamental da articulação entre sophrosýne e andreía, única capaz de apreender em uníssono o Belo uno, o Bom uno e o Justo uno. 355 Trans/Form/Ação, Marília, v. 44, n. 4, p. 353-356, Out./Dez., 2021 Recebido: 04/6/2021 Aceito: 09/6/2021 BOCAYUVA, I. A. Ora, os mitos escatológicos da imortalidade da alma implicam e configuram o âmbito divino, separado, imperecível, puro, onde habitam as Formas unas. A alma remêmora tem acesso justamente a elas e só por isso conhece. Esse mundo separado, onde habitam as unidades, inicialmente sustentado por mitos e depois consolidado como realidade necessária, tem, em Platão, uma função ao mesmo tempo educativa e política, o que nos faz lembrar a República, onde é sugerido que o rei-filósofo, único conhecedor do Bem, para um convencimento amplo e pacífico da população, deva apresentar- lhe uma mentira útil, mostrando, num mito, que uns nasceram com o coração de ouro, outros de prata, outros de bronze. No Fédon, mas também no Górgias, como igualmente no mito de Er, da República, os mitos escatológicos, comprometidos de algum modo com a tese da imortalidade da alma, parecem mesmo ter a função de pelo menos assustar o ouvinte, endereçando-o a uma conduta exemplar, não reprovável aos olhos dos deuses, mas, com isso, também direcioná-lo àquele mesmo um, comum aos que sabem em uníssono do Bem, do Belo, do Justo. Eis algumas questões em que a leitura do artigo de Rachid me fizeram pensar. Trans/Form/Ação, Marília, v. 44, n. 4, p. 353-356, Out./Dez., 2021 Referência RACHID, R. J. R. Reminiscência e alma remêmora no Fédon de Platão. Trans/form/ ação: revista de filosofia da Unesp, v. 44, n. 4, p. 327-348, 2021. 356
https://openalex.org/W2804972113	https://europepmc.org/articles/pmc5983634?pdf=render	English	null	Silencing Stem Cell Factor Gene in Fibroblasts to Regulate Paracrine Factor Productions and Enhance c-Kit Expression in Melanocytes on Melanogenesis	International journal of molecular sciences	2,018	cc-by	12,985	Received: 5 March 2018; Accepted: 10 May 2018; Published: 16 May 2018 Abstract: Melanogenesis is a complex physiological mechanism involving various paracrine factors. Skin cells such as keratinocytes, ﬁbroblasts, and melanocytes communicate with one another through secreted regulators, thereby regulating the melanocytes’ bio-functions. The stem cell factor (SCF) is a paracrine factor produced by ﬁbroblasts, and its receptor, c-kit, is expressed on melanocytes. Binding of SCF to c-kit activates autophosphorylation and tyrosine kinase to switch on its signal transmission. SCF inhibition does not suppress ﬁbroblast proliferation in MTT assay, and SCF silencing induced mRNA expressions of paracrine factor genes, HGF, NRG-1, and CRH in qPCR results. Following UVB stimulation, gene expressions of HGF, NRG, and CRH were higher than homeostasis; in particular, HGF exhibited the highest correlation with SCF variations. We detected ﬁbroblasts regulated SCF in an autocrine-dependent manner, and the conditioned medium obtained from ﬁbroblast culture was applied to treat melanocytes. Melanogenesis-related genes, tyrosinase and pmel17, were upregulated under conditioned mediums with SCF silencing and exposed to UVB treatments. Melanin quantities in the melanocytes had clearly increased in the pigment content assay. In conclusion, SCF silencing causes variations in both ﬁbroblast paracrine factors and melanocyte melanogenesis, and the differences in gene expressions were observed following UVB exposure. Keywords: stem cell factor; ﬁbroblasts; paracrine factors; melanin; melanocytes Silencing Stem Cell Factor Gene in Fibroblasts to Regulate Paracrine Factor Productions and Enhance c-Kit Expression in Melanocytes on Melanogenesis Pin-Hui Li 1,†, Li-Heng Liu 2,†, Cheng-Chung Chang 3, Rong Gao 4, Chung-Hang Leung 5, Dik-Lung Ma 6 and Hui-Min David Wang 3,7,8,* ID 1 Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwa s27159@gmail.com 1 Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung City 807, Taiw s27159@gmail.com 1 Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwan; s27159@gmail.com 2 Department of Life Science, National Chung Hsing University, Taichung City 402, Taiwan; arthur851213@gmail.com 2 Department of Life Science, National Chung Hsing University, Taichung City 402, Taiwan; arthur851213@gmail.com 3 Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung City 402, Taiw ccchang555@dragon.nchu.edu.tw 4 Jiaxing Deqin Biotechnology Department, Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, Zhejiang, China; gaorong709@naver.com g j g g g 5 State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau 999078, China; duncanleung@umac.mo g j g g g 5 State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, y p g 6 Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong 22100, China edmondma@hkbu.edu.hk 7 Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung City 807, Taiwan Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung City 807, Taiwan 8 College of Oceanology and Food Science, Quanzhou Normal University, Quanzhou 362000, China * Correspondence: davidw@dragon.nchu.edu.tw; Tel.: +886-4-22840733 (ext. 651); Fax: +886-4-22852242 † Th th t ib t d ll t thi k Ce e o S e Ce ese c , o s g e c U e s y, o s g C y 80 , 8 College of Oceanology and Food Science, Quanzhou Normal University, Quanzhou 362000, China * Correspondence: davidw@dragon.nchu.edu.tw; Tel.: +886-4-22840733 (ext. 651); Fax: +886-4-22852242 8 College of Oceanology and Food Science, Quanzhou Normal University, Quanzhou 362000, China † These authors contributed equally to this work. † These authors contributed equally to this work. International Journal of Molecular Sciences International Journal of Molecular Sciences www.mdpi.com/journal/ijms 1. Introduction The human skin is the largest organ covering the entire body and is made up of three layers, the epidermis, dermis, and subcutaneous layer [1]. The epidermis is distinguished by ﬁve layers—the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and the stratum basale Int. J. Mol. Sci. 2018, 19, 1475; doi:10.3390/ijms19051475 www.mdpi.com/journal/ijms 2 of 18 Int. J. Mol. Sci. 2018, 19, 1475 (stratum germinativum), from its exterior to the interior. The basal cells in the stratum basale divide continuously, and move toward the stratum corneum [2]. Besides the keratinocytes, the epidermis contains other cells, such as Merkel cells, Langerhans cells, and melanocytes. The dermis is made up of connective tissue and it contains some extracellular matrix components, such as glycosaminoglycan and hyaluronic acid. The dermis possesses sweat glands, sebaceous glands [1,3,4], hair roots, nerves, lymph, and blood vessels. It contains three major cells—the ﬁbroblasts, mast cells, and macrophages. There is a subcutaneous layer under the dermis containing several adipose cells that store fat and maintain body temperature [5]. The skin has certain physiological functions, including protection from pathogens and the external environment, perception of pain or sentience, synthesis of vitamin D, temperature adjustment, absorption, and water resistance [6]. Paracrine factors connect between keratinocytes, ﬁbroblasts, and melanocytes within the skin and play a vital role in ultraviolet (UV)-induced pigmentation and melanocyte activity. More recently, dermal ﬁbroblasts were demonstrated to regulate cutaneous melanin production through secrevarious cytokines. In dark human skin, neuregulin (NRG)-1 is highly expressed by ﬁbroblasts, suggesting its potential role in constitutive human skin color regulation. Human palmoplantar area is thicker and lighter in color than the nonpalmoplantar area. It has been demonstrated that ﬁbroblasts produce abundant Dikkopf-1 (DKK-1) in the palms and soles, and then suppress the growth and function of melanocytes by arresting the Wnt-signaling pathway [7,8]. In addition, ﬁbroblasts also secrete numerous melanogenic factors, such as stem cell factor (SCF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), corticotropin-releasing hormone (CRH), endothelin-1 (ET-1), interferon-γ (IFN-γ), and interleukin-1 (IL-1) [9–13]. Several studies have suggested dermal ﬁbroblasts play a central role in regulating melanocyte functions and inﬂuencing human skin pigmentation and constitutive color regulation. The stratum basale layer of the epidermis, hair, and iris contain a type of cells with the ability to produce melanin for the human body, which are known as melanocytes. 1. Introduction Melanocytes are derived from embryonic cells and neural crest cells (NCC), and undergo several life cycles involving melanocyte differentiation from NCC into melanoblasts, the precursor cells. Melanocytes are reported to migrations and proliferations to the target site, differentiations and maturations into melanocytes, transportations and releases of melanosomes ﬁlled with melanin to keratinocytes, and cell death [14]. The skin suffers some stresses such as inﬂammation and free radical accumulation, especially the UV effect on melanin production for defense against injury from the external or internal environment [15]. Melanogenesis is a complex mechanism and process involving intercommunication between melanocytes, keratinocytes, and ﬁbroblasts, thereby regulating signal transduction by secreting paracrine factors and cytokines. There are numerous paracrine factors which regulate melanin production, such as the proopiomelanocortin (POMC)-derived hormone, [(α-MSH and adrenocorticotropic hormone (ACTH)], ET-1, HGF, CRH, nitric oxide, and some inﬂammatory cytokines (IL-1β, -6, and -10, and TNF-α) [6,16]. Most paracrine factors modulate melanogenesis by upregulating or downregulating the expression of the microphthalmia-associated transcription factor (MITF), which ultimately is the primary transcription factor for tyrosinase, tyrosinase-related protein-1 (TRP-1), and dopachrome tautomerase (tyrosinase related protein-2, TRP-2). Melanin synthesis is accompanied by melanosome maturation, the special cellular organelles in melanocytes. Tyrosinase plays a role as a central and rate-limiting enzyme because it initiates the reaction of tyrosine hydroxylation to L-3,4-dihydroxyphenylalanine (L-DOPA) and oxidization into DOPA quinone. In the presence of thiols such as cysteine or glutathione, DOPA quinone reacts with it and generates cysteinyl-DOPA, turning it into yellow-red pheomelanin. On the other hand, spontaneous cyclization of DOPA quinone to DOPA chrome occurs, followed by a spontaneous loss of carboxylic acid to form 5,6-dihydroxyindole (DHI). The dark-brown eumelanin is converted into indole-5,6-quinone by DHI oxidization via TRP-1 and polymerization. If the DOPA chrome is catalyzed by TRP-2 to DHI-2-carboxylic acid and then oxidized to form indole-5,6-quinone-2-carboxylic acid, its polymerization results in lighter brown eumelanin [17]. The ﬁnal step of skin pigmentation 3 of 18 Int. J. Mol. Sci. 2018, 19, 1475 is the transport of mature melanosomes from melanocytes along with cytoskeletal elements to dendrites, followed by internalization by adjacent keratinocytes contacting with melanocytes [18,19], and 30–40 associated keratinocytes. The mature melanosomes are transported to the adjacent [20,21]. and 30 40 associated keratinocytes. The mature melanosomes are transported to the adjacent [20,21]. SCF is a paracrine cytokine produced by ﬁbroblasts, keratinocytes, and endothelial cells [22]. 1. Introduction SCF and its receptor c-kit play an essential role in hemopoiesis and maintaining hematopoietic stem cell survival, mast cell stimulation, mediating the function of the pancreas, and melanogenesis [23]. A mutation in SCF could cause anemia, lack of mast cells, immaturity and loss of functions of pancreatic endocrine cells, and disappearance of pigmentation, as well as the absence of melanocytes [24,25]. c-Kit is a receptor tyrosine kinase expressed on melanocytes. It has two domains containing a glycosylated extracellular ligand-binding domain, comprising ﬁve Ig-like domains, and a cytoplasmic region, which comprises the protein tyrosine kinase domain. At the ectodomain, ﬁve Ig-like domains are separated from the ﬁrst three domains, constituting a ligand-binding area, and two other domains act on monomerization or dimerization of c-kit. Another part, the phosphorylation site, is situated in the intracellular region, consisting of the juxtamembrane area and a large kinase-insert region [26]. Binding of SCF to c-kit stimulates dimerization and initiation of catalytic activities of autophosphorylation and tyrosine kinase to complete its signal transmission. Y721 phosphorylation of c-kit is involved in the phosphoinositide 3-kinase (PI3K) pathway. PI3K does not only regulate cell survival but also cause pigmentation by increasing the serine/threonine-speciﬁc protein kinase AKT activity, which can produce phosphorylate glycogen synthase kinase 3β (GSK-3β) and lead to β-catenin accumulation, resulting in translocation to the nucleus to increase MITF activity [27]. c-Kit phosphorylation at Y703 and Y936 activates the mitogen-activated protein kinase (MAPK) pathway and leads to cell proliferation, differentiation, and melanin alteration [28]. The activated extracellular signal-related kinase ERK and c-Jun N-terminal kinase JNK, the members of the MAPK family, upregulate the transcription activity or ubiquitin-dependent degradation of MITF as a feedback mechanism of melanin production. On the other hand, the activated p38 induces phosphorylation of CREB and then activates MITF to promote tyrosinase transcription [29]. The other phosphorylation sites such as Y586, Y570, Y900, Y568, Y570, and Y730 are involved in cell proliferation, survival, adhesion, and differentiation, and Y586, Y570, and Y936 participate in receptor downregulation. Some studies even indicated clearly increased expression of SCF was observed after UV light exposure, especially UVB, in both keratinocytes and ﬁbroblasts, which facilitated melanogenesis behavior [13,30]. In addition, SCF is released in greater levels by ﬁbroblasts than by keratinocytes [31]. 1. Introduction Several studies have demonstrated the functions of SCF, irrespective of its secretion by keratinocytes or ﬁbroblasts, in proliferation, differentiation, and melanogenesis of melanocytes; however, the importance of SCF and its effect on other paracrine factors are yet to be determined [32,33]. Furthermore, ﬁbroblasts play an important role in regulating skin color, and the expression level of SCF in ﬁbroblasts is more than that in keratinocytes. Therefore, it is of particular interest to understand the inﬂuence on SCF in terms of whether its dysfunction is occurring in ﬁbroblasts and then the alteration of other cytokines produced by ﬁbroblasts, as well as melanogenesis in melanocytes. This study examined whether SCF silencing inﬂuences other paracrine factors secreted by ﬁbroblasts and the changes in melanogenesis of melanocytes. 2.2. The Proliferation of Fibroblasts Was Not Suppressed by SCF Gene Silencing 2.2. The Proliferation of Fibroblasts Was Not Suppressed by SCF Gene Silencing We next examined if SCF inhibited by siRNA caused increased or decreased proliferation of ﬁbroblasts. The MTT method was used to demonstrate ﬁbroblast proliferation. Compared with the vehicle control group, the cell viability showed no difference between the control (100%) and SCF gene knockdown ﬁbroblasts (103%). Figure 1b demonstrates neither cellular morphology nor cell conﬂuency were altered. Similar results were observed regarding ﬁbroblast morphology, where without cellular atrophy damage, ﬂattened or low conﬂuency were observed in Figure 1c. The data indicated the lack of an SCF gene did not affect cell growth and cellular morphology. We next examined if SCF inhibited by siRNA caused increased or decreased proliferation of fibroblasts. The MTT method was used to demonstrate fibroblast proliferation. Compared with the vehicle control group, the cell viability showed no difference between the control (100%) and SCF gene knockdown fibroblasts (103%). Figure 1b demonstrates neither cellular morphology nor cell confluency were altered. Similar results were observed regarding fibroblast morphology, where without cellular atrophy damage, flattened or low confluency were observed in Figure 1c. The data indicated the lack of an SCF gene did not affect cell growth and cellular morphology. Figure 1. Validation of fibroblasts after SCF targeted siRNA 25 nM transfection for 48 h. (a) The 75% gene knockdown efficiency of SCF in fibroblasts; (b) Fibroblasts were treated with 25 nM SCF-targeted siRNA for 48 h, and the proliferation was assessed by MTT assay; (c) Phase-contrast images compared between control and SCF gene knockdown fibroblasts (100×). The data are presented as means ± SD; n = 3, * p < 0.05. Figure 1. Validation of ﬁbroblasts after SCF targeted siRNA 25 nM transfection for 48 h. (a) The 75% gene knockdown efﬁciency of SCF in ﬁbroblasts; (b) Fibroblasts were treated with 25 nM SCF-targeted siRNA for 48 h, and the proliferation was assessed by MTT assay; (c) Phase-contrast images compared between control and SCF gene knockdown ﬁbroblasts (100×). The data are presented as means ± SD; n = 3, * p < 0.05. Figure 1. Validation of fibroblasts after SCF targeted siRNA 25 nM transfection for 48 h. (a) The 75% gene knockdown efficiency of SCF in fibroblasts; (b) Fibroblasts were treated with 25 nM SCF-targeted siRNA for 48 h, and the proliferation was assessed by MTT assay; (c) Phase-contrast images compared between control and SCF gene knockdown fibroblasts (100×). 2.1. The Validation of Short Interfering RNA Knockdown Efﬁciency Transfection was used to introduce siRNA into human ﬁbroblasts. In the attempt to knockdown SCF gene expression, it was necessary to validate how successful its genetic knockdown was. Comparisons between the controlled, regular ﬁbroblast cells, and the same ﬁbroblast cells with introduced 25 nM siRNA delineated the productive knockdown in the mRNA expression levels. Figure 1 shows the qPCR-validated knockdown efﬁciency of SCF mRNA levels. qPCR is a quantiﬁcation technique used to determine the mRNA expression levels of, in this case, the ampliﬁed SCF gene. 4 of 18 Int. J. Mol. Sci. 2018, 19, 1475 Transfection of Hs68 human ﬁbroblasts with 25 nM SCF siRNA showed a signiﬁcant decrease in SCF gene expression 48 h after the transfection compared with regular Hs68 ﬁbroblasts. Using the transfection method and the qPCR conﬁrmation, the results indicated that a successful >75% of SCF mRNA expression levels were effectively silenced in Hs68 cells. Int. J. Mol. Sci. 2018, 19, x 4 of 18 Using the transfection method and the qPCR confirmation, the results indicated that a successful >75% of SCF mRNA expression levels were effectively silenced in Hs68 cells. 2.2. The Proliferation of Fibroblasts Was Not Suppressed by SCF Gene Silencing 2.2. The Proliferation of Fibroblasts Was Not Suppressed by SCF Gene Silencing The data are presented as means ± SD; n = 3, * p < 0.05. Figure 1. Validation of ﬁbroblasts after SCF targeted siRNA 25 nM transfection for 48 h. (a) The 75% gene knockdown efﬁciency of SCF in ﬁbroblasts; (b) Fibroblasts were treated with 25 nM SCF-targeted siRNA for 48 h, and the proliferation was assessed by MTT assay; (c) Phase-contrast images compared between control and SCF gene knockdown ﬁbroblasts (100×). The data are presented as means ± SD; n = 3, * p < 0.05. 2.4. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts Using th Stimulated Model 2.4. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts Using the UVB-Stimulated Model 2.4. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts Using t Stimulated Model 2.4. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts Using the UVB-Stimulated Model 2.4. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts Using t Stimulated Model 2.4. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts Using the UVB-Stimulated Model Paracrine factors showed alterations in their mRNA expression levels when the SCF ge expression was inhibited. Similar experiments under identical conditions were conducted again w the addition of UVB exposure. Exposure to UVB is known to induce expression of SCF, so its effe on mRNA levels compared to the levels without UVB exposure would be a necessary observatio Comparisons between the paracrine factors with and without SCF knockdown, and with or witho UVB, emphasize the individual gene expression variations not only in terms of SCF differences b also in determining whether UVB causes certain factors to react and express differently. In Figure the results show the upregulation gene expressions of SCF, HGF, NRG, and CRH with UVB exposu to regular fibroblasts compared with the control group, which did not undergo UVB stimulation. T ET-1 gene was downregulated, and no significant difference was observed with DKK-1. Followi the determination of the influence of UVB on regular fibroblasts, the different outcome in fibrobla with SCF gene knockdown under UVB stimulation was tested. A comparison between regu fibroblasts with UVB treatment and SCF gene knockdown fibroblasts with the same UVB treatme is presented. The data presented the upregulation mRNA expression of HGF, NRG, and CRH a their expression levels when the fibroblast cells, specifically with stable SCF silencing, were expos to UVB. Compared to the regular fibroblast gene expressions with UVB exposure, HGF, CRH, a NRG with SCF knockdowns all had their mRNA expressions increased, even with the same UV treated situation. HGF demonstrated a particularly dramatic increase in gene expression levels cells with SCF genetic knockdown, while DKK-1 and ET-1 were the SCF-silenced genes that we downregulated when treated with UVB. SCF gene alteration influenced the fibroblast paracri system and the condition differed with or without UVB stimulation. Paracrine factors showed alterations in their mRNA expression levels when the SCF gene expression was inhibited. Similar experiments under identical conditions were conducted again with the addition of UVB exposure. 2.3. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts 2.3. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts We evaluated whether SCF affected the fibroblasts and the mRNA paracrine factor levels from fibroblasts. To determine how differently the paracrine factors reacted due to SCF silencing, the controlled gene expressions were compared to the gene expression with SCF knockdown. In Figure 2, the data showed a significant increase in HGF, NRG, and CRH genes when SCF was stably silenced 48 h post transfection. The downregulated mRNA levels with DKK-1 and ET-1 were affected because of SCF knockdown in fibroblast cells. ET-1 was the most severely affected gene, while the paracrine growth factor HGF was the least affected of all the genes. SCF gene alteration affected the fibroblast paracrine system and its knockdown increased or decreased all the specific paracrine gene expression levels. We evaluated whether SCF affected the ﬁbroblasts and the mRNA paracrine factor levels from ﬁbroblasts. To determine how differently the paracrine factors reacted due to SCF silencing, the controlled gene expressions were compared to the gene expression with SCF knockdown. In Figure 2, the data showed a signiﬁcant increase in HGF, NRG, and CRH genes when SCF was stably silenced 48 h post transfection. The downregulated mRNA levels with DKK-1 and ET-1 were affected because of SCF knockdown in ﬁbroblast cells. ET-1 was the most severely affected gene, while the paracrine growth factor HGF was the least affected of all the genes. SCF gene alteration affected the ﬁbroblast paracrine system and its knockdown increased or decreased all the speciﬁc paracrine gene expression levels. 5 of 18 5 of Int. J. Mol. Sci. 2018, 19, 1475 Int. J. Mol. Sci. 2018, 19, x Figure 2. The influences on paracrine factors secreted by fibroblasts were compared between th vehicle control, UVB only, SCF knockdown, and SCF knockdown with UVB exposure groups. Th data are presented as means ± SD; n = 3, * p < 0.05. Figure 2. The inﬂuences on paracrine factors secreted by ﬁbroblasts were compared between the vehicle control, UVB only, SCF knockdown, and SCF knockdown with UVB exposure groups. The data are presented as means ± SD; n = 3, * p < 0.05. Figure 2. The influences on paracrine factors secreted by fibroblasts were compared between t vehicle control, UVB only, SCF knockdown, and SCF knockdown with UVB exposure groups. T data are presented as means ± SD; n = 3, * p < 0.05. Figure 2. 2.4. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts Using th Stimulated Model 2.4. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts Using the UVB-Stimulated Model Exposure to UVB is known to induce expression of SCF, so its effect on mRNA levels compared to the levels without UVB exposure would be a necessary observation. Comparisons between the paracrine factors with and without SCF knockdown, and with or without UVB, emphasize the individual gene expression variations not only in terms of SCF differences but also in determining whether UVB causes certain factors to react and express differently. In Figure 2, the results show the upregulation gene expressions of SCF, HGF, NRG, and CRH with UVB exposure to regular ﬁbroblasts compared with the control group, which did not undergo UVB stimulation. The ET-1 gene was downregulated, and no signiﬁcant difference was observed with DKK-1. Following the determination of the inﬂuence of UVB on regular ﬁbroblasts, the different outcome in ﬁbroblasts with SCF gene knockdown under UVB stimulation was tested. A comparison between regular ﬁbroblasts with UVB treatment and SCF gene knockdown ﬁbroblasts with the same UVB treatment is presented. The data presented the upregulation mRNA expression of HGF, NRG, and CRH and their expression levels when the ﬁbroblast cells, speciﬁcally with stable SCF silencing, were exposed to UVB. Compared to the regular ﬁbroblast gene expressions with UVB exposure, HGF, CRH, and NRG with SCF knockdowns all had their mRNA expressions increased, even with the same UVB treated situation. HGF demonstrated a particularly dramatic increase in gene expression levels in cells with SCF genetic knockdown, while DKK-1 and ET-1 were the SCF-silenced genes that were downregulated when treated with UVB. SCF gene alteration inﬂuenced the ﬁbroblast paracrine system and the condition differed with or without UVB stimulation. 2.3. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts 2.3. The Variations in Paracrine Factor Expressions through SCF Silencing in Fibroblasts The inﬂuences on paracrine factors secreted by ﬁbroblasts were compared between the vehicle control, UVB only, SCF knockdown, and SCF knockdown with UVB exposure groups. The data are presented as means ± SD; n = 3, * p < 0.05. 2.5. The Influence on Melanocytes When Treated with Conditioned Medi 2.5. The Inﬂuence on Melanocytes When Treated with Conditioned Medium Among them, tyrosinase, MITF, ERK-1, ERK-2, Myo5a, and c-kit were upregulated, and Pmel17 was downregulated when the conditioned medium from ﬁbroblasts undergoing UVB exposure and SCF silencing was applied to treat melanocytes, compared with the same medium with only UVB stimulation. These ﬁndings indicated SCF silencing caused the variations in melanocytes and regulated the melanogenesis-related gene expression using the conditioned medium treatment method. Int. J. Mol. Sci. 2018, 19, x 6 of 1 conditioned medium with UVB compared with the homeostatic condition. In both treatments of UV exposure with SCF inhibition group, the data showed the alterations in gene expressions i melanocytes when the conditioned medium was used for treatment. Among them, tyrosinase, MITF ERK-1, ERK-2, Myo5a, and c-kit were upregulated, and Pmel17 was downregulated when th conditioned medium from fibroblasts undergoing UVB exposure and SCF silencing was applied t treat melanocytes, compared with the same medium with only UVB stimulation. These finding indicated SCF silencing caused the variations in melanocytes and regulated the melanogenesis related gene expression using the conditioned medium treatment method. Pmel17 increased, while MITF, ERK-1, ERK-2, myosin Va (Myo5a), and c-kit decreased. The genes of tyrosinase, ERK-1, ERK-2, Myo5a, and c-kit were upregulated when treated in the conditioned medium with UVB compared with the homeostatic condition. In both treatments of UVB exposure with SCF inhibition group, the data showed the alterations in gene expressions in melanocytes when the conditioned medium was used for treatment. Among them, tyrosinase, MITF, ERK-1, ERK-2, Myo5a, and c-kit were upregulated, and Pmel17 was downregulated when the conditioned medium from ﬁbroblasts undergoing UVB exposure and SCF silencing was applied to treat melanocytes, compared with the same medium with only UVB stimulation. These ﬁndings indicated SCF silencing caused the variations in melanocytes and regulated the melanogenesis-related gene expression using the conditioned medium treatment method. Int. J. Mol. Sci. 2018, 19, x 6 of 1 conditioned medium with UVB compared with the homeostatic condition. In both treatments of UV exposure with SCF inhibition group, the data showed the alterations in gene expressions i melanocytes when the conditioned medium was used for treatment. Among them, tyrosinase, MITF ERK-1, ERK-2, Myo5a, and c-kit were upregulated, and Pmel17 was downregulated when th conditioned medium from fibroblasts undergoing UVB exposure and SCF silencing was applied t treat melanocytes, compared with the same medium with only UVB stimulation. 2.5. The Influence on Melanocytes When Treated with Conditioned Medi 2.5. The Inﬂuence on Melanocytes When Treated with Conditioned Medium These finding indicated SCF silencing caused the variations in melanocytes and regulated the melanogenesis related gene expression using the conditioned medium treatment method. Figure 3. The effects of different conditioned media on the mRNA expression levels were compared with regular fibroblasts, fibroblasts simulated UVB, fibroblasts with SCF knockdown, and fibroblasts exposed UVB with SCF gene silenced. The data are presented as means ± SD; n = 3, * p < 0.05. Figure 3. The effects of different conditioned media on the mRNA expression levels were compared with regular ﬁbroblasts, ﬁbroblasts simulated UVB, ﬁbroblasts with SCF knockdown, and ﬁbroblasts exposed UVB with SCF gene silenced. The data are presented as means ± SD; n = 3, * p < 0.05. Figure 3. The effects of different conditioned media on the mRNA expression levels were compare with regular fibroblasts, fibroblasts simulated UVB, fibroblasts with SCF knockdown, and fibroblas exposed UVB with SCF gene silenced. The data are presented as means ± SD; n = 3, * p < 0.05. Figure 3. The effects of different conditioned media on the mRNA expression levels were compared with regular ﬁbroblasts, ﬁbroblasts simulated UVB, ﬁbroblasts with SCF knockdown, and ﬁbroblasts exposed UVB with SCF gene silenced. The data are presented as means ± SD; n = 3, * p < 0.05. 2.6. The Variation in Melanin Content in Melanocyte 2.6. The Variation in Melanin Content in Melanocytes 2.5. The Influence on Melanocytes When Treated with Conditioned Medi 2.5. The Inﬂuence on Melanocytes When Treated with Conditioned Medium To examine the changes in melanocytes caused by fibroblasts with SCF gene alteration, the gen related to melanogenesis were analyzed. After the fibroblast cells with or without SCF gene we treated with or without UVB, the conditioned medium was collected after 24 h culture and then us to treat melanocytes for 24 h. The experimental conditioned medium obtained from fibroblasts w divided into four experimental groups: fibroblasts without both UVB exposure and SCF inhibitio with only UVB exposure, with only SCF inhibition, and with both UVB exposure and SCF inhibitio These secretions were used as culturing mediums for melanocytes to analyze their differe expression levels when cultured in mediums with SCF or without SCF. In Figure 3, SCF ge knockdown, the two conditioned medium groups including the one from regular fibroblasts an SCF-silenced fibroblasts, were used to treat melanocytes separately. The gene expression level To examine the changes in melanocytes caused by ﬁbroblasts with SCF gene alteration, the genes related to melanogenesis were analyzed. After the ﬁbroblast cells with or without SCF gene were treated with or without UVB, the conditioned medium was collected after 24 h culture and then used to treat melanocytes for 24 h. The experimental conditioned medium obtained from ﬁbroblasts was divided into four experimental groups: ﬁbroblasts without both UVB exposure and SCF inhibition, with only UVB exposure, with only SCF inhibition, and with both UVB exposure and SCF inhibition. These secretions were used as culturing mediums for melanocytes to analyze their different expression levels when cultured in mediums with SCF or without SCF. In Figure 3, SCF gene knockdown, the two conditioned medium groups including the one from regular ﬁbroblasts and SCF-silenced ﬁbroblasts, were used to treat melanocytes separately. The gene expression level of tyrosinase and 6 of 18 Int. J. Mol. Sci. 2018, 19, 1475 Pmel17 increased, while MITF, ERK-1, ERK-2, myosin Va (Myo5a), and c-kit decreased. The genes of tyrosinase, ERK-1, ERK-2, Myo5a, and c-kit were upregulated when treated in the conditioned medium with UVB compared with the homeostatic condition. In both treatments of UVB exposure with SCF inhibition group, the data showed the alterations in gene expressions in melanocytes when the conditioned medium was used for treatment. 2.6. The Variation in Melanin Content in Melanocyte 2.6. The Variation in Melanin Content in Melanocytes After culturing the melanocyte cells in the conditioned mediums containing the fibroblast ce secretions, the foreign medium could potentially have affected melanocytes and melanin production The alterations in melanin quantities in the melanocytes were confirmed using the melanin conten method. Fibroblasts with exposure to UVB, SCF gene knockdown, and SCF gene knockdown wit exposure to UVB were compared to figure out the actual variation in melanin content in melanocyte due to the different culturing mediums. In Figure 4a, the melanin production in melanocyte increased by 23.5% when treated in the conditioned medium from SCF fibroblasts knockdow compared to the normal fibroblasts and increased 27.3% under SCF fibroblasts knockdown with UV exposure. In Figure 4b, the morphology of melanocytes was altered by treatment with th conditioned medium of fibroblasts with SCF knockdown and UVB exposure, resulting in th melanocytes getting more synapses than UVB only. The result was consistent with Figure 2 indicating SCF silencing caused an essential variation in melanin quantities in the melanocytes. After culturing the melanocyte cells in the conditioned mediums containing the ﬁbroblast cell secretions, the foreign medium could potentially have affected melanocytes and melanin production. The alterations in melanin quantities in the melanocytes were conﬁrmed using the melanin content method. Fibroblasts with exposure to UVB, SCF gene knockdown, and SCF gene knockdown with exposure to UVB were compared to ﬁgure out the actual variation in melanin content in melanocytes due to the different culturing mediums. In Figure 4a, the melanin production in melanocytes increased by 23.5% when treated in the conditioned medium from SCF ﬁbroblasts knockdown compared to the normal ﬁbroblasts and increased 27.3% under SCF ﬁbroblasts knockdown with UVB exposure. In Figure 4b, the morphology of melanocytes was altered by treatment with the conditioned medium of ﬁbroblasts with SCF knockdown and UVB exposure, resulting in the melanocytes getting more synapses than UVB only. The result was consistent with Figure 2, indicating SCF silencing caused an essential variation in melanin quantities in the melanocytes. 7 of 18 Int. J. Mol. Sci. 2018, 19, 1475 Int. J. Mol. Sci. 2018, 19, x 7 of 18 Figure 4. (a) The melanin content in melanocytes after being treated with conditioned medium from fibroblast cells for 24 h; (b) The morphology of melanocytes was altered (bar = 125 μm). Red arrows showed the melanocytes with dendricity shape morphology. g g Using qPCR, we observed fibroblasts had a c-kit receptor that c 2.7. c-Kit Activation and SCF Regulation through Autocrine Mechanism 2 7 c-Kit Activation and SCF Regulation through Autocrine Mechanism under different conditions. Figure 5a shows c-kit gene expression was increased with UVB stimulation, even under SCF gene silencing by siRNA transfection compared with regular fibroblasts. In an attempt to demonstrate the existence of an autocrine system in fibroblast cells, different tests were conducted. In Figure 5b, the fibroblast’s inner SCF levels were downregulated when treated with 30 ng of recombinant SCF, while the fibroblast’s inner SCF levels were upregulated when treated with 3 μM of masitinib, which is an inhibitor of SCF and c-kit. When combined, 30 ng of SCF and 3 μM of masitinib produced a result similar to the controlled fibroblast’s inner SCF level. When treated with inhibiting or facilitative SCF environments, fibroblast cells varied their inner SCF levels to balance out their inner and outer levels, confirming the existence of an autocrine signaling system. Using qPCR, we observed ﬁbroblasts had a c-kit receptor that changed the expression level under different conditions. Figure 5a shows c-kit gene expression was increased with UVB stimulation, even under SCF gene silencing by siRNA transfection compared with regular ﬁbroblasts. In an attempt to demonstrate the existence of an autocrine system in ﬁbroblast cells, different tests were conducted. In Figure 5b, the ﬁbroblast’s inner SCF levels were downregulated when treated with 30 ng of recombinant SCF, while the ﬁbroblast’s inner SCF levels were upregulated when treated with 3 µM of masitinib, which is an inhibitor of SCF and c-kit. When combined, 30 ng of SCF and 3 µM of masitinib produced a result similar to the controlled ﬁbroblast’s inner SCF level. When treated with inhibiting or facilitative SCF environments, ﬁbroblast cells varied their inner SCF levels to balance out their inner and outer levels, conﬁrming the existence of an autocrine signaling system. g g Using qPCR, we observed fibroblasts had a c-kit receptor that changed the expression level under different conditions. Figure 5a shows c-kit gene expression was increased with UVB stimulation, even under SCF gene silencing by siRNA transfection compared with regular fibroblasts. In an attempt to demonstrate the existence of an autocrine system in fibroblast cells, different tests were conducted. In Figure 5b, the fibroblast’s inner SCF levels were downregulated when treated with 30 ng of recombinant SCF, while the fibroblast’s inner SCF levels were upregulated when treated with 3 μM of masitinib, which is an inhibitor of SCF and c-kit. 2.6. The Variation in Melanin Content in Melanocyte 2.6. The Variation in Melanin Content in Melanocytes The data are presented as means ± SD; n = 3, * p < 0.05. Figure 4. (a) The melanin content in melanocytes after being treated with conditioned medium from ﬁbroblast cells for 24 h; (b) The morphology of melanocytes was altered (bar = 125 µm). Red arrows showed the melanocytes with dendricity shape morphology. The data are presented as means ± SD; n = 3, * p < 0.05. nt. J. Mol. Sci. 2018, 19, x 7 of 18 Figure 4. (a) The melanin content in melanocytes after being treated with conditioned medium from fibroblast cells for 24 h; (b) The morphology of melanocytes was altered (bar = 125 μm). Red arrows showed the melanocytes with dendricity shape morphology. The data are presented as means ± SD; n = 3 * p < 0 05 Figure 4. (a) The melanin content in melanocytes after being treated with conditioned medium from fibroblast cells for 24 h; (b) The morphology of melanocytes was altered (bar = 125 μm). Red arrows showed the melanocytes with dendricity shape morphology. The data are presented as means ± SD; n = 3, * p < 0.05. Figure 4. (a) The melanin content in melanocytes after being treated with conditioned medium from ﬁbroblast cells for 24 h; (b) The morphology of melanocytes was altered (bar = 125 µm). Red arrows showed the melanocytes with dendricity shape morphology. The data are presented as means ± SD; n = 3, * p < 0.05. Figure 4. (a) The melanin content in melanocytes after being treated with conditioned medium from fibroblast cells for 24 h; (b) The morphology of melanocytes was altered (bar = 125 μm). Red arrows showed the melanocytes with dendricity shape morphology. The data are presented as means ± SD; 3 * < 0 05 3. Discussion and Conclusions In human skin, melanosomes are released from melanocyte dendrites and taken directly up by keratinocytes through endocytosis or phagocytosis. Little was known concerning the relationships between the morphology and the melanin content or tyrosinase activities of the melanocytes. In previous keratinocyte and melanocyte co-culture studies, we found that as melanocytes lose their characteristic dendritic structures and adopt ﬁbroblast-like bipolar forms, the cell–cell contact between melanocytes and keratinocytes is considerably reduced, resulting in a reduction in pigment transfer [34]. Our previous two studies suggested that there might be some connections between the dendritic morphology changes and physiological properties of melanocytes [12,33]. These potential relationships should be further investigated to understand their physiological signiﬁcances. SCF is a paracrine cytokine that plays an essential role in hematopoiesis, maintenance of cell survival, proliferation, and activation, as well as in melanogenesis. Although several studies demonstrated the functions and mechanisms of SCF in the melanogenesis process, those studies mostly focused on pigment production only. This study examined the role of SCF in paracrine factors in ﬁbroblasts and its subsequent inﬂuence on melanocytes under a loss of SCF function in ﬁbroblasts. RNA interference (RNAi) is a physiologically mediated mechanism that induces sequence-speciﬁc gene degradation. The double-strand RNA undergoes the dicer processes into the siRNA. Binding of siRNA to the target gene causes mRNA degradation directly or arrests the speciﬁc protein translation; therefore, the phenomenon is known as gene silencing [35]. Consequently, siRNA is suitable for observing the gene inﬂuences, protein functions, and cell developments when the target gene is knocked down and for understanding the role of the target gene. This study used the SCF-targeted siRNA-cooperated transfection method to introduce siRNA into the intracellular environment and inhibit SCF gene expression in ﬁbroblasts. In this study, all the experiments processed the validation and kept the transfection efﬁciency at a stable inhibitory state. After siRNA transfected into the intracellular environment and induced cellular SCF gene inhibition, the ﬁrst aspect to conﬁrm was whether the gene silencing caused cell death or a morphological change, since SCF was responsible for cell survival and proliferation. In MTT assay data, it presented no signiﬁcant changes in ﬁbroblast cell proliferations, and similar results were observed regarding ﬁbroblast morphology, where without cellular atrophy damage, ﬂattened or low conﬂuency were observed. Fibroblast proliferation did not rely on SCF, even though it was silenced. g g Using qPCR, we observed fibroblasts had a c-kit receptor that c 2.7. c-Kit Activation and SCF Regulation through Autocrine Mechanism 2 7 c-Kit Activation and SCF Regulation through Autocrine Mechanism When combined, 30 ng of SCF and 3 μM of masitinib produced a result similar to the controlled fibroblast’s inner SCF level. When treated with inhibiting or facilitative SCF environments, fibroblast cells varied their inner SCF levels to balance out their inner and outer levels, confirming the existence of an autocrine signaling system. Figure 5. Autocrine of SCF on fibroblasts. (a) The expression of c-kit on fibroblast; (b) Fibroblasts treated with SCF 30 ng, mastinib 3 μM, and both of mastinib and SCF. The data are presented as means ± SD; n = 3. * p < 0.015. Figure 5. Autocrine of SCF on fibroblasts. (a) The expression of c-kit on fibroblast; (b) Fibroblasts treated with SCF 30 ng, mastinib 3 μM, and both of mastinib and SCF. The data are presented as means ± SD; n = 3. * p < 0.015. Figure 5. Autocrine of SCF on ﬁbroblasts. (a) The expression of c-kit on ﬁbroblast; (b) Fibroblasts treated with SCF 30 ng, mastinib 3 µM, and both of mastinib and SCF. The data are presented as means ± SD; n = 3. * p < 0.015. gure 5. Autocrine of SCF on fibroblasts. (a) The expression of c-kit on fibroblast; (b) Fibrobl eated with SCF 30 ng, mastinib 3 μM, and both of mastinib and SCF. The data are presented eans ± SD; n = 3. * p < 0.015. p Figure 5. Autocrine of SCF on fibroblasts. (a) The expression of c-kit on fibroblast; (b) Fibroblasts treated with SCF 30 ng, mastinib 3 μM, and both of mastinib and SCF. The data are presented as means ± SD; n = 3. * p < 0.015. Figure 5. Autocrine of SCF on ﬁbroblasts. (a) The expression of c-kit on ﬁbroblast; (b) Fibroblasts treated with SCF 30 ng, mastinib 3 µM, and both of mastinib and SCF. The data are presented as means ± SD; n = 3. * p < 0.015. Int. J. Mol. Sci. 2018, 19, 1475 8 of 18 3. Discussion and Conclusions Although SCF inhibition did not cause ﬁbroblast death, the inﬂuence on ﬁbroblasts was unclear. To explore the effect on ﬁbroblasts when the SCF gene was silenced, some paracrine factors secreted by ﬁbroblasts showing effects on melanocyte cell survival and melanin production were determined, including HGF, DKK-1, NRG-1, CRH, and ET-1. HGF is a polypeptide growth factor that can bind to the MET receptor expressed on melanocytes, with HGF stimulation leading to MITF-dependent Met message and protein induction [36]. The MET proto-oncogene encodes for the hepatocyte growth factor (HGF) receptor, a plasma membrane tyrosine kinase that is involved in melanocyte growth and melanoma development. When the ligand binds to the receptor, MET processes autophosphorylation on tyrosine residues produce the docking site for PI3K to promote growth and differentiation of melanocytes, thereby inﬂuencing melanogenesis via activation of the cyclic adenosine monophosphate (cAMP) pathway [37]. DKK-1 secreted by ﬁbroblasts mostly exists in the palmoplantar skin compared with the nonpalmoplantar skin [38]. DKK-1 has the ability to inhibit melanocyte growth via regulating the Wnt signaling pathway in which DKK-1 suppresses β-catenin accumulation and then decreases MITF activity, thereby inﬂuencing the functions of the melanocytes. In a previous study, Wonseon Choi et al. used the microarray analysis to indicate in dark skin, and NRG-1 was the highly expressed factor secreted by ﬁbroblasts and increased skin pigmentation [6]. NRG-1 mediates melanogenesis through the PI3K pathway, increasing melanocyte proliferation and survival and melanin production. CRH is regulated by UV or as a response to stress. CRH can bind to its receptors, CRH-R1 and CRH-R2, and then induce the POMC-derived hormones, including MSH and ACTH released to inﬂuence the skin phenotype system [39]. ET-1 functions as Int. J. Mol. Sci. 2018, 19, 1475 9 of 18 a mitogenic factor for melanocytes and facilitates melanogenesis. ET-1 can bind to its endothelin receptors A (ETA) and activate the cAMP pathway that increases protein kinase C activation to stimulate MITF expression. Akira Hachiya et al. demonstrated the responses of SCF and ET-1 to UVB irradiation, in which SCF responded in the early stage and ET-1 in the later phase of UVB-induced melanin production [40,41]. p Through SCF-targeted siRNA inhibition, we demonstrated HGF, NRG, and CRH gene expressions were signiﬁcantly increased; especially, HGF gene level had the highest change while DKK-1 and ET-1 gene expressions were decreased. 3. Discussion and Conclusions The results demonstrated an autoregulation phenomenon between gene networks, inducing a feedback expression when the SCF gene was silenced in ﬁbroblasts. Cellular autoregulation is frequently observed in gene networks through either negative or positive feedback adjustments [42]. Among them, HGF showed 20-fold high expression in ﬁbroblasts after SCF gene silencing compared with regular ﬁbroblasts. Some experiments demonstrated a similar result that SCF expressed in murine stromal cells was activated and induced an increased expression when treated with extra HGF [43–45]. This demonstration indicated the relationship between HGF and SCF. Hence, in this case, SCF gene knockdown induced transcription of HGF for facilitating the expression of SCF to balance the gene networks. NRG and CRH involved in melanogenesis also increased their gene expressions under the condition of SCF gene knockdown. On the other hand, DKK-1, a Wnt-antagonist, was downregulated after SCF was inhibited. Gherghe et al. indicated Wnt treatment signiﬁcantly activated HGF expression in human endothelial progenitor cells [46]. In this case, the Wnt-antagonist DKK-1 was downregulated to avoid the Wnt signaling system inhibition, thereby increasing the expression of HGF. The other downregulated gene, ET-1, was indicated for the mechanism of synergistic effect on ET-1 and SCF that led to melanogenesis. Therefore, SCF inhibition inﬂuences ET-1 gene expression. Under the homeostatic status, some paracrine factors are changed or their expression is induced by cells suffering from different stresses such as UV. SCF plays a potential role in regulating melanogenesis under homeostatic and stimulatory statuses, especially in response to UVB irradiation [47]. It is known that UVB upregulates transcription, increases the expressions of c-kit and SCF, and stimulates melanogenic activity in melanocytes. To understand whether the stimulation with UVB when SCF was silenced would inﬂuence the paracrine factors in ﬁbroblasts, an experiment with gene knockdown and then treatment with UVB were performed. The results showed ﬁbroblasts under UVB exposure increase the mRNA expression levels of SCF, HGF, DKK-1, NRG, and CRH related to the melanogenesis gene. Under the same condition, ET-1 was downregulated, as shown in previous studies that reported ET-1 was responsible for a later stage of UVB-induced melanin production. Then, comparison between only UVB-treated and both SCF gene knockdown and UVB-stimulated ﬁbroblasts showed similar results but increasing levels of HGF, NRG, and CRH gene expressions under an SCF-silenced status. 3. Discussion and Conclusions 36H downregulated protein expression for Myo5a and might prevent a darkening of skin color. Melanin is deposited on these ﬁbers and melanosomes become mature in the melanocytes [54]. Melanocytes are activated through multiple paracrine factors secreted from adjacent cells, such as keratinocytes and ﬁbroblasts. The paracrine factors bind to the speciﬁc receptors expressed on melanocytes and then accelerate signal transductions to initiate melanin production. For example, SCF binds to the c-kit receptor, and MAPK family, ERK1/2, JNK, and p38, are activated in the melanocytes, thereby altering MITF activities and functions through phosphorylation or dephosphorylation. MITF is a speciﬁc transcription factor of tyrosinase, TRP-1 and TRP-2, and the mutant of MITF leads to a deﬁciency in melanocytes. Melanin is the end product of the multistep conversion of tyrosine. Tyrosinase, a rate-limiting enzyme, catalyzes tyrosine into DOPA, and subsequently DOPA undergoes a separate pathway to convert to pheomelanin or eumelanin. After maturation of melanin, it is transferred to keratinocytes along with microtubules and dendrites by the kinesin and dynein motor proteins. Actin-based motor protein myosinVa attaches to melanosomes and moves via interaction with Rab27a and melanophilin to the surrounding keratinocytes. In this work, to avoid direct UVB exposure and for observing the inﬂuence from ﬁbroblasts, culture conditioned mediums from ﬁbroblasts were harvested and used to treat melanocytes. Using the conditioned medium from regular ﬁbroblasts and ﬁbroblasts with UVB treatment, ERK-1, ERK-2, Myo5a, and c-kit genes were upregulated while MITF and Pmel17 were downregulated when treated with UVB-exposed FB medium. According to the above results, UVB-induced SCF, HGF, NRG, and CRH increased. ERK-1 and ERK-2 were signiﬁcantly increased, attributable to both the SCF and HGF signaling transduction pathways. UVB induced SCF production, so the receptor c-kit expressed on melanocytes increased. In contrast, MITF gene expression was downregulated under UVB-exposed ﬁbroblast medium treatment. McGill et al. indicated MET, the HGF receptor, was regulated by MITF, since MET was a transcriptional target of MITF. In this case, HGF was increased from the conditioned medium and caused high MET expression in the melanocytes, so MITF decreased to reduce MET overexpression [55]. After treatment with the conditioned medium from ﬁbroblasts with SCF silencing, the melanocytes showed downregulation of MITF, ERK-1, ERK-2, Myo5a, and c-kit, while tyrosinase and Pmel17 were upregulated, compared with treatment with regular ﬁbroblast medium. 3. Discussion and Conclusions Among them, HGF showed more than a 3-fold change compared to only UVB-treated ﬁbroblasts and more than a 20-fold change compared to regular ﬁbroblasts. It was demonstrated SCF was important for UVB-induced paracrine secretion, so HGF needed to strengthen its expression to balance the defect from SCF silencing. In addition, SCF and HGF showed a high correlation in ﬁbroblasts with either UVB stimulation or the homeostatic condition [48,49]. Silencing SCF affected paracrine factor expressions in the ﬁbroblasts. Melanocytes receive the signals generated from ﬁbroblasts and then activate melanin production. In subsequent experiments, we investigated whether SCF-silenced ﬁbroblasts further inﬂuenced melanocytes for melanogenesis, and the gene expression in melanocytes was determined. Melanin biosynthesis is a complex process occurring in melanocytes within particular membrane-bound organelles, known as melanosomes [39]. The major component of the melanosome is Pmel17, which is a transmembrane protein and essential for melanin synthesis and deposition to form the ﬁbrillar matrix. Rab27a, melanophilin (MLPH), and Myo5a form a tri-protein complex to bind melanosomes at the melanocytes peripheries. In the process of melanosome transport, the ternary complex is the connection between actin cytoskeleton and melanosome. A lack of these proteins affects the transport, and melanoregulin (Mreg) drives 10 of 18 Int. J. Mol. Sci. 2018, 19, 1475 melanosome transfer from melanocytes to keratinocytes via a regulated shedding mechanism. Human skin melanin is driven by the intercellular movement of melanin-containing melanosomes from the extremities of human melanocytes dendrites to neighboring keratinocytes. When it is carried by the actin ﬁlament, melanosome moves to the dendritic tail section, through exocytosis, and is transported into keratinocytes [50]. The greater the amount of melanin that is transferred into keratinocytes, the darker is the color of the skin [18]. The movement on the microtubule depends on the dynein–dynactin motor complex. Mreg forms a complex with Rab-interacting lysosomal protein and p150 (Glued), which is a subunit of dynactin [51]. Mreg adjusts a shedding system which makes melanosome transport from human melanocytes to keratinocytes. The shedding process from human melanocytes of melanosome-rich packages is undergoing the phagocytosis of keratinocytes. The shedding not only takes place principally at dendritic extremities, but also around the center areas, having the adhesion to keratinocytes, tightening behind the forming packages, and apparent self-abscissions [52]. The movement on the actin ﬁlament requires Myo5a, Rab27a, and MLPH as the connecting bridge [53]. 3. Discussion and Conclusions Under this situation, SCF was inhibited by siRNA, so the expression was followed by a decrease, and then the melanocytes treated with this medium showed decreased gene expression of ERK-1, ERK-2, and SCF receptor c-kit. Comparing regular ﬁbroblasts exposed to UVB and SCF-inhibited ﬁbroblasts treated with UVB stimulation, the melanocytes cultured in the conditioned medium showed increased gene expression of tyrosinase, MITF, Myo5a, and c-kit. According to the above results, HGF, NRG, and CRH were upregulated in SCF-silenced ﬁbroblasts with UVB exposure compared with ﬁbroblasts treated with only UVB, so the melanocytes accepted more stimulation to increase the melanogenesis-related gene expression level. SCF silencing also caused ERK-1 downregulation, but ERK-2 showed no signiﬁcant Int. J. Mol. Sci. 2018, 19, 1475 11 of 18 change in its gene level compared to regular ﬁbroblasts exposed to UVB in melanocytes. In addition, this result is observed when the SCF gene is knocked down, the gene Myo5a is downregulated, and Pmel17 is upregulated whether there is UVB exposure or not. To conﬁrm the essential changes in the melanocyte, the quantity of melanin production was examined. The melanin content assay showed melanocytes cultured with conditioned medium from SCF-silenced ﬁbroblasts exposed to UVB increased the amount of melanin by about 18%, compared with regular ﬁbroblasts exposed to UVB. This result was consistent with the abovementioned data that melanogenesis-related genes were upregulated. Based on these ﬁndings, indeed, SCF silencing caused variations in ﬁbroblasts to paracrine factors, and the variation changed if ﬁbroblasts suffered UVB stimulation. The results from different ﬁbroblasts affected melanocyte activities via melanogenesis-related gene expression. Especially under the condition of ﬁbroblasts with SCF silencing and then exposure to UVB, the melanocytes cultured in ﬁbroblast secretions exhibited increased melanogenesis-related gene levels, and the phenomenon was conﬁrmed by their increased melanin quantities [36]. Analysis of the SCF receptor c-kit mRNA expression level in the ﬁbroblasts showed the c-kit gene level increased under SCF inhibition in ﬁbroblasts and further increased when ﬁbroblasts with SCF inhibition were exposed to UVB. Although c-kit showed increased expression following UVB exposure with or without SCF gene knockdown, the underlying mechanisms need to be conﬁrmed. A probable reason would be that as SCF and c-kit were induced by UVB stimulation and SCF was knocked down, c-kit increased its expression to improve the binding of the ligand to the receptor. 4.1. Reagents and Materials Cell culture material 4.1. Reagents and Materials Cell culture materials Medium 254, Medium 254 supplement, fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), and antibiotics were obtained from Gibco (Waltham, MA, USA). Short interfering RNA (siRNA) transfection reagent, Trizol reagent, 1-bromo-3- chloropropane (BCP), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical (St. Louis, MO, USA). ToolScript MMLV RT kit and TOOLS 2× SYBR quantitative real-time polymerase chain reaction (qPCR) Mix was purchased from Biotools (Taipei, Taiwan). Cell culture materials Medium 254, Medium 254 supplement, fetal bovine serum (FBS), Dulbecco’s modiﬁed Eagle medium (DMEM), and antibiotics were obtained from Gibco (Waltham, MA, USA). Short interfering RNA (siRNA) transfection reagent, Trizol reagent, 1-bromo-3-chloropropane (BCP), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical (St. Louis, MO, USA). ToolScript MMLV RT kit and TOOLS 2× SYBR quantitative real-time polymerase chain reaction (qPCR) Mix was purchased from Biotools (Taipei, Taiwan). 4.2. Cell Cultures 4.2. Cell Cultures The neonatal foreskin human melanocytes purchased from Cascade Biologics (C-102-5C, Gibco) were maintained in Medium 254 supplemented with human melanocyte growth supplement containing basic FGF (3 ng/mL), insulin (5 μg/mL), transferrin (5 μg/mL), bovine pituitary extract (0.2%), FBS (0.5%), heparin (3 μg/mL), hydrocortisone (0.18 μg/mL), and phorbol 12-myristate 13- acetate (10 ng/mL). The human fibroblasts Hs68 cells derived from male foreskin (ATCC® CRL-1635™) were obtained from the Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan) and cultured in DMEM supplemented with 10% FBS and 1% antibiotic. All cells were incubated at 37 °C under 5% CO2 [8,13]. The neonatal foreskin human melanocytes purchased from Cascade Biologics (C-102-5C, Gibco) were maintained in Medium 254 supplemented with human melanocyte growth supplement containing basic FGF (3 ng/mL), insulin (5 µg/mL), transferrin (5 µg/mL), bovine pituitary extract (0.2%), FBS (0.5%), heparin (3 µg/mL), hydrocortisone (0.18 µg/mL), and phorbol 12-myristate 13-acetate (10 ng/mL). The human ﬁbroblasts Hs68 cells derived from male foreskin (ATCC® CRL-1635™) were obtained from the Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan) and cultured in DMEM supplemented with 10% FBS and 1% antibiotic. All cells were incubated at 37 ◦C under 5% CO2 [8,13]. 3. Discussion and Conclusions Autocrine signaling is a type of cell regulation in which cells secrete and respond to their particular growth factors. According to ﬁbroblast autoregulated gene expression under SCF gene knockdown and c-kit expression on ﬁbroblasts, the ﬁbroblasts regulated SCF produced in an autocrine manner. Addition of recombinant SCF resulted in a decreased expression level, showing a natural feedback in that the ﬁbroblast cells stopped producing as much SCF [8]. Similarly, adding masitinib inhibited the binding between SCF and c-kit, and so the expression level was increased. The fact that adding both masitinib and SCF produces similar expression levels as the control indicated the automatic balancing of the inner SCF levels, which is the role of an autocrine signaling system. A similar SCF autocrine effect was demonstrated in several tumors [56,57]. These results further showed the existence of an autocrine system with co-expression of SCF and c-kit in ﬁbroblasts. The present study revealed that although SCF is involved in several processes such as cellular differentiation, proliferation, and survival, it does not inﬂuence ﬁbroblast proliferation. Once SCF is silenced in ﬁbroblasts, the autoregulation in gene networks balances the SCF defects in an autocrine regulatory manner. Investigation of the paracrine factors HGF, DKK-1, NRG, CRH, and ET-1 showed HGF exhibited an especially high correlation with SCF. Fibroblasts with SCF dysfunction inﬂuenced their own paracrine systems and further affected melanocytes to alter melanin production, particularly under UVB stimulation (Figure 6). Altogether, SCF plays an essential role in ﬁbroblast paracrine factors and in the melanocyte cellular melanogenesis effect. 12 of 18 Int. J. Mol. Sci. 2018, 19, 1475 Figure 6. The mRNA pathway of SCF in fibroblasts affects the melanogenesis in melanocytes. 4 M t i l d M th d Figure 6. The mRNA pathway of SCF in ﬁbroblasts affects the melanogenesis in melanocytes. Figure 6. The mRNA pathway of SCF in fibroblasts affects the melanogenesis in melanocytes. Figure 6. The mRNA pathway of SCF in ﬁbroblasts affects the melanogenesis in melanocytes. 4.5. UVB Exposure to Fibroblasts and Melanocytes Treated with Conditioned Medium To induce SCF secretion from ﬁbroblasts and simulate the condition of light stimulation, UVB was used to complete the test. This test was performed according to the method, with some modiﬁcations, described by Shin et al. [13]. Fibroblasts with or without previous siRNA transfection were cultured in a 6-well plate. Before UV exposure, the medium was replaced by PBS during that time. UVB exposure was performed using Ultraviolet Crosslinkers CL-1000 (UVP, Upland, CA, USA) with UVB radiation (302 nm) at a nontoxic dosage of 5 mJ/cm2 3 times at intervals of 5 h. After UVB treatment and culturing for 24 h, the medium was collected into the conditioned medium and used for treating melanocytes previously cultured in the 6-well plate for 24 h. Experimental cells were harvested for subsequent testing. Target sequence 1: GGAAUCGUGUGACUAUAA Target sequence 2: AUAAGUAUGUUGCAAGAGA Target sequence 3: UAAGCGAGAUGGUAGUACA Target sequence 4: GCUUUAUAGUUGCCGAUUA Target sequence 1: GGAAUCGUGUGACUAUAA Target sequence 2: AUAAGUAUGUUGCAAGAGA Target sequence 3: UAAGCGAGAUGGUAGUACA Target sequence 4: GCUUUAUAGUUGCCGAUUA 4.6. Total RNA Isolation and Extraction The total RNA was extracted using Trizol RNA isolation reagent, which can break down cell lysates and isolate RNA, DNA, and proteins. First, 1 mL Trizol reagent was added to each and transferred to a 1.5-mL microtube at room temperature for 5 min. Then, 200 µL BCP per mL of Trizol reagent was added and mixed vigorously. Following incubation for 2 min, the samples were centrifuged at 14,000× g for 15 min. The sample homogenates formed two phases, from which the aqueous phase on the top of the homogenate was transferred to a new Eppendorf tube. To precipitate RNA, an equal volume of isopropanol was added and mixed. The mixture was centrifuged at 14,000× g for 15 min and the supernatant was removed. The RNA pellet was washed with 1 mL of 75% ethanol to remove the residual salts. Finally, the mixture was centrifuged at 12,000× g for 5 min and the RNA pellet was dried and dissolved with 50 µL diethylpyrocarbonate (DEPC)-treated water. The concentration and quality of the RNA extracts were determined by NanoDrop (Thermo Fisher, Waltham, MA, USA). 4.3. Transfection of SCF-Targeted siRNA into Fibroblasts 4.3. Transfection of SCF-Targeted siRNA into Fibroblasts Fibroblasts were seeded in a 6-well plate at a density of 2 × 105 cells and then cultured for 24 h until the cells reached about 80%–90% confluence. Then, the siRNA mixture was prepared for further experiments. SCF-targeted pooled siRNA supplied by Dharmacon (GE Dharmacon, Lafayette, CO, USA) was diluted to 25 nM in 100 μL per well of serum-free DMEM and 20 μL per well transfection reagent was added into the diluted siRNA solution. After mixing and incubation for 20 min, 100 μL Fibroblasts were seeded in a 6-well plate at a density of 2 × 105 cells and then cultured for 24 h until the cells reached about 80%–90% conﬂuence. Then, the siRNA mixture was prepared for further experiments. SCF-targeted pooled siRNA supplied by Dharmacon (GE Dharmacon, Lafayette, CO, USA) was diluted to 25 nM in 100 µL per well of serum-free DMEM and 20 µL per well transfection reagent was added into the diluted siRNA solution. After mixing and incubation for 20 min, 100 µL 13 of 18 Int. J. Mol. Sci. 2018, 19, 1475 of the mixture was added to each well and incubated for 3 days at 37 ◦C under 5% CO2. The control group was ﬁbroblasts transfected reagent without siRNA. The transgene expression efﬁciency was detected by qPCR. The siRNA sequences used in the experiment are shown in Table 1. of the mixture was added to each well and incubated for 3 days at 37 ◦C under 5% CO2. The control group was ﬁbroblasts transfected reagent without siRNA. The transgene expression efﬁciency was detected by qPCR. The siRNA sequences used in the experiment are shown in Table 1. Table 1. SCF-targeted siRNA sequences used in this study. Table 1. SCF-targeted siRNA sequences used in this study. Table 1. SCF-targeted siRNA sequences used in this study. 4.4. MTT Assay in Cell Viability MTT assay was used to evaluate cell viability and proliferation. MTT, a tetrazole with a yellow color, is taken up by living cells and then converted into a purple formazan-reduced state by mitochondrial dehydrogenase. Cells were seeded in a 96-well plate at a density of 8 × 103 cells per well and cultured for 24 h. Following attachment, cells were replaced in fresh medium with siRNA and transfection reagent complex and cultured for 48 h. Then, the medium was changed to fresh medium containing 100 µL of 0.5 mg/mL MTT and then cultured for 2 h at 37 ◦C under 5% CO2. Then, the medium was discarded and 100 µL DMSO was added to dissolve the formazan. The absorbance was measured at 595 nm [15]. 4.5. UVB Exposure to Fibroblasts and Melanocytes Treated with Conditioned Medium 4.7. Detection of mRNA Gene Expression Using qPCR Before qPCR analysis, RNA should be processed by a reverse transcription step to obtain [8] complementary DNA (cDNA). cDNA synthesis was accomplished by ToolScript MMLV RT kit. Brieﬂy, 1 µg total RNA of each sample was mixed with 2 µL oligo dT, 2 µL dNTP, and distillation-distillation 14 of 18 14 of 18 Int. J. Mol. Sci. 2018, 19, 1475 H2O to bring the total volume to 14.5 µL. To open the RNA secondary structure, the RNA mixture was heated at 70 ◦C for 5 min and then cooled on ice for 2 min. To the cooled mixture were added 4 µL reverse buffer, 0.5 µL RNasin, and 1 µL MMLV. Afterward, the mixture was incubated at 42 ◦C for 50 min to synthesize cDNA and then heated at 95 ◦C for 5 min to inactivate the MMLV. Further, the cDNA products were processed on qPCR using TOOLS 2× SYBR qPCR Mix. First, a reaction solution was prepared containing 10 µL 2× SYBR qPCR mix, 0.1 µL forward primer, 0.1 µL reverse primer, 1 µL cDNA template, 2 µL of 50× ROX reference dye, and distillation–distillation H2O to bring the total volume to 20 µL per well. Next, the qPCR machine (ABITM StepOneTM Plus, Thermo Fisher) was set up. The initial denaturation temperature was 95 ◦C for 15 min by one cycle, followed by a denaturation temperature of 95 ◦C for 10 s, annealing at 60 ◦C for 20 s, and extension at 72 ◦C for 30 s, with the total PCR stage being 40 cycles. The designed forward and reverse primers from 5′ to 3′ used in this experiment are shown in (Table 2) [8]. Table 2. The primer sequences used in this study. SCF Forward: 5′-CTGCCAACGATCCTATCTTCCT-3′ Reverse: 5′-GGTTATGTCCAATGGGTGCATT-3′ HGF Forward: 5′-TCCCTACCTCTCTCGCTGTCT-3′ Reverse: 5′-GGGTAAGGGCCAGCATGTA-3′ DKK-1 Forward: 5′-GCGGGAATAAGTACCAGACCAT-3′ Reverse: 5′-TGCAGGCGAGACAGATTTG-3′ NRG Forward: 5′-AGGCCAGGACCCTATTATTTC-3′ Reverse: 5′-TTCAGATTGAGCCCTAGAGACA-3′ CRH Forward: 5′-AGGCACCGGAGAGAGAAAG-3′ Reverse: 5′-GTTTCCTGTTGCTGTGAGCTT-3′ ET-1 Forward: 5′-TGGTTCCTGACTGGCAAAG-3′ Reverse: 5′-GGAAGCCAGTGAAGATGGTT-3′ Tyrosinase Forward: 5′-CTGCCAACGATCCTATCTTCCT-3′ Reverse: 5′-GGTTATGTCCAATGGGTGCATT-3′ MITF Forward: 5′-TTGGTGCCACCTAAAACATTGT-3′ Reverse: 5′-CCGTTGGGCTTGCTGTATG-3′ ERK-1 Forward: 5′-CAACACCACCTGCGACCTT-3′ Reverse: 5′-GCCACATACTCCGTCAGGAA-3′ ERK-2 Forward: 5′-CGGTGTTCTTCTTCCCAGTTC-3′ Reverse: 5′-AAAGCCACAACTACCAGAAACC-3′ Pmel17 Forward: 5′-GGATGGTACAGCCACCTTAAGG-3′ Reverse: 5′-CAGGATCTCGGCACTTTCAATAC-3′ Myo5a Forward: 5′-GCCCAGATTGTGAAAGTGTTGA-3′ Reverse: 5′-CCTGTCTCGTAAACGCATCTGT-3′ Table 2. The primer sequences used in this study. 4.7. Detection of mRNA Gene Expression Using qPCR SCF Forward: 5′-CTGCCAACGATCCTATCTTCCT-3′ Reverse: 5′-GGTTATGTCCAATGGGTGCATT-3′ HGF Forward: 5′-TCCCTACCTCTCTCGCTGTCT-3′ Reverse: 5′-GGGTAAGGGCCAGCATGTA-3′ DKK-1 Forward: 5′-GCGGGAATAAGTACCAGACCAT-3′ Reverse: 5′-TGCAGGCGAGACAGATTTG-3′ NRG Forward: 5′-AGGCCAGGACCCTATTATTTC-3′ Reverse: 5′-TTCAGATTGAGCCCTAGAGACA-3′ CRH Forward: 5′-AGGCACCGGAGAGAGAAAG-3′ Reverse: 5′-GTTTCCTGTTGCTGTGAGCTT-3′ ET-1 Forward: 5′-TGGTTCCTGACTGGCAAAG-3′ Reverse: 5′-GGAAGCCAGTGAAGATGGTT-3′ Tyrosinase Forward: 5′-CTGCCAACGATCCTATCTTCCT-3′ Reverse: 5′-GGTTATGTCCAATGGGTGCATT-3′ MITF Forward: 5′-TTGGTGCCACCTAAAACATTGT-3′ Reverse: 5′-CCGTTGGGCTTGCTGTATG-3′ ERK-1 Forward: 5′-CAACACCACCTGCGACCTT-3′ Reverse: 5′-GCCACATACTCCGTCAGGAA-3′ ERK-2 Forward: 5′-CGGTGTTCTTCTTCCCAGTTC-3′ Reverse: 5′-AAAGCCACAACTACCAGAAACC-3′ Pmel17 Forward: 5′-GGATGGTACAGCCACCTTAAGG-3′ Reverse: 5′-CAGGATCTCGGCACTTTCAATAC-3′ Myo5a Forward: 5′-GCCCAGATTGTGAAAGTGTTGA-3′ Reverse: 5′-CCTGTCTCGTAAACGCATCTGT-3′ Table 2. The primer sequences used in this study. SCF Forward: 5′-CTGCCAACGATCCTATCTTCCT-3′ Reverse: 5′-GGTTATGTCCAATGGGTGCATT-3′ HGF Forward: 5′-TCCCTACCTCTCTCGCTGTCT-3′ Reverse: 5′-GGGTAAGGGCCAGCATGTA-3′ DKK-1 Forward: 5′-GCGGGAATAAGTACCAGACCAT-3′ Reverse: 5′-TGCAGGCGAGACAGATTTG-3′ NRG Forward: 5′-AGGCCAGGACCCTATTATTTC-3′ Reverse: 5′-TTCAGATTGAGCCCTAGAGACA-3′ CRH Forward: 5′-AGGCACCGGAGAGAGAAAG-3′ Reverse: 5′-GTTTCCTGTTGCTGTGAGCTT-3′ ET-1 Forward: 5′-TGGTTCCTGACTGGCAAAG-3′ Reverse: 5′-GGAAGCCAGTGAAGATGGTT-3′ Tyrosinase Forward: 5′-CTGCCAACGATCCTATCTTCCT-3′ Reverse: 5′-GGTTATGTCCAATGGGTGCATT-3′ MITF Forward: 5′-TTGGTGCCACCTAAAACATTGT-3′ Reverse: 5′-CCGTTGGGCTTGCTGTATG-3′ ERK-1 Forward: 5′-CAACACCACCTGCGACCTT-3′ Reverse: 5′-GCCACATACTCCGTCAGGAA-3′ ERK-2 Forward: 5′-CGGTGTTCTTCTTCCCAGTTC-3′ Reverse: 5′-AAAGCCACAACTACCAGAAACC-3′ Pmel17 Forward: 5′-GGATGGTACAGCCACCTTAAGG-3′ Reverse: 5′-CAGGATCTCGGCACTTTCAATAC-3′ Myo5a Forward: 5′-GCCCAGATTGTGAAAGTGTTGA-3′ Reverse: 5′-CCTGTCTCGTAAACGCATCTGT-3′ Table 2. The primer sequences used in this study. Int. J. Mol. Sci. 2018, 19, 1475 15 of 18 4.8. Melanin Content Assessment Estimation of melanin content is a method for cellular melanin determination. At ﬁrst, cells were seeded in a 6-well plate at a density of 2 × 105 cells and cultured for 24 h. Then, the medium was changed to conditioned medium obtained from ﬁbroblasts cultured for 24 h. Afterward, the cell pellets were harvested and dissolved in 2.0 N NaOH, and then heated at 95 ◦C for 1 h. The absorbance of melanin was measured at 490 nm [30]. 4.9. Statistical Analysis All the experiments in each platform were carried out in triplicate and presented as mean ± standard error. For statistical analysis, all data were analyzed by Student’s t-test for multiple comparisons. A signiﬁcant difference (*) was deﬁned as p < 0.05. Author Contributions: P.-H.L. and L.-H.L. performed the experiments; P.-H.L., L.-H.L., C.-C.C., R.G., C.-H.L., D.-L.M. and H.-M.D.W. analyzed the data; C.-C.C. and H.-M.D.W. contributed reagents/materials/analysis tools; C.-C.C., R.G., C.-H.L., D.-L.M. and H.-M.D.W. wrote the paper. Acknowledgments: This work was supported by grants from the Ministry of Science and Technology, Taiwan (MOST 104-2221-E-005-096-MY2, MOST 104-2628-E-005-004-MY3and MOST 106-2622-E-005-002-CC2). We also thank the projects of Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan, KMU-TP104G00, KMU-TP104G01 and KMU-TP104G02-05. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Wang, H.D.M.; Li, X.C.; Lee, D.J.; Chang, J.S. Potential biomedical applications of marine algae. Bioresour. Technol. 2017, 244, 1407–1415. [CrossRef] [PubMed] as, P.M. The how, why and clinical importance of stratum corneum acidiﬁcation. Exp. Dermatol. 2017, 26, 9–1003. 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https://openalex.org/W1974705183	https://bmcpsychiatry.biomedcentral.com/track/pdf/10.1186/s12888-015-0396-y	English	null	BMC Psychiatry reviewer acknowledgement 2014	BMC psychiatry	2,015	cc-by	6,605	BMC Psychiatry reviewer acknowledgement 2014 Catherine M Rice* and Diana M Marshall REVIEWER ACKNOWLEDGEMENT Open Access © 2015 Rice and Marshall; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rice and Marshall BMC Psychiatry (2015) 15:54 DOI 10.1186/s12888-015-0396-y Rice and Marshall BMC Psychiatry (2015) 15:54 DOI 10.1186/s12888-015-0396-y Contributing reviewers The editors of BMC Psychiatry would like to thank all our reviewers who have contributed to the journal in Volume 14 (2014). 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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Contributing reviewers Page 2 of 15 Page 2 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Istvan Bitter Hungary Simon Blackwell UK Sergio Blay Brazil Olof Blix Sweden D Blumberger Canada Sune Bo Denmark Jed Boardman UK Petr Bob Czech Republic Julio Bobes Spain Iwona Bojar Poland Ilaria Bonoldi UK Rhonda Booth UK Rami Bou Khalil France Aurore Boulard Belgium Jennifer Boyd USA Laurent Boyer France Lindy-Lou Boyette Netherlands Abby Braden USA Joanne Brady USA Nicholas Breitborde USA Benjamin Brent USA David Brent USA Stefano Baroni Italy Giovanni Battista Bartolucci Italy Judith Bass USA Deborah Bassett USA Daniel Bateman USA Stephanie Bauer Germany Thomas Beblo Germany Margherita Bechi Italy Arthur Becker-Weidman USA Hassan Bella Saudi Arabia Martino Belvederi Murri Italy Raoul Belzeaux France Susan Mary Benbow UK Maneeton Benchalak Thailand Kristina Bennert UK Dror Ben-Zeev USA Yemane Berhane Ethiopia Kalpana Bhaskaran Singapore Kamaldeep Bhui UK Andargachew Kassa Biratu Ethiopia Victoria Bird UK Carl Birmingham Canada ucci n i atu Istvan Bitter Hungary Simon Blackwell UK Sergio Blay Brazil Olof Blix Sweden D Blumberger Canada Sune Bo Denmark Jed Boardman UK Petr Bob Czech Republic Julio Bobes Spain Iwona Bojar Poland Ilaria Bonoldi UK Rhonda Booth UK Rami Bou Khalil France Aurore Boulard Belgium Jennifer Boyd USA Laurent Boyer France Lindy-Lou Boyette Netherlands Abby Braden USA Joanne Brady USA Nicholas Breitbord USA Benjamin Brent USA David Brent USA Stefano Baroni Italy Giovanni Battista Bartolucci Italy Judith Bass USA Deborah Bassett USA Daniel Bateman USA Stephanie Bauer Germany Thomas Beblo Germany Margherita Bechi Italy Arthur Becker-Weidman USA Hassan Bella Saudi Arabia Martino Belvederi Murri Italy Raoul Belzeaux France Susan Mary Benbow UK Maneeton Benchalak Thailand Kristina Bennert UK Dror Ben-Zeev USA Yemane Berhane Ethiopia Kalpana Bhaskaran Singapore Kamaldeep Bhui UK Andargachew Kassa Biratu Ethiopia Victoria Bird UK Carl Birmingham Canada Stefano Baroni Italy Giovanni Battista Bartolucci Italy Judith Bass USA Deborah Bassett USA Daniel Bateman USA Stephanie Bauer Germany Thomas Beblo Germany Margherita Bechi Italy Arthur Becker-Weidman USA Hassan Bella Saudi Arabia Martino Belvederi Murri Italy Raoul Belzeaux France Susan Mary Benbow UK Maneeton Benchalak Thailand Kristina Bennert UK Dror Ben-Zeev USA Yemane Berhane Ethiopia Kalpana Bhaskaran Singapore Kamaldeep Bhui UK Andargachew Kassa Biratu Ethiopia Victoria Bird UK Carl Birmingham Canada Istvan Bitter Hungary Simon Blackwell UK Sergio Blay Brazil Olof Blix Sweden D Blumberger Canada Sune Bo Denmark Jed Boardman UK Petr Bob Czech Republic Julio Bobes Spain Iwona Bojar Poland Ilaria Bonoldi UK Rhonda Booth UK Rami Bou Khalil France Aurore Boulard Belgium Jennifer Boyd USA Laurent Boyer France Lindy-Lou Boyette Netherlands Abby Braden USA Joanne Brady USA Nicholas Breitborde USA Benjamin Brent USA David Brent USA Joshua Breslau USA Jeffrey Bridge USA William Brinkman USA Teresita Briones USA Markus Britschgi Switzerland June Brown UK Margaret Brown Australia Martin Bruene Germany Scott Brunero Australia Andre Brunoni Brazil Fabrizio Bruschi Italy Kelly Buck USA Kyle Burghardt USA Julius Burkauskas Lithuania Lisa Burke Australia Christopher Bushe UK Andrew Bushmakin USA Casimiro Cabrera Abre Canada Raffaele Cacciaglia Spain John Cacioppo USA Kristin Cadenhead USA Joseph Calabrese USA Page 3 of 15 Page 3 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Samuele Cortese USA William Coryell USA Philippe Courtet France Kay Lorraine Cox Australia Paul Crits-Christoph USA Anselm Crombach Germany Gary Cuddeback USA Alexis Cullen UK Kathryn Cullen USA Larry Culpepper USA Caroline Dalton UK Gerhard Dammann Switzerland Louise Danielsson Sweden Alison Darcy USA Carl D'Arcy Canada Renaud David France Larry Davidson USA Darlene Davis USA Derek De Beurs Netherlands Nicola De Carlo Italy Giovanni De Girolamo Italy Peter De Jonge Netherlands Chetty da oat n m a ou mas t Cloninger am s pola coran ing Samuele Cortese USA William Coryell USA Philippe Courtet France Kay Lorraine Cox Australia Paul Crits-Christoph USA Anselm Crombach Germany Gary Cuddeback USA Alexis Cullen UK Kathryn Cullen USA Larry Culpepper USA Caroline Dalton UK Gerhard Dammann Switzerland Louise Danielsson Sweden Alison Darcy USA Carl D'Arcy Canada Renaud David France Larry Davidson USA Darlene Davis USA Derek De Beurs Netherlands Nicola De Carlo Italy Giovanni De Girolamo Italy Peter De Jonge Netherlands Alison Calear Australia Michael Caligiuri USA John Cape UK Alejandra Caqueo-Urizar Chile R. Contributing reviewers Nicholas Carleton Canada Hannah Carliner USA Vaughan Carr Australia Giovanni Castellini Italy Carl Castro USA Ferrán Catalá-López Spain Pascal Cathebras France Hilal Ceit Netherlands Eduardo Chachamovich Canada Subhajit Chakravorty USA Lai Fong Chan Malaysia Raymond Chan China Kristine Chapleau USA Robert Chaplin UK Gregory Chasson USA Imran Chaudhry UK Huafu Chen China Chih-Ying Chen USA Manoranjenni Chetty UK Dixon Chibanda Zimbabwe Howard Chilcoat USA Weng Yee Chin Hong Kong Dan Chisholm Switzerland Pratap Chokka Canada Yuan-Hwa Chou Taiwan David Christmas UK Quetzal Class USA Claude Robert Cloninger USA Laurie Clune Canada Vanessa Cobham Australia Alex Cohen UK Moran Cohn Netherlands Hannie Comijs Netherlands David Conn Canada Amy Conrad USA Andrew Cook USA Maurizio Coppola Italy Rhiannon Corcoran UK Angel Correa Spain Deborah Corring Canada Page 4 of 15 Page 4 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Flora De La Barra Chile Jose De Leon USA Jesus 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Johannes Fellinger Austria Liang Feng Singapore Christopher Ferguson USA Jilles Fermont UK Ana Fernandez Spain Maria Isabel Fernández San Martín Spain Fernando Fernandez-Aranda Spain Mark Ferro Canada Mathew Fetzner Canada Jess Fiedorowicz USA Andrea Fiorillo Italy Sarah Fischer USA Ellen Fitzsimmons-Craft USA Amos Fleischmann Israel Marie-Josee Fleury Canada Leon Flicker Australia Fei Du USA Ke-Lin Du China Arnaud Duhoux Canada Sophie Duranceau Canada Nicola Dusi Italy John Dziak USA Bjorn Ebdrup Denmark Lara Ebenfeld Germany David Daniel Ebert Germany Chad Ebesutani Korea, South Takashi Ebisawa Japan Thomas Ehring Germany Solvig Ekblad Sweden Thomas Elbert Germany Jennifer Elliott USA Graham Emslie USA Jerome Endrass Switzerland Harald Engler Germany Linda Ercoli USA Pascale Esch Luxembourg Ozlem Eylem Netherlands Harris Eyre Australia De La Barra De Leon De Pedro-Cuesta ni De Silva nka olas Deakin erine Deeprose Delapp ppe Delespaul rlands Deng alia h Deng us Denier erland Derntl any Dervaux e d Diamond t Diefenbacher any i Dima n Dobscha Donker alia Doucette da n Dougherty Draskovic rlands Drozdek rlands Fei Du USA Ke-Lin Du China Arnaud Duhoux Canada Sophie Duranceau Canada Nicola Dusi Italy John Dziak USA Bjorn Ebdrup Denmark Lara Ebenfeld Germany David Daniel Ebert Germany Chad Ebesutani Korea, South Takashi Ebisawa Japan Thomas Ehring Germany Solvig Ekblad Sweden Thomas Elbert Germany Jennifer Elliott USA Graham Emslie USA Jerome Endrass Switzerland Harald Engler Germany Linda Ercoli USA Pascale Esch Luxembourg Ozlem Eylem Netherlands Harris Eyre Australia Thomas Ehring Germany Solvig Ekblad Sweden Thomas Elbert Germany Jennifer Elliott USA Jerome Endrass Switzerland Page 5 of 15 Page 5 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Tim Fong USA Eduardo Fonseca Pedrer Spain David Forbes Australia Thomas Forkmann Germany Tomislav Franic Croatia Kenneth Freedland USA Richard Frye USA Sanae Fukuda Japan Sadaaki Fukui USA Daniel Fung Singapore Adel 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Silvia Gallagher Ireland Juan Gallego USA Orsola Gambini Italy Matthew Gambino USA David Garcia Switzerland Ricardo García-Mayor Spain Genevieve Gariepy Canada Andrew Garratt Norway Faye Gary USA Fiona Gaughran UK Caterina Gawrilow Germany Measho Gebregziabher Ethiopia James Gedra USA Bizu Gelaye USA Dejan Georgiev UK Adam Gerace Australia Adam Geraghty UK Lilian Ghandour Lebanon Lucio Ghio Italy Domenico Giacco UK Katrin Giel Germany Itzhak Gilat Israel Stephen Gilman USA Damiano Girardi Italy Ragy Girgis USA Lydia Gisle Belgium Melissa Gladstone UK Heide Glaesmer Germany Joseph Glass USA Robert Göder Germany Betty Goguikian Ratcliff Switzerland Carlos Gois Portugal Lutz Goldbeck Germany David Goldberg UK Hadass Goldblatt Israel Andrea Goldschmidt USA Jesus Gomar USA Marcus Gomes Brazil Rafael Gonzalez UK Robert Gonzalez USA Alexandre Gonzalez-Rodriguez Spain Peter Goossens Netherlands Jessica Goren USA Carla Maria Gramaglia Italy Iria Grande Spain Michael Grandner USA Kelly Green USA 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Katrin Giel Germany Itzhak Gilat Israel Stephen Gilman USA Damiano Girardi Italy Ragy Girgis USA Lydia Gisle Belgium Melissa Gladstone UK Heide Glaesmer Germany Joseph Glass USA Robert Göder Germany Betty Goguikian Ratcliff Switzerland Fiona Gaughran UK Caterina Gawrilow Germany Measho Gebregziabher Ethiopia James Gedra USA Bizu Gelaye USA Dejan Georgiev UK Adam Gerace Australia Adam Geraghty UK Lilian Ghandour Lebanon Lucio Ghio Italy Domenico Giacco UK Katrin Giel Germany Itzhak Gilat Israel Stephen Gilman USA Damiano Girardi Italy Ragy Girgis USA Lydia Gisle Belgium Melissa Gladstone UK Heide Glaesmer Germany Joseph Glass USA Robert Göder Germany Betty Goguikian Ratclif Switzerland Page 6 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Sarah Honaker USA Lisa Horowitz USA Samantha Horswill Canada Chiung-Yu Huang Taiwan Teresa Hudson USA Libby Hughes Australia Annemiek Huisman Netherlands Benjamin Hummelen Norway Rene Hurlemann Germany Tuula Hurtig Finland Gerard Hutchinson Trinidad and Tobago Mari Hysing Norway Felice Iasevoli Italy Benjamin Iffland Germany Yoshito Igarashi Japan Masumi Iida USA Masatoshi Inagaki Japan Pasquale Innominato France Makoto Ishitobi Japan Mariko Iwayama Japan Nadja Jacob Switzerland Sabrina Anne Jacob Malaysia Kenji Hashimoto Japan Ilanit Hasson-Ohayon Israel Tobias Hecker Germany Craig Anne Heflinger USA Markus Heinimaa Finland Martin Hellmich Germany Claire Henderson UK Urs Hepp Switzerland Andres Herane Vives UK Philipp Herzberg Germany Klaus Hesse Germany Morten Hesse Denmark Terrence Hill USA Hubertus Himmerich Germany Andreas Hinz Germany Erin Hoare Australia Robert Hodapp USA Nicolas Hoertel France Michael Höfler Germany Tobias Hofmann Germany Marjan Holloway USA Mikael Holma Finland Martin Grunwald Germany Heinz Grunze UK Maria Rosaria Gualano Italy Cristiana Guetti Italy Mai-Britt Guldin Denmark Emilio Gutierrez Spain Yari Gvion Israel Noami Hadas- Lidor Israel Mark Haddad UK Hyeouk Chris Hahm USA Helinä Hakko Finland Mark Hamer UK David Hanauer USA Tonelle Handley Australia Lars Hansson Sweden Sheila Hardy UK Michelle Harley Ireland Ashley Harrison USA Christopher Harte USA Sigan Hartley USA Daniel Hartung USA Carol Harvey Australia David Hanauer USA Page 7 of 15 Page 7 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Alice Keski-Valkama Finland Ronald Kessler USA Hari-Mandir Khalsa USA Judi Kidger UK Jae-Min Kim Korea, South Yong Sik Kim Korea, South David Kingdon UK John David Kinzie USA Julia Kirkham Canada Anne Kleinsasser USA Elizabeth Klerman USA Sören Kliem Germany Stefan Kloiber Germany Christine Knaevelsrud Germany Jeroen Knipscheer Netherlands Sarah Knowles UK Kathrin Koch Germany Manami Kodaka Japan Stephan Köhler Germany Michael Kohn Australia Tsuyoshi Kondo Japan Barna Konkoly Thege Canada Frank Jacobi Germany Susanne Jaeger Germany Anthony James UK Josee L Jarry Canada Rachel Jenkins UK Tawanchai Jirapramukpitak Thailand Stefan Johansson Norway Bjorn Johnson Sweden Mandy Johnstone UK Sheila Jones Denmark Ruud Jongedijk Netherlands Andreas Joos Germany Rikke Jørgensen Denmark Georg Juckel Germany Minyoung Jung Korea, South Mario Juruena Brazil Colette Kabrita Lebanon Michael Kaess Germany Kirsti Kähärä Finland Ondrej Kalina Slovakia Bengt Källén Sweden Eva Kaltenthaler UK Inge Kamp-Becker Germany Olli Kampman Finland Richard Antony Alexander Kan UK Yukiko Kano Japan Joshua Kantrowitz USA Manav Kapoor USA Nestor Damian Kapusta Austria Nilamadhab Kar UK Susanne Karch Bosnia and Herzegovina Andrea Kass USA Takahiro Kato Japan Marcia Kauer-Sant'Anna Brazil Brooke Kauffman USA Anne Kavanagh Australia Yoshitaka Kawashima Japan Robert Keeley USA Matthew Keller USA Friederike Kendel Germany Harry Kennedy Ireland Martin Kennedy New Zealand Michelle Kermode Australia Sarah Kertz USA Inge Kamp-Becker Germany Olli Kampman Finland Richard Antony Alexander Kanaan UK Yukiko Kano Japan Joshua Kantrowitz USA Manav Kapoor USA Nestor Damian Kapusta Austria Nilamadhab Kar UK Susanne Karch Bosnia and Herzegovina Andrea Kass USA Takahiro Kato Japan Marcia Kauer-Sant'Anna Brazil Brooke Kauffman USA Anne Kavanagh Australia Yoshitaka Kawashima Japan Robert Keeley USA Matthew Keller USA Friederike Kendel Germany Harry Kennedy Ireland Martin Kennedy New Zealand Michelle Kermode Australia Sarah Kertz USA Alice Keski-Valkama Finland Ronald Kessler USA Hari-Mandir Khalsa USA Judi Kidger UK Jae-Min Kim Korea, South Yong Sik Kim Korea, South David Kingdon UK John David Kinzie USA Julia Kirkham Canada Anne Kleinsasser USA Elizabeth Klerman USA Sören Kliem Germany Stefan Kloiber Germany Christine Knaevelsrud Germany Jeroen Knipscheer Netherlands Sarah Knowles UK Kathrin Koch Germany Manami Kodaka Japan Stephan Köhler Germany Michael Kohn Australia Tsuyoshi Kondo Japan Barna Konkoly Thege Canada Inge Kamp-Becker Germany Olli Kampman Finland Richard Antony Alexander Kanaan UK Yukiko Kano Japan Joshua Kantrowitz USA Manav Kapoor USA Nestor Damian Kapusta Austria Nilamadhab Kar UK Susanne Karch Bosnia and Herzegovina Andrea Kass USA Takahiro Kato Japan Marcia Kauer-Sant'Anna Brazil Brooke Kauffman USA Anne Kavanagh Australia Yoshitaka Kawashima Japan Robert Keeley USA Matthew Keller USA Friederike Kendel Germany Harry Kennedy Ireland Martin Kennedy New Zealand Michelle Kermode Australia Sarah Kertz USA Alice K Finland Ronald USA Hari-M USA Judi Kid UK Jae-Min Korea, S Yong Si Korea, S David K UK John D USA Julia Ki Canada Anne K USA Elizabe USA Sören K German Stefan K German Christin German Jeroen Netherl Sarah K UK Kathrin German Manam Japan Stephan German Michae Australi Tsuyosh Japan Barna K Canada Inge Kamp-Becker Germany Olli Kampman Finland Richard Antony Alexander Kanaan UK Yukiko Kano Japan Joshua Kantrowitz USA Manav Kapoor USA Frank Jacobi Germany Susanne Jaeger Germany Anthony James UK Josee L Jarry Canada Rachel Jenkins UK Tawanchai Jirapramukpitak Thailand Stefan Johansson Norway Bjorn Johnson Sweden Mandy Johnstone UK Sheila Jones Denmark Ruud Jongedijk Netherlands Andreas Joos Germany Rikke Jørgensen Denmark Georg Juckel Germany Minyoung Jung Korea, South Mario Juruena Brazil Colette Kabrita Lebanon Michael Kaess Germany Kirsti Kähärä Finland Ondrej Kalina Slovakia Bengt Källén Sweden Eva Kaltenthaler UK Inge Kamp-Becker Germany Olli Kampman Finland Richard Antony Alexander Kana UK Yukiko Kano Japan Joshua Kantrowitz USA Manav Kapoor USA Nestor Damian Kapusta Austria Nilamadhab Kar UK Susanne Karch Bosnia and Herzegovina Andrea Kass USA Takahiro Kato Japan Marcia Kauer-Sant'Anna Brazil Brooke Kauffman USA Anne Kavanagh Australia Yoshitaka Kawashima Japan Robert Keeley USA Matthew Keller USA Friederike Kendel Germany Harry Kennedy Ireland Martin Kennedy New Zealand Michelle Kermode Australia Sarah Kertz USA Page 8 of 15 Page 8 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Dimitrios Kontis Greece Hans Koppeschaar Netherlands Jyrki Korkeila Finland Hirotaka Kosaka Japan Ernst Koster Belgium Diana Koszycki Canada Leda Kovatsi Greece Kate Krajci USA Ueli Kramer Switzerland Levente Kriston Germany Kurt Kroenke USA Jesper Krogh Denmark Ziad Kronfol Qatar Hans Kroon Netherlands Tillmann Kruger Germany Robert Kumsta Afghanistan Matthew Kurtz USA Philipp Kuwert Germany Jeoung A Kwon Korea, South Cornbelis Laban Netherlands Jean Lachaine Canada Daniel Lai Canada Dimitrios Kontis Greece Hans Koppeschaar Netherlands Jyrki Korkeila Finland Hirotaka Kosaka Japan Ernst Koster Belgium Diana Koszycki Canada Leda Kovatsi Greece Kate Krajci USA Ueli Kramer Switzerland Levente Kriston Germany Kurt Kroenke USA Jesper Krogh Denmark Ziad Kronfol Qatar Hans Kroon Netherlands Tillmann Kruger Germany Robert Kumsta Afghanistan Matthew Kurtz USA Philipp Kuwert Germany Jeoung A Kwon Korea, South Cornbelis Laban Netherlands Jean Lachaine Canada Daniel Lai Canada Brian Lakey USA Marie-Eve Lamontagne Canada Carol Landis USA Nils Inge Landrfø Norway Pierre Lau Belgium Chi-Kin Law Hong Kong Robyne Le Brocque Australia Monique Lebourgeois USA Daniel Lebouthillier Canada David Ledgerwood USA William Lee UK Robert Leeman USA Seblewengel Lemma Ethiopia Bethany Leonhardt USA Karolina Leopold Germany Stefan Leucht Germany Catharine J. Contributing reviewers Lewis Germany Jun Li China Kaigang Li USA Pesach Lichtenberg Israel Belinda Liddell Australia Shih-Ku Lin Taiwan Brian Lakey USA Marie-Eve Lamontagne Canada Carol Landis USA Nils Inge Landrfø Norway Pierre Lau Belgium Chi-Kin Law Hong Kong Robyne Le Brocque Australia Monique Lebourgeois USA Daniel Lebouthillier Canada David Ledgerwood USA William Lee UK Robert Leeman USA Seblewengel Lemma Ethiopia Bethany Leonhardt USA Karolina Leopold Germany Stefan Leucht Germany Catharine J. Lewis Germany Jun Li China Kaigang Li USA Pesach Lichtenberg Israel Belinda Liddell Australia Shih-Ku Lin Taiwan Jean-Pierre Lindenmayer USA Jamey Lister USA Xianhua Liu China Zhongchun Liu China James Livingston Canada Brynmor Lloyd-Evans UK Alexandre Loch Brazil Steven Lockley USA Paul Lombroso USA Barbara Lopes Cardozo USA Jorge Lopez-Castroman Spain Lee-Fay Low Australia Evan Lutkenhoff USA Sean Lynch UK John Lyne Ireland Corey Mackenzie Canada Helen Mactier UK Franziska Maier Germany Thomas Maier Switzerland Pallab Majumder UK Marelign Malaju Ethiopia Sumaya Mall USA Dimitrios Kontis Greece Hans Koppeschaar Netherlands Jyrki Korkeila Finland Hirotaka Kosaka Japan Ernst Koster Belgium Diana Koszycki Canada Leda Kovatsi Greece Kate Krajci USA Ueli Kramer Switzerland Levente Kriston Germany Kurt Kroenke USA Jesper Krogh Denmark Ziad Kronfol Qatar Hans Kroon Netherlands Tillmann Kruger Germany Robert Kumsta Afghanistan Matthew Kurtz USA Philipp Kuwert Germany Jeoung A Kwon Korea, South Cornbelis Laban Netherlands Jean Lachaine Canada Daniel Lai Canada Brian Lakey USA Marie-Eve Lamontagne Canada Carol Landis USA Nils Inge Landrfø Norway Pierre Lau Belgium Chi-Kin Law Hong Kong Robyne Le Brocque Australia Monique Lebourgeois USA Daniel Lebouthillier Canada David Ledgerwood USA William Lee UK Robert Leeman USA Seblewengel Lemma Ethiopia Bethany Leonhardt USA Karolina Leopold Germany Stefan Leucht Germany Catharine J. Contributing reviewers Lewis Germany Jun Li China Kaigang Li USA Pesach Lichtenberg Israel Belinda Liddell Australia Shih-Ku Lin Taiwan Tillmann Kruger Germany Robert Kumsta Afghanistan Matthew Kurtz USA Jeoung A Kwon Korea, South Page 9 of 15 Page 9 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Jerome Maller Australia Stefania Mannarini Italy Donatella Marazziti Italy Angelo Giovanni Icro Maremma Italy Dominik Marin Germany Michel Maron France Colin Martin UK Leticia Martínez González Spain Jose Martinez-Raga Spain Francesca Martino Italy Matteo Martino Italy Enrica Marzola Italy Susan Marzolini Canada Holly Mash USA Julie Maslowsky USA Davide Massidda Italy Aleksandra Matanov UK Faith Matcham UK Catherine Mathews South Africa Yuki Matsumoto Japan Fermin Mayoral Spain Patrick Mccabe USA Jerome Maller Australia Stefania Mannarini Italy Donatella Marazziti Italy Angelo Giovanni Icro Maremmani Italy Dominik Marin Germany Michel Maron France Colin Martin UK Leticia Martínez González Spain Jose Martinez-Raga Spain Francesca Martino Italy Matteo Martino Italy Enrica Marzola Italy Susan Marzolini Canada Holly Mash USA Julie Maslowsky USA Davide Massidda Italy Aleksandra Matanov UK Faith Matcham UK Catherine Mathews South Africa Yuki Matsumoto Japan Fermin Mayoral Spain Patrick Mccabe USA Rosemarie Mccabe UK Terence Mccann Australia Kevin Mccarthy USA Troy Mcewan Australia Gary Mclean UK Hamish Mcleod UK Caitlin Mcomish Australia Melissa Mcpheeters USA Nick Meader UK Denis Meadows Australia Ybe Meesters Netherlands Alison Menatti USA Itiana Castro Menezes Brazil Andreas Menke Germany Fiona Mensah UK Lisa Merlo USA Thomas Messer Germany Ricarda Mewes Germany Patrick Michaels USA Makoto Michikawa Japan Daniela Mier Germany Jouko Miettunen Finland Rafael T Mikolajczyk Germany Brian Miller USA Alessandra Minelli Italy Silvia Minozzi Italy Regina Miranda USA Mansha Mirza USA Mitsuhiro Miyashita Japan Toshiki Mizuno Japan Shakeh Moamrtin Australia Sara Modig Sweden Roy Moncayo Austria Akira Monji Japan Christiane Montag Germany Ali Montazeri Iran Galia Moran Israel Carlos Renato Moreira Maia Brazil Kevin Morgan UK Elaine Morrato USA Richard Morriss UK Anett Mueller-Alcazar Germany Gerry Mugford Canada Amin Muhammad Gadit Canada Jerome Maller Australia Stefania Mannarini Italy Donatella Marazziti Italy Angelo Giovanni Icro Maremmani Italy Dominik Marin Germany Michel Maron France Colin Martin UK Leticia Martínez González Spain Jose Martinez-Raga Spain Francesca Martino Italy Matteo Martino Italy Enrica Marzola Italy Susan Marzolini Canada Holly Mash USA Julie Maslowsky USA Davide Massidda Italy Aleksandra Matanov UK Faith Matcham UK Catherine Mathews South Africa Yuki Matsumoto Japan Fermin Mayoral Spain Patrick Mccabe USA Jerome Maller Australia Stefania Mannarini Italy Donatella Marazziti Italy Angelo Giovanni Icro Maremmani Italy Dominik Marin Germany Michel Maron France Colin Martin UK Jerome Maller Australia Stefania Mannarini Italy Donatella Marazziti Italy Angelo Giovanni Icro Maremmani Italy Dominik Marin Germany Michel Maron France Colin Martin UK Leticia Martínez González Spain Jose Martinez-Raga Spain Francesca Martino Italy Matteo Martino Italy Enrica Marzola Italy Susan Marzolini Canada Holly Mash USA Julie Maslowsky USA Davide Massidda Italy Aleksandra Matanov UK Faith Matcham UK Catherine Mathews South Africa Yuki Matsumoto Japan Fermin Mayoral Spain Patrick Mccabe USA Rosemarie Mccabe UK Terence Mccann Australia Kevin Mccarthy USA Troy Mcewan Australia Gary Mclean UK Hamish Mcleod UK Caitlin Mcomish Australia Melissa Mcpheeters USA Nick Meader UK Denis Meadows Australia Ybe Meesters Netherlands Alison Menatti USA Itiana Castro Menezes Brazil Andreas Menke Germany Fiona Mensah UK Lisa Merlo USA Thomas Messer Germany Ricarda Mewes Germany Patrick Michaels USA Makoto Michikawa Japan Daniela Mier Germany Jouko Miettunen Finland Rosemarie Mccabe UK Terence Mccann Australia Kevin Mccarthy USA Troy Mcewan Australia Gary Mclean UK Hamish Mcleod UK Caitlin Mcomish Australia Melissa Mcpheeters USA Nick Meader UK Denis Meadows Australia Ybe Meesters Netherlands Alison Menatti USA Itiana Castro Menezes Brazil Andreas Menke Germany Fiona Mensah UK Lisa Merlo USA Thomas Messer Germany Ricarda Mewes Germany Patrick Michaels USA Makoto Michikawa Japan Daniela Mier Germany Jouko Miettunen Finland Aleksandra Matanov UK Catherine Mathews South Africa Yuki Matsumoto Japan Page 10 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Killian O'Rourke Ireland Maciej Owecki Poland Shane Owens USA Yuji Ozeki Japan Seline Ozer UK Chi-Un Pae Korea, South Andrew Page Australia Daniel Pagnin Brazil Utpal Pajvani USA Ståle Pallesen Norway Maria Panagioti UK Ananda Pandurangi USA Igor Pantic Serbia A-La Park UK Sohee Park USA Barbara Parry USA Timo Partonen Finland Augusto Pasini Italy Sue Patterson Australia Lenin Pavon Mexico Nalin Payakachat USA Martha Payne USA d aus mura nt meier n nor Dea ard Odenwald ghue a mura va ucarella ny dvari mere y a Killian O'Rourke Ireland Maciej Owecki Poland Shane Owens USA Yuji Ozeki Japan Seline Ozer UK Chi-Un Pae Korea, South Andrew Page Australia Daniel Pagnin Brazil Utpal Pajvani USA Ståle Pallesen Norway Maria Panagioti UK Ananda Pandurangi USA Igor Pantic Serbia A-La Park UK Sohee Park USA Barbara Parry USA Timo Partonen Finland Augusto Pasini Italy Sue Patterson Australia Lenin Pavon Mexico Nalin Payakachat USA Martha Payne USA Matthias J Müller Germany Astrid Müller Germany Gioia Mura Italy Stuart Murray Australia Jochen Mutschler Switzerland Maria Muzik USA Inez Myin-Germeys Netherlands Makalani Myrtveit Norway Michael Nadorff USA Farooq Naeem Pakistan Akiko Nakagawa Japan Takashi Nakamae Japan Hideyuki Nakane Japan Ora Nakash Israel Kristin Naragon-Gainey USA Davide Nardo Italy Carryl Navalta USA Manuela Neuman Canada Kelly Newell Australia James Newham UK Christian Hans Nickel Switzerland Angela Nickerson USA Inga Niedtfeld Germany Dana Jh Niehaus South Africa Katsuji Nishimura Japan Maria Nobile Italy Egil Nygaard Norway Shwe Zin Nyunt Singapore Michael Obermeier Germany Louise O'Brien USA Daniel O'Connor Australia Bridianne O'Dea Australia Michael Gerhard Odenwald Germany Brian O’Donoghue Australia Jeneva Ohan Australia Takashi Okada Japan Yasuyuki Okumura Japan Francesco Oliva Italy Javier Oltra-Cucarella Spain Joyce O'Mahony Canada Sepideh Omidvari Iran Juliana Onwumere UK Claire O'Reilly Australia Vasiliki Orgeta UK d Killian O'Rourke Ireland Maciej Owecki Poland Shane Owens USA Yuji Ozeki Japan Seline Ozer UK Chi-Un Pae Korea, South Andrew Page Australia Daniel Pagnin Brazil Utpal Pajvani USA Ståle Pallesen Norway Maria Panagioti UK Ananda Pandurangi USA Igor Pantic Serbia A-La Park UK Sohee Park USA Barbara Parry USA Timo Partonen Finland Augusto Pasini Italy Sue Patterson Australia Lenin Pavon Mexico Nalin Payakachat USA Martha Payne USA Inga Niedtfeld Germany Dana Jh Niehaus South Africa Katsuji Nishimura Japan Maria Nobile Italy Egil Nygaard Norway Shwe Zin Nyunt Singapore Michael Obermeier Germany Louise O'Brien USA Daniel O'Connor Australia Bridianne O'Dea Australia Michael Gerhard Odenwald Germany Brian O’Donoghue Australia Jeneva Ohan Australia Takashi Okada Japan Yasuyuki Okumura Japan Francesco Oliva Italy Javier Oltra-Cucarella Spain Joyce O'Mahony Canada Sepideh Omidvari Iran Juliana Onwumere UK Claire O'Reilly Australia Vasiliki Orgeta UK Page 11 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Carsten Bøcker Pedersen Denmark Kirsi Peltonen France Cinzia Perlini Italy Jane Persons USA Inge Petersen South Africa Anett Pfeiffer Uganda Andrea Pfennig Germany Ruth Pidsley Australia Anilkumar Pillai USA Martin Pinquart Germany Melissa Pinto USA Yehuda Pollak Israel Nunzio Pomara USA Maurizio Pompili Italy Alexander Ponizovsky Israel Piero Porcelli Italy Maj-Britt Posserud Norway Robert Powers USA Sarah Pratt USA Peter Pregelj Slovenia Antonio Preti Italy Stefan Priebe UK Lucia Reisch Denmark Javier Rejas Spain Solomon Renati India Leslie Rescorla USA Petra Retz-Junginger Germany Valdo Ricca Italy Derek Richards Ireland Debra Rickwood Australia Philipp S. Contributing reviewers Ritter Germany Patricio Riva-Posse USA Brandy Roane USA Laurence Inès Martine Robel France Pasquale Roberge Canada David Roberts USA Seren Roberts UK Jo Robinson Australia Dan Robotham UK James Roerig USA Paul Rohde USA Suely Roizenblatt Brazil Maria Graciela Rojas Castillo Chile Martha Romero Mexico Petroula Proitsi UK David Pulsford UK Stephen Puntis UK Marianna Purgato Italy Bernd Puschner Germany Eli Puterman USA Terry Quinn UK Daniel Racey UK Mikael Rahmqvist Sweden Mahesh Rajasuriya Sri Lanka Anto P. Rajkumar Denmark Padmavati Ramachandran India Hugh Ramsay Ireland Beverley Raphael Australia Carla Rash USA Silvia Ravera Netherlands Ursula Read UK Nicola Reavley Australia Elliott Rees UK Susan Rees Australia Gayle Reiber USA Kathryn Reid USA Petroula Proitsi UK David Pulsford UK Stephen Puntis UK Marianna Purgato Italy Bernd Puschner Germany Eli Puterman USA Terry Quinn UK Daniel Racey UK Mikael Rahmqvist Sweden Mahesh Rajasuriya Sri Lanka Anto P. Rajkumar Denmark Padmavati Ramachandran India Hugh Ramsay Ireland Beverley Raphael Australia Carla Rash USA Silvia Ravera Netherlands Ursula Read UK Nicola Reavley Australia Elliott Rees UK Susan Rees Australia Gayle Reiber USA Kathryn Reid USA Alexander Ponizovsky Israel Page 12 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Roberto Rona UK Kathlyn Ronaldson Australia Rita Roncone Italy Jennifer Rose USA Roderick Rose USA Irwin Rosenfarb USA Daniel Rossignol USA Francesco Rotella Italy Cecile Rousseau Canada Guy Rousseau Canada Michael Roy USA Abraham Rudnick Canada Montserrat Rue Spain Reiner Rugulies Denmark M Angeles Ruiperez Spain Miguel Ruiz-Veguilla Spain Dan Rujescu Germany Zlatka Russinova USA Jennifer Rusted UK Torleif Ruud Norway Joanne Ryan Australia Janusz Rybakowski Poland Marc Schmid Switzerland Regien Schoemaker Netherlands Georg Schomerus Germany Daniel Schöttle Germany Beate M. Schulze Switzerland Jan Scott UK Bridie Scott-Parker Bahamas Dallas Seitz Canada Carmen Senra Spain Michael Sernyak USA Argentina Servin USA Emanuel Severus Germany Asim Shah USA Katherine Sharkey USA Ann Sharpley UK Taiwo Lateef Sheikh Nigeria Yukihiko Shirayama Japan Dvora Shmulewitz USA Michael Shuman USA Tianmei Si China Etienne Sibille USA Ingrid Sibitz Austria Roberto Rona UK Kathlyn Ronaldson Australia Rita Roncone Italy Jennifer Rose USA Roderick Rose USA Irwin Rosenfarb USA Daniel Rossignol USA Francesco Rotella Andrew Ryder Canada Ad Sad Bahrain Hans Inge Sævareid Norway Yoichi Sakakihara Japan Joao Salgado Brazil Raimo K. R. Contributing reviewers Salokangas Finland Harshal Salve India Virginio Salvi Italy Zainab Samaan Canada Stephen Sammut USA Marsal Sanches USA Marco Sarchiapone Italy Jerome Sarris Australia Tsuyoshi Sasaki Japan Anna Sasdelli Italy Cathrin Sauer Germany Broderick Sawyer USA Andrea Sboner USA Aart Schene Netherlands Jeffrey Scherrer USA Robert Schlack Germany Jann E Schlimme Germany Andrew Ryder Canada Ad Sad Bahrain Hans Inge Sævareid Norway Yoichi Sakakihara Japan Joao Salgado Brazil Raimo K. R. Contributing reviewers Salokangas Finland Harshal Salve India Virginio Salvi Italy Zainab Samaan Canada Stephen Sammut USA Marsal Sanches USA Marco Sarchiapone Italy Jerome Sarris Australia Tsuyoshi Sasaki Japan Anna Sasdelli Italy Cathrin Sauer Germany Broderick Sawyer USA Andrea Sboner USA Aart Schene Netherlands Jeffrey Scherrer USA Robert Schlack Germany Jann E Schlimme Germany Cecile Rousseau Canada Michael Sernyak USA Guy Rousseau Canada Emanuel Severus Germany Montserrat Rue Spain Asim Shah USA Katherine Sharkey USA M Angeles Ruiperez Spain Taiwo Lateef Sheikh Nigeria Miguel Ruiz-Veguilla Spain Yukihiko Shirayama Japan Zlatka Russinova USA Dvora Shmulewitz USA Page 13 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Cecilia Sighinolfi Italy Roberta Siliquini Italy Derrick Silove Australia Kang Sim Singapore Magenta Simmons Australia Claudia Simons Netherlands Jay Singh Norway Mark Sinyor Canada Bonnie Wm Siu Hong Kong Anna Smith UK Patrick Smith USA Rebecca Smith UK Shubulade Smith UK David G Smithard UK Marcel Smits Netherlands Moria Smoski USA Anne Söderlund Sweden Daya Somasundaram Sri Lanka Lin Sørensen Norway Julia Friederike Sowislo Switzerland Francesco Spallotta Germany Mona Srivastava India Paul St John-Smith UK Wouter Staal Netherlands David Stacey Australia Victoria Stanhope USA Jack Stevens USA Jon Stone UK Martin Strassnig USA Barbara Stringer Netherlands Brendon Stubbs UK Paula Suárez-Pinilla Spain Hajime Sueki Japan Koreaki Sugimoto Japan Sarah Sullivan UK Jaana Suvisaari Finland Takefumi Suzuki Japan Bengt Svensson Sweden Piotr Switaj Poland Andras Szekely Hungary Eskinder Tafesse USA Yueh-Ming Tai Taiwan Ricardo Taipa Portugal Mutsuko Takahashi Japan Cecilia Sighinolfi Italy Roberta Siliquini Italy Derrick Silove Australia Kang Sim Singapore Magenta Simmons Australia Claudia Simons Netherlands Jay Singh Norway Mark Sinyor Canada Bonnie Wm Siu Hong Kong Anna Smith UK Patrick Smith USA Rebecca Smith UK Shubulade Smith UK David G Smithard UK Marcel Smits Netherlands Moria Smoski USA Anne Söderlund Sweden Daya Somasundaram Sri Lanka Lin Sørensen Norway Julia Friederike Sowislo Switzerland Francesco Spallotta Germany Mona Srivastava India Taro Takeshima Japan Qingrong Tan China Ilaria Tarricone Italy Cumhur Tas Turkey Alvin Kuowei Tay Australia David Taylor UK Kate Tchanturia UK Michelle Teale Sapach Canada Ghazel Tellawi USA Henk Temmingh South Africa Martin Teufel Germany Michel Thibodeau Canada Henning Tiemeier Netherlands Nickolai Titov Australia Arun Tiwari Canada Carmine Tomasetti Italy Ulysses Torres Brazil Lurdes Tse Canada Wan-Ling Tseng USA Marco Tuccori Italy James Tung Canada Alyna Turner Australia Taro Takeshima Japan Qingrong Tan China Ilaria Tarricone Italy Cumhur Tas Turkey Alvin Kuowei Tay Australia David Taylor UK Kate Tchanturia UK Michelle Teale Sapach Canada Ghazel Tellawi USA Henk Temmingh South Africa Martin Teufel Germany Michel Thibodeau Canada Henning Tiemeier Netherlands Nickolai Titov Australia Arun Tiwari Canada Carmine Tomasetti Italy Ulysses Torres Brazil Lurdes Tse Canada Wan-Ling Tseng USA Marco Tuccori Italy James Tung Canada Alyna Turner Australia Paul St John-Smith UK Wouter Staal Netherlands David Stacey Australia Victoria Stanhope USA Jack Stevens USA Jon Stone UK Martin Strassnig USA Barbara Stringer Netherlands Brendon Stubbs UK Paula Suárez-Pinilla Spain Hajime Sueki Japan Koreaki Sugimoto Japan Sarah Sullivan UK Jaana Suvisaari Finland Takefumi Suzuki Japan Bengt Svensson Sweden Piotr Switaj Poland Andras Szekely Hungary Eskinder Tafesse USA Yueh-Ming Tai Taiwan Ricardo Taipa Portugal Mutsuko Takahashi Japan Shubulade Smith UK David G Smithard UK Page 14 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Marjolein Verhoeven Netherlands Jan Vevera Czech Republic Philippe Vincent Canada Benedetto Vitiello USA David Vogel USA Jen Vohs USA Katharina Voigt Germany Corrine Voils USA Eberhard Voit USA Umberto Volpe Italy Silke Von Esenwein USA Martin Voracek Austria Tracey Wade Australia Margda Waern Sweden Akio Wakabayashi Japan Fredrik A. Contributing reviewers Walby Norway Wei Wang China Yuan-Pang Wang Brazil Jia Wang China Qiang Wang China Norio Watanabe Japan Rafal Watrowski Germany Florian Weck Germany Peter M. Wehmeier Germany Aviv Weinstein Israel Abraham Weizman Israel David M Wellsted UK Benedict Weobong Ghana Perla Werner Israel Ruud Wetzels Netherlands Amanda Wheeler Australia Ross White UK Richard Whittington UK Johanna Wigman Netherlands Dirk Wildgruber Germany Sarah Wilker Germany Alishia Williams Australia Amanda Williams UK Monnica Williams USA Tony Winefield Australia Tracy Witte USA Katrin Woitecki Germany Owen Wolkowitz USA Miranda Wolpert UK Hiroyuki Uchida Japan Alp Ucok Turkey Rudolf Uher Canada Zsolt Unoka Hungary Dominique Valade France Tom Van Daele Belgium Geurt Van De Glind Netherlands Fons Van De Vijver Netherlands Marjan Van Den Akker Netherlands Rutger Jan Van Der Gaaag Netherlands Peggy Van Der Pol Netherlands Louk Van Der Post Netherlands Annemarie Van Elburg Netherlands Harm Van Marwijk Netherlands Bregje Van Spijker Australia Cornelia Van Uden-Kraan Netherlands Benjamin Van Voorhees USA Erik Vanderlip USA Gustavo Vazquez Argentina Per Vendsborg Denmark Peter Ventevogel Netherlands Lena Verdeli USA Marjolein Verhoeven Netherlands Jan Vevera Czech Republic Philippe Vincent Canada Benedetto Vitiello USA David Vogel USA Jen Vohs USA Katharina Voigt Germany Corrine Voils USA Eberhard Voit USA Umberto Volpe Italy Silke Von Esenwein USA Martin Voracek Austria Tracey Wade Australia Margda Waern Sweden Akio Wakabayashi Japan Fredrik A. Walby Norway Wei Wang China Yuan-Pang Wang Brazil Jia Wang China Qiang Wang China Norio Watanabe Japan Rafal Watrowski Germany er Gaaag rg Kraan ees Marjolein Verhoeven Netherlands Jan Vevera Czech Republic Philippe Vincent Canada Benedetto Vitiello USA David Vogel USA Jen Vohs USA Katharina Voigt Germany Corrine Voils USA Eberhard Voit USA Umberto Volpe Italy Silke Von Esenwein USA Martin Voracek Austria Tracey Wade Australia Margda Waern Sweden Akio Wakabayashi Japan Fredrik A. Contributing reviewers Walby Norway Wei Wang China Yuan-Pang Wang Brazil Jia Wang China Qiang Wang China Norio Watanabe Japan Rafal Watrowski Germany Hiroyuki Uchida Japan Alp Ucok Turkey Rudolf Uher Canada Zsolt Unoka Hungary Dominique Valade France Tom Van Daele Belgium Geurt Van De Glind Netherlands Fons Van De Vijver Netherlands Marjan Van Den Akker Netherlands Rutger Jan Van Der Gaaag Netherlands Peggy Van Der Pol Netherlands Louk Van Der Post Netherlands Annemarie Van Elburg Netherlands Harm Van Marwijk Netherlands Bregje Van Spijker Australia Cornelia Van Uden-Kraan Netherlands Benjamin Van Voorhees USA Erik Vanderlip USA Gustavo Vazquez Argentina Per Vendsborg Denmark Peter Ventevogel Netherlands Lena Verdeli USA Dirk Wildgruber Germany Sarah Wilker Germany Alishia Williams Australia Amanda Williams UK Monnica Williams USA Tracy Witte USA Owen Wolkowitz USA Page 15 of 15 Rice and Marshall BMC Psychiatry (2015) 15:54 Xiang Rong Zhang China Huiping Zhang USA Han Zhang China Bin Zhang China Jie Zhang USA Min Zhao China Jiang-Ning Zhou China Gang Zhu China Tim Ziermans Netherlands Stephan Zipfel Germany Phillip Zoladz USA Jon-Kar Zubieta USA Maria Victoria Zunzunegui Canada Eugene Wong UK Jesse Wright USA Yin Wu UK Til Wykes UK Brian Wymbs USA Shifu Xiao China Kirsten Yaffe USA Amit Yamin Israel Chih Chieh Yang USA Yen Kuang Yang Taiwan Philip Yanos USA Shuqiao Yao China Norio Yasui-Furukori Japan Wai-Ying Wendy Yau USA Barbara Yawn USA Ksenija Yeeles UK Cheng-Fang Yen Taiwan Yuan Yong Gui China Ki-Bong Yoo Korea, South Nicole Yuan USA Kai Yuan China Xiaojun Yuan USA David Yusko USA Clement Zai Canada Yu-Feng Zang China Faika Zanjani USA Yuliya Zaytseva Germany Patrizia Zeppegno Italy Barbara Yawn USA Ksenija Yeeles UK Cheng-Fang Yen Taiwan Yuan Yong Gui China Ki-Bong Yoo Korea, South Nicole Yuan USA Kai Yuan China Xiaojun Yuan USA David Yusko USA Clement Zai Canada Yu-Feng Zang China Faika Zanjani USA Yuliya Zaytseva Germany Patrizia Zeppegno Italy Eugene Wong UK Jesse Wright USA Yin Wu UK Til Wykes UK Brian Wymbs USA Shifu Xiao China Kirsten Yaffe USA Amit Yamin Israel Chih Chieh Yang USA Yen Kuang Yang Taiwan Philip Yanos USA Shuqiao Yao China Norio Yasui-Furukori Japan Wai-Ying Wendy Yau USA Xiang Rong Zhang China Huiping Zhang USA Han Zhang China Bin Zhang China Jie Zhang USA Min Zhao China Jiang-Ning Zhou China Gang Zhu China Tim Ziermans Netherlands Stephan Zipfel Germany Phillip Zoladz USA Jon-Kar Zubieta USA Maria Victoria Zunzu Canada Xiang Rong Zhang China Huiping Zhang USA Han Zhang China Bin Zhang China Jie Zhang USA Min Zhao China Jiang-Ning Zhou China Gang Zhu China Tim Ziermans Netherlands Stephan Zipfel Germany Phillip Zoladz USA Jon-Kar Zubieta USA Maria Victoria Zunzu Canada
https://openalex.org/W2965593074	https://hal.inrae.fr/hal-02627910/file/2019_Levakova_Journal%20of%20the%20Royal%20Society%20Interface_1.pdf	English	null	Adaptive integrate-and-fire model reproduces the dynamics of olfactory receptor neuron responses in a moth	Journal of the Royal Society interface	2,019	cc-by	9,485	Adaptive integrate-and-fire model reproduces the dynamics of olfactory receptor neuron responses in a moth Adaptive integrate-and-fire model reproduces the dynamics of olfactory receptor neuron responses in a moth Marie Levakova, Lubomir Kostal, Christelle Monsempes, Philippe Lucas, Ryota Kobayashi Marie Levakova, Lubomir Kostal, Christelle Monsempes, Philippe Lucas, Ryota Kobayashi Marie Levakova, Lubomir Kostal, Christelle Monsempes, Philippe Lucas, Ryota Kobayashi To cite this version: Marie Levakova, Lubomir Kostal, Christelle Monsempes, Philippe Lucas, Ryota Kobayashi. Adaptive integrate-and-fire model reproduces the dynamics of olfactory receptor neuron responses in a moth. Journal of the Royal Society Interface, 2019, 16 (157), 10.1098/rsif.2019.0246. hal-02627910 Adaptive integrate-and-fire model reproduces the dynamics of olfactory receptor neuron responses in a moth Distributed under a Creative Commons Attribution 4.0 International License Keywords: Keywords: olfactory receptor neuron, integrate-and-fire model, adaptive threshold HAL Id: hal-02627910 https://hal.inrae.fr/hal-02627910v1 Submitted on 26 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Adaptive integrate-and-fire model reproduces the dynamics of olfactory receptor neuron responses in a moth royalsocietypublishing.org/journal/rsif royalsocietypublishing.org/journal/rsif Marie Levakova1, Lubomir Kostal1, Christelle Monsempès2, Philippe Lucas2 and Ryota Kobayashi3,4 ML, 0000-0001-9104-4962; PL, 0000-0003-2166-8248 ML, 0000-0001-9104-4962; PL, 0000-0003-2166-8248 http://dx.doi.org/10.1098/rsif.2019.0246 In order to understand how olfactory stimuli are encoded and processed in the brain, it is important to build a computational model for olfactory recep- tor neurons (ORNs). Here, we present a simple and reliable mathematical model of a moth ORN generating spikes. The model incorporates a simpli- fied description of the chemical kinetics leading to olfactory receptor activation and action potential generation. We show that an adaptive spike threshold regulated by prior spike history is an effective mechanism for reproducing the typical phasic–tonic time course of ORN responses. Our model reproduces the response dynamics of individual neurons to a fluctu- ating stimulus that approximates odorant fluctuations in nature. The parameters of the spike threshold are essential for reproducing the response heterogeneity in ORNs. The model provides a valuable tool for efficient simulations of olfactory circuits. Received: 2 April 2019 Accepted: 24 June 2019 Received: 2 April 2019 Accepted: 24 June 2019 Subject Category: Life Sciences–Mathematics interface 1. Introduction Many animals rely on olfaction for detecting food, natural predators and mating partners. The odorant is initially recognized by olfactory receptor neurons (ORNs). The information is then transferred to a secondary region, either the antennal lobe in insects or olfactory bulb in vertebrates. Projections from the secondary region extend to higher order brain regions, the mushroom body and lateral horn in insects and the orbitofrontal cortex, amygdala, entorhinal cortex and ventral striatum in vertebrates. The architecture of the olfactory circuit differs from that of other sensory modalities (for a review, see [1,2]); for example, the olfactory circuit consists of fewer layers. Therefore concepts derived from experimental and theoretical studies on other systems may not be applicable to olfaction. Computational models that can replicate the behaviour of real ORNs during odorant stimulation may generate testable hypotheses on mechanisms underlying olfactory transduction and encoding. Research 1Department of Computational Neuroscience, Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic 2Institute of Ecology and Environmental Sciences, INRA, route de St Cyr, 78000 Versailles, France 3Principles of Informatics Research Division, National Institute of Informatics, 2-1-2 Hitotsubashi, Chiyoda-ku, Tokyo, Japan 4Department of Informatics, SOKENDAI (The Graduate University for Advanced Studies), 2-1-2 Hitotsubashi, Chiyoda-ku, Tokyo, Japan 1Department of Computational Neuroscience, Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic Cite this article: Levakova M, Kostal L, Monsempès C, Lucas P, Kobayashi R. 2019 Adaptive integrate-and-fire model reproduces the dynamics of olfactory receptor neuron responses in a moth. J. R. Soc. Interface 16: 20190246. g p 2Institute of Ecology and Environmental Sciences, INRA, route de St Cyr, 78000 Versailles, France 3Principles of Informatics Research Division, National Institute of Informatics, 2-1-2 Hitotsubashi, Chiyoda-ku Tokyo, Japan 4Department of Informatics, SOKENDAI (The Graduate University for Advanced Studies), 2-1-2 Hitotsubashi, Chiyoda-ku, Tokyo, Japan ML, 0000-0001-9104-4962; PL, 0000-0003-2166-8248 Author for correspondence: Marie Levakova e-mail: marie.levakova@fgu.cas.cz Author for correspondence: Marie Levakova e-mail: marie.levakova@fgu.cas.cz Indeed, computational models have enhanced our understanding of the mechanisms underlying odorant detection in both invertebrates and vertebrates [3–8] and facilitated investigations of olfactory pathway functions [9–12]. Such models have also been used to clarify the coding properties of ORNs such as the stimulus–response relationship of the ORNs [13,14] and the implications of the efficient coding hypothesis [15]. © 2019 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. Receptor activation. We describe the process of receptor activation by the following chemical reactions, derived by Kaissling and coworkers [15,30,31] Pheromone detection in moth ORNs occurs in two stages: receptor activation by the odorant and action potential (spike) generation. Odorant molecules are first absorbed by the sen- sillum lymph, where they initiate a cascade of complex biochemical interactions. Receptor activation and related downstream signalling cascades leading to membrane depolarization have been described by various mathematical models [3,14,16], including detailed biophysical models [4–7,17,18]. To understand the mechanisms of pheromone detection, it is essential to develop a computational model that replicates odorant-evoked ORN responses. 2 Lair ! ki L (2:1) nL þ R O k1 k1 RL O k2 k2 R (2:2) and L þ N O k3 k3 NL ! k4 P þ N: (2:3) Lair ! ki L (2:1) nL þ R O k1 k1 RL O k2 k2 R (2:2) and L þ N O k3 k3 NL ! k4 P þ N: (2:3) (2:3) Equation (2.1) describes an absorption of odorant molecules in the air Lair by the sensillum lymph at a rate ki, which yields odorant molecules at the receptor site L. Equation (2.2) describes the binding of n molecules of odorant L to a receptor. Odorant molecules L reversibly bind to free recep- tors R at rates k1 and k−1, which yields the receptor–ligand complex RL. Then, the complexes RL are reversibly activated (R) at a rate k2 and k−2. Finally, equation (2.3) describes the kinetics of odorant degradation at the receptor site by an odorant degrading enzyme N. The odorant and enzyme reversibly form a complex NL according to rate constants k3 and k−3, and the complex is degraded into an inactive pro- duct P at a rate k4. to pheromone Experimental data were obtained from ORNs by applying different pheromone doses to antennae of the moth Agrotis ipsilon (see Methods for details). To simulate the fluctuating odorant concentration in a natural environment [27], the pheromone was applied in short intermittent pulses ( puffs) separated by stimulus-free periods (blanks) of random duration (figure 1a). IR(t) ¼ gR(t)(V ER), (2:5) (2:5) where R(t) is the concentration of activated receptors R* at time t, ER is the reversal potential of the receptor current and γ represents the conductance induced by a single activated receptor R. A spike is generated when the membrane poten- tial V(t) reaches a threshold θ(t). After each spike, the membrane potential is reset to a value Vreset. In the following sections, we consider two types of spike thresholds, a constant threshold and an adaptive threshold. Responses of different ORNs to the same pheromone pulse exhibited marked cell-to-cell variability (figure 1b) as reported in previous studies [28,29]. This response heterogen- eity of ORNs might be caused, for example, by differences in the density of olfactory receptors (ORs), odorant-binding pro- teins and odorant-degrading enzymes among ORNs. Nonetheless, averaged responses across cells demonstrated a typical phasic–tonic time course regardless of pheromone dose (figure 1c–f). From a baseline rate near 0 Hz, the firing rates increased rapidly (phasic period), reaching a peak around 100 ms after stimulus onset, and then slowly decaying toward a steady-state firing rate that was higher than the spontaneous firing rate (tonic period). The peak firing rate increased with pheromone dose, but the delay of the peak firing rate (latency) and the phasic–tonic response time course did not change. Author for correspondence: Marie Levakova e-mail: marie.levakova@fgu.cas.cz The chemical kinetics (2.1)–(2.3) can be described by a system of differential equations (see Methods, equations (4.1)–(4.6)). Reduced neuronal models, such as the leaky integrate- and-fire (LIF) neuron [19–21], can be good approximations of real neurons [22,23] and therefore useful tools for simulat- ing and investigating prominent features of network dynamics [24,25]. A few models incorporating receptor acti- vation into a simple spike generation mechanism based on the LIF model have been developed [13,26] in order to study steady-state ORN behaviour. However, the LIF model cannot accurately replicate the response dynamics. Here, we develop a computational model for individual ORNs that generates spikes in response to dynamic odorant stimulation. We demonstrate that an adaptation mechanism in spike threshold is necessary to reproduce the response dynamics of ORNs. The mathematical tractability and simpli- city of the proposed model allows for efficient simulations and analysis of ORN spiking activity. Spike generation. We describe the ORN by a single- compartment model. The membrane potential V(t) evolves according to [32] Cm dV dt ¼ gL(V EL) þ IR(t), (2:4) (2:4) 2.3. Model with constant spike threshold cannot reproduce the response dynamics of an olfactory receptor neuron First, we considered the model with a constant spike threshold, θ(t) = θ0, known as the leaky integrate-and-fire (LIF) model [32]. We investigated whether the LIF model with receptor dynamics (2.1)–(2.5) can reproduce the average response of ORNs to a pheromone pulse stimulus (figure 1c–f). We observed that the firing rates of the model increase monotoni- cally, whereas the firing rates of ORNs always exhibited a peak followed by a slower decline to steady state (phasic–tonic response) (figure 3a). The model firing rates increase monoto- nically because the number of activated receptors R(t) increases during the stimulation period. Thus, the model based on (2.1)–(2.5) with a constant spike threshold cannot reproduce the time course of the average ORN response. 2. Results where Cm is the cell capacitance, gL is the leak conductance and EL is the reversal potential of the leak current. The cur- rent from the odorant receptors IR(t) is determined by the quantity of activated receptors according to [13] 2.2. Model of an olfactory receptor neuron Experimental data for the responses of olfactory receptor neurons (ORNs) to pheromone stimulation. (a) ORNs were stimulated by intermittent delivery of the sex pheromone (four pheromone doses ranging from 1 to 1000 pg) to mimic fluctuating odorant concentration in a pheromone plume. (b) Examples of spike trains generated by two ORNs (cells A and B) in response to 0.5 s of constant pheromone stimulation at 100 pg. Top: The average firing rate of each cell. Bottom: Raster plots of 10 trials (rows) from each cell. Note the heterogeneity in firing rates between the two ORNs despite stimulation by the same pheromone pulse. (c–f) The average firing rate across cells in response to the same 0.5 s pulse stimulus of pheromone at different doses (1–1000 pg). The shaded area represents the range between the lower and upper quartile trajectory. (Online version in colour.) Figure 1. Experimental data for the responses of olfactory receptor neurons (ORNs) to pheromone stimulation. (a) ORNs were stimulated by intermittent delivery of the sex pheromone (four pheromone doses ranging from 1 to 1000 pg) to mimic fluctuating odorant concentration in a pheromone plume. (b) Examples of spike trains generated by two ORNs (cells A and B) in response to 0.5 s of constant pheromone stimulation at 100 pg. Top: The average firing rate of each cell. Bottom: Raster plots of 10 trials (rows) from each cell. Note the heterogeneity in firing rates between the two ORNs despite stimulation by the same pheromone pulse. (c–f) The average firing rate across cells in response to the same 0.5 s pulse stimulus of pheromone at different doses (1–1000 pg). The shaded area represents the range between the lower and upper quartile trajectory. (Online version in colour.) Except for non-decreasing firing rate profiles, the model has another issue of being able to reproduce correctly only either the peak firing rate or the first-spike latency, but not both of them simultaneously. This problem could only be numerically resolved by allowing an unphysiologically long refractory period after each spike. Figure 3a shows a compro- mise fit that could be achieved with a realistic 3 ms refractory period, where both the peak firing rate and the first-spike latency are much larger than in real ORNs. The model parameters are summarized in tables 1 and 2. 2.2. Model of an olfactory receptor neuron Most of them were adopted from previous studies [7,15,16,30–32,37], while the two rate constants for receptor activation (equation (2.3)), k3 and k4, were chosen to achieve rapid deactivation of L. The remaining four parameters (n, τ, Δ and γ) were determined by minimizing the integrated squared error between the average response of ORNs and the model response (see Methods). 2.2. Model of an olfactory receptor neuron The proposed ORN model (figure 2) consists of two main parts: (i) receptor activation due to pheromone stimulation and (ii) spike generation according to an integrate-and-fire mechanism. odorant source puff blank stimulus course spikes time 0 0.1 0.2 0.3 0.4 0 20 40 60 firing rate (Hz) cell A cell B stimulus onset 0 0.1 0.2 0.3 0.4 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 time (s) 0 0.1 0.2 0.3 0.4 time (s) 0 0.1 0.2 0.3 0.4 time (s) time (s) 0 0.1 0.2 0.3 0.4 time (s) firing rate (Hz) 1 pg 10 pg 102 pg 103 pg (e) ( f ) (b) (a) (c) (d) Figure 1. Experimental data for the responses of olfactory receptor neurons (ORNs) to pheromone stimulation. (a) ORNs were stimulated by intermittent delivery of he sex pheromone (four pheromone doses ranging from 1 to 1000 pg) to mimic fluctuating odorant concentration in a pheromone plume. (b) Examples of spike rains generated by two ORNs (cells A and B) in response to 0.5 s of constant pheromone stimulation at 100 pg. Top: The average firing rate of each cell. Bottom: Raster plots of 10 trials (rows) from each cell. Note the heterogeneity in firing rates between the two ORNs despite stimulation by the same pheromone pulse. (c–f) The average firing rate across cells in response to the same 0.5 s pulse stimulus of pheromone at different doses (1–1000 pg). The shaded area represents the range between the lower and upper quartile trajectory. (Online version in colour.) odorant source puff blank stimulus course spikes time (a) 3 (a) stimulus onset 0 20 40 60 80 0 0.1 0.2 0.3 0.4 time (s) 102 pg e) ( stimulus onset 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 0.1 0.2 0.3 0.4 time (s) 0 0.1 0.2 0.3 0.4 time (s) 0 0.1 t 10 pg 102 pg (e) ( f ) (d) 0 20 40 60 80 0 0.1 0.2 0.3 0.4 time (s) 10 pg ( (d) 0 0.1 0.2 0.3 0.4 0 20 40 60 80 time (s) firing rate (Hz) 1 pg (c) (c) time (s) Figure 1. Experimental data for the responses of olfactory receptor neurons (ORNs) to pheromone stimulation. (a) ORNs were stimulat Figure 1. 2.4. Model with an adaptive spike threshold reproduces the response dynamics of an olfactory receptor neuron In the natural environment, odorant concentrations fluctuate rapidly; therefore, it is crucial to replicate the response dynamics of an ORN to such stimulation. To mimic the natu- ral pheromone plume under experimental conditions, we stimulated the antennae by intermittent delivery of the pheromone [41,42]. The firing rates of individual ORNs were then compared with those generated by the model with the adaptive spike threshold. olfactory receptor neuron The model provides spike times from which the firing rate can be calculated. (Online version in colour.) input: stimulus stimulus course L R extracellular voltage dynamics intracellular receptor controlled membr. potential threshold Figure 2. Proposed model of an olfactory receptor neuron (ORN). Stimulus. The odorant concentration fluctuating in time is the input to the model neuron. (1) Receptor activation. The odorant molecules in the air Lair are adsorbed in the lymph at the receptor site. The adsorbed molecules L either bind to receptors R resulting in activated receptors R* or they are degraded by an enzyme N, which converts them into an inactive product P. (2) Spike generation. Activated receptors R* induce a receptor current in a single-compartment model. The model neuron generates action potentials when the membrane potential reaches a threshold θ(t). Note that a time-dependent spike threshold model (dotted) can reproduce experimentally observed ORN responses. Response. The model provides spike times from which the firing rate can be calculated. (Online version in colour.) improvement compared with heterogeneity only in (Δ, τ) and the median prediction error was even slightly lower (median 0.59, inter-quartile range: 0.47 to 0.66), most likely because too many free parameters led to overfitting. γ); second, we let the pair of threshold parameters Δ and τ be cell specific (heterogeneity in (Δ, τ)); and third, we fitted all three parameters γ, Δ and τ to each neuron (heterogeneity in (γ, Δ, τ)). Finally, we examined the prediction performance of each heterogeneous model by the coefficient of determination (see Methods). Therefore, we concluded that the cell-to-cell heterogeneity among ORNs is best captured by fitting the threshold par- ameters (Δ and τ) to the experimental recording of each individual ORN, since this yields a significant improvement in the prediction performance over the homogeneous model (Wilcoxon’s rank sum test, p < 0.001, n = 84). Figure 6a illus- trates an example of the model fit to recordings of two neurons. While the temporal pattern of the observed responses is similar, the amplitudes are different. The model with the adaptive spike threshold reproduces the response time course of the two neurons accurately. The distribution of the response time course of the fitted model neurons (n = 84) to the same stimulus is shown in figure 6b. olfactory receptor neuron Since the LIF model with constant spike threshold could not replicate the qualitative characteristics of ORN responses, it was modified by including an adaptive spike threshold [33–36], which depends on previous spike times. The threshold θ(t) increases by Δ/τ after each spike and decreases exponen- tially to an asymptotic level θ0 with the time constant τ. The parameter Δ represents the strength of adaptation (see Methods for a formal mathematical description). Since we wanted to reproduce the activity of individual ORNs, we had to take into account a cell-to-cell variability in ORN responses (figure 1b). The heterogeneity among ORNs can be captured by fitting some of the model par- ameters to the experimental recording of each individual ORN (see Methods), while keeping all the other parameters fixed as in tables 1 and 2. As for the choice of which par- ameters should be allowed to vary across the cells, we tested three options. First, we let γ vary (heterogeneity in Unlike the LIF model, the model with the adaptive spike threshold is able to accurately reproduce the time course of the average ORN responses under each odorant concentration (figure 3b). In addition, the model captures the dependence of the response characteristics of ORNs, i.e. the peak firing rate (figure 4b) and the first-spike latency (figure 4c), on the odorant concentration over a wide range of odorant doses (1000-fold). royalsocietypublishing.org/journal/rsif J. R. Soc. Interface 16: 20190246 4 input: stimulus 1. receptor activation 2. spike generation voltage dynamics threshold output: spikes stimulus course Lair L R RL R* N NL P R* receptor controlled extracellular intracellular leak gL gR* ER EL Cm time membr. potential spike train Figure 2. Proposed model of an olfactory receptor neuron (ORN). Stimulus. The odorant concentration fluctuating in time is the input to the model neuron. (1) Receptor activation. The odorant molecules in the air Lair are adsorbed in the lymph at the receptor site. The adsorbed molecules L either bind to receptors R resulting in activated receptors R* or they are degraded by an enzyme N, which converts them into an inactive product P. (2) Spike generation. Activated receptors R* induce a receptor current in a single-compartment model. The model neuron generates action potentials when the membrane potential reaches a threshold θ(t). Note that a time-dependent spike threshold model (dotted) can reproduce experimentally observed ORN responses. Response. 3.1. Heterogeneity of olfactory receptor neurons heterogeneity in threshold parameters, the amplitudes of the responses are highly variable among the model neurons, but the temporal patterns of the responses remain similar. Figure 5a shows the threshold parameters obtained from all ORNs. The mean values (+/−the standard deviation) of the parameters are 1.2 ± 0.38 s for the threshold time constant τ and 0.5 ± 0.23 mV s for the adaptation level Δ. Values of τ and Δ are negatively correlated (correlation coefficient −0.48). This finding can be intuitively explained as that these two par- ameters can compensate for each other to some extent. A similar firing rate may be achieved by combining either a small step increase and a slow relaxation time or a big increase and fast relaxation. Although the threshold parameters exhibit high variability among the ORNs, they are comparable to the parameters fitted to the average response (table 2). heterogeneity in threshold parameters, the amplitudes of the responses are highly variable among the model neurons, but the temporal patterns of the responses remain similar. Figure 5a shows the threshold parameters obtained from all ORNs. The mean values (+/−the standard deviation) of the parameters are 1.2 ± 0.38 s for the threshold time constant τ and 0.5 ± 0.23 mV s for the adaptation level Δ. Values of τ and Δ are negatively correlated (correlation coefficient −0.48). This finding can be intuitively explained as that these two par- ameters can compensate for each other to some extent. A similar firing rate may be achieved by combining either a small step increase and a slow relaxation time or a big increase and fast relaxation. Although the threshold parameters exhibit high variability among the ORNs, they are comparable to the parameters fitted to the average response (table 2). g y y p The response heterogeneity of moth ORNs, manifested by different dose–response properties among cells, and its impact on neuronal coding were thoroughly studied by Rospars et al. [29]. In addition, cell-to-cell response variability among ORNs has been investigated in other animal species such as mice [43]. This variability is captured in our model by setting different threshold parameters, i.e. the strength and the time constant of the adaptation. Previous works [36,44] suggested that the biophysical origins of the adaptive threshold are the slow K+ currents in the neuron, such as the Ca2+-activated K+ current [39] and M-type K+ current. olfactory receptor neuron Owing to the The prediction performances of the three heterogeneous models with cell-specific parameters were compared with the model where all parameters were fixed for all cells as in table 2 (homogeneous model); see figure 5a. The median pre- diction performance of the homogeneous model was 0.13 (inter-quartile range: −0.02 to 0.30). Fitting only γ led to a mild improvement in the prediction performance (median 0.26, inter-quartile range: 0.18 to 0.35). The prediction per- formance improved substantially with heterogeneous τ and Δ (median 0.6, inter-quartile range: 0.46 to 0.67). Having all three parameters γ, τ, Δ heterogeneous did not bring any 0 0.1 0.2 0.3 0.4 0 100 200 stimulus stimulus time (s) 0 0.1 0.2 0.3 0.4 time (s) firing rate (Hz) constant threshold 0 20 40 60 adaptive threshold model (concentration) Lair= 10−1 pM Lair= 1 pM Lair= 10 pM Lair= 102 pM observed (dose) 1 pg 10 pg 102 pg 103 pg (b) (a) Figure 3. Model with an adaptive spike threshold can reproduce the phasic–tonic response of ORNs to a pulse odorant stimulation. Average responses of ORNs (dashed lines) were compared with the responses of the model neurons (solid lines), i.e. the model with a constant threshold (a) and the model with an adaptive threshold (b). The unit receptor conductance was γ = 41 nS · μM−1 in (a) and γ = 99 nS · μM−1 in (b). Each spike generated by the model with a constant threshold (a) was followed by a 3 ms refractory period. The pheromone concentration in the air, Lair, was set to 0.1 pM, 1 pM, 10 pM, 100 pM for the pheromone doses 1 pg, 10 pg, 100 pg, 1000 pg, respectively. See tables 1 and 2 for the other parameters. (Online version in colour.) stimulus 0 0.1 0.2 0.3 0.4 time (s) 0 20 40 60 adaptive threshold model (concentration) Lair= 10−1 pM Lair= 1 pM Lair= 10 pM Lair= 102 pM observed (dose) 1 pg 10 pg 102 pg 103 pg (b) 0 0.1 0.2 0.3 0.4 0 100 200 stimulus time (s) firing rate (Hz) constant threshold (a) royalsocietypublishing.org/journal/rsif J. R. Soc. Interface 16: 20190246 5 Figure 3. Model with an adaptive spike threshold can reproduce the phasic–tonic response of ORNs to a pulse odorant stimulation. Average responses of ORNs (dashed lines) were compared with the responses of the model neurons (solid lines), i.e. olfactory receptor neuron the model with a constant threshold (a) and the model with an adaptive threshold (b). The unit receptor conductance was γ = 41 nS · μM−1 in (a) and γ = 99 nS · μM−1 in (b). Each spike generated by the model with a constant threshold (a) was followed by a 3 ms refractory period. The pheromone concentration in the air, Lair, was set to 0.1 pM, 1 pM, 10 pM, 100 pM for the pheromone doses 1 pg, 10 pg, 100 pg, 1000 pg, respectively. See tables 1 and 2 for the other parameters. (Online version in colour.) –1 0 1 2 Lair (log10 pM) 0 0.05 0.10 latency (s) (c) peak rate latency time (a) peak rate latency time –1 0 1 2 0 20 40 60 80 Lair (log10 pM) –1 0 1 2 Lair (log10 pM) peak rate (Hz) 0 0.05 0.10 latency (s) (b) (a) (c) Figure 4. Model with an adaptive spike threshold can reproduce the odorant response characteristics of ORNs. (a) A scheme illustrating two salient characteristics of the response time course: the peak firing rate and the first-spike latency. (b,c) The effect of odorant concentration on the response characteristics. The peak firing rate (b) and the first-spike latency (c) obtained from experimental data (dashed blue, mean with inter-quartile range) were compared with those obtained from the model (solid black). (Online version in colour.) –1 0 1 2 0 20 40 60 80 Lair (log10 pM) peak rate (Hz) (b) ( Figure 4. Model with an adaptive spike threshold can reproduce the odorant response characteristics of ORNs. (a) A scheme illustrating two salient characteristics of the response time course: the peak firing rate and the first-spike latency. (b,c) The effect of odorant concentration on the response characteristics. The peak firing rate (b) and the first-spike latency (c) obtained from experimental data (dashed blue, mean with inter-quartile range) were compared with those obtained from the model (solid black). (Online version in colour.) 3.2. Advantages of the proposed model The model presented here serves as an efficient tool for simu- lating moth ORN responses. First, the model captures the typical response properties observed experimentally, particu- larly the phasic–tonic response pattern characterized by a rapid increase and a slow decay to a steady-state firing rate, as well as the effect of odorant concentration on the peak firing rate and first-spike latency. Second, our model can simulate cell-to-cell response variability among ORNs by indi- vidually setting only two parameters controlling the adaptive spike threshold. Third, our model provides the spike times, unlike linear–nonlinear models, which can capture only the 3.1. Heterogeneity of olfactory receptor neurons Thus, our results imply that differences in the slow K+ current density might contribute to the response heterogeneity among ORNs. 3. Discussion We present a computational model of a moth ORN that reproduces the firing rate dynamics of an ORN under inter- mittent pheromone stimulation over a 1000-fold range of concentrations. Further, our model captures cell-to-cell response variability of ORNs by tuning only two model par- ameters controlling the spike threshold. The model is less accurate for longer stimulations, where the model firing rate increases more slowly than the true firing rate. The model also mildly underestimates maximal spike rates. Table 2. Parameters for the model of spike generation. Table 1. Parameters for the model of receptor activation (equations (2.1)–(2.3)). Table 1. Parameters for the model of receptor activation (equations (2.1)–(2.3)). 6 p g value unit ﬁtted/ﬁxed Cm 0.00144 nF ﬁxed [7,38] gL 1.44 nS ﬁxed [7,38] γ 99.27 nS · μM−1 ﬁtted EL −62 mV ﬁxed [7,32,39,40] ER 0 mV ﬁxed [7] Vreset −62 mV ﬁxed [7,32] θ0 −55 mV ﬁxed [32] Δ 0.77 mV s ﬁtted τ 0.58 s ﬁtted value unit ﬁtted/ﬁxed Rtot 1.64 μM ﬁxed [16,30,31] Ntot 1 μM ﬁxed [16,30,31] ki 106 s−1 ﬁxed [37] k1 0.209 s−1 · μM−1 ﬁxed [16,30,31] k−1 7.9 s−1 ﬁxed [16,30,31] k2 16.8 s−1 ﬁxed [16,30,31] k−2 98 s−1 ﬁxed [16,30,31] k3 100 s−1 · μM−1 ﬁxed k−3 98.9 s−1 ﬁxed [16,30,31] k4 40 000 s−1 ﬁxed n 0.056 ﬁtted temporal structure of odour plumes to be encoded independent of their concentration [14]. In spite of its simplicity, our model effectively captures the adaptation process, since it can predict the response dynamics of ORNs recorded in experiments. However, the feedback mechanism of our model might be fundamentally different from that induced by the second messenger signal- ling pathways. For instance, the adaptation process due to the adaptive spike threshold model depends solely on previous spike history and is different from the adaptation process in real ORNs caused by Ca2+ influx and the following transduc- tion cascade [7]. An investigation of more physiological feedback mechanisms could allow for further improvements of the model. One possibility may be to include explicit for- mulae describing the interaction of OR–Orco complexes and the adaptation of the rates of switching between the inactive and the active state, such as in the model by Gorur-Shandilya et al. [14]. firing rates [41,42,45]. 3.3. Limitations and future works Experimental evidence suggests that adaptation occurs at the level of both the receptor potential and action potential gen- erators [50,51]. This is effectively achieved in our model by including the chemical kinetics of activated receptors, which is dependent on the stimulation history, and by the adaptive threshold dependent on the spiking history. However, the proposed model does not consider detailed biochemical path- ways downstream of odorant-receptor binding that also play a role in adaptive processes, since a comprehensive picture of the olfactory transduction does not emerge yet and since it is notoriously difficult to fit parameters of detailed biophysical models from limited experimental data. In such cases, even slight differences in initial parameter settings can lead to highly disparate results [52,53]. 3. Discussion Hence, our model could be useful for investigating the possibility of latency coding in olfactory information processing [46,47] and the role of spike-timing- dependent plasticity in olfaction [12,48,49]. Consequently, the proposed model can be applied to simulate a network of heterogeneous ORNs in order to investigate how ORN populations process olfactory information in the moth. 4.1.1. Receptor activation Receptor activation by the pheromone (2.1)–(2.3) is described by the following reaction-rate equations: Sliding adjustment of odour response threshold and kin- etics has several molecular actors, such as ion channels, second messengers and ORs. ORs make non-selective cation channels, which are permeable also for Ca2+. First, adaptation in Drosophila OR-expressing ORNs is mediated by the Ca2+ influx during odour responses [54] and Ca2+-dependent channels may also serve for odour adaptation as in vertebrate ORNs [55]. Second, G-protein signalling cascades can both increase or decrease the ORN sensitivity [56,57]. Finally, ORs also adjust their sensitivity according to previous odour detections [58,59]. Insect ORs are formed by an odour-specific OrX protein and an odorant co-receptor, Orco, which plays a central role in both downregulating and upregulating the ORN sensitivity. In moth pheromone- sensitive ORNs, Orco was proposed to function as a pace- maker channel, controlling the kinetics of the pheromone responses [60]. 4.1. Model of an olfactory receptor neuron 4.1. Model of an olfactory receptor neuron 4.1. Model of an olfactory receptor neuron Here, we provide the details of the proposed neuron model. y p Here, we provide the details of the proposed neuron mo 4.1.2. Spike generation g The membrane voltage V(t) of an ORN is described by the fol- lowing equation: Electrophysiology. Insects were immobilized with the head protruding. One antenna was fixed with adhesive tape on a small support and a tungsten electrode (TW5-6; Science Pro- ducts, Hofheim, Germany) was inserted at the base of a long pheromone-responding sensillum trichodeum located on an antennal branch. The reference electrode was inserted in the antennal stem. The electrical signal was amplified (×1000) and band-pass filtered (10 Hz to 5 kHz) with an ELC-03X (NPI elec- tronic, Tamm, Germany), and sampled at 10 kHz by a 16-bit acquisition board (NI-9215; National Inst., Nanterre, France) under Labview (National Inst.). One sensillum was recorded per insect. Cm dV dt ¼ gL(V EL) gR(t)(V ER), (4:13) (4:13) where Cm is the cell capacitance, gL is the leak conductance, γ is the unit receptor conductance, R(t) is the concentration of acti- vated receptor, and EL and ER are the reversal potentials of the leak and the receptor currents, respectively (parameter values shown in table 2). The model neuron generates a spike when the voltage V(t) reaches the spike threshold θ(t), and, then, the voltage is instan- taneously reset to a value Vreset. We consider two descriptions for the threshold. In the first description, the threshold is constant, θ(t) = θ0. This description is equivalent to the standard LIF model [13,32]. In the second description, the spike threshold is modulated by previous spikes and is formally described as follows [33,35,36]. p Stimulation. ORNs were stimulated with the major A. ipsilon sex pheromone, (Z)-7-dodecenyl acetate (Z7-12:Ac). Pheromone was diluted in decadic steps in hexane and applied to a filter paper introduced in a Pasteur pipette. The antenna was constantly super- fused by a humidified and charcoal-filtered air stream (70 l · h−1). Air puffs (10 l · h−1) were delivered through a calibrated capillary (ref. 11762313; Fisher Scientific, France) positioned 1 mm from the antenna and containing the odorant-loaded filter paper (10 × 2 mm). An electrovalve (LHDA-1233215-H; Lee Company, France) was controlled by custom-made Labview programs read- ing sequences generated by Matlab scripts. The time resolution of the sequence was 1 ms. The characteristic response time of the valves, i.e. the time to switch from open to closed or closed to open, was less than 5 ms. The durations of the pheromone puffs and pauses were randomized. 4.2. Experiments Insects. Experiments were performed with laboratory-reared 4–5- day-old (sexually mature) adult male Agrotis ipsilon fed 20% sucrose solution ad libitum [62]. Pupae were sexed, and males and females were kept separately at 22°C under an inversed light–dark cycle (16–18 h light–dark photoperiod). The model parameters are listed in table 1. The model parameters are listed in table 1. 4.1.1. Receptor activation One or a combination of mechanisms of modulation of ORN sensitivity may contribute to expand the dynamic range of olfactory detection and thus allow the dL dt ¼ kiLair nk1LnR þ nk1RL k3LN þ k3NL, (4:1) dR dt ¼ k1LnR þ k1RL, (4:2) dRL dt ¼ k1LnR (k1 þ k2)RL þ k2R, (4:3) dR dt ¼ k2RL k2R, (4:4) dN dt ¼ k3LN þ (k3 þ k4)NL (4:5) dNL dt ¼ k3LN (k3 þ k4)NL, (4:6) dL dt ¼ kiLair nk1LnR þ nk1RL k3LN þ k3NL, (4:1) dR dt ¼ k1LnR þ k1RL, (4:2) dRL dt ¼ k1LnR (k1 þ k2)RL þ k2R, (4:3) dR dt ¼ k2RL k2R, (4:4) dN dt ¼ k3LN þ (k3 þ k4)NL (4:5) dNL dt ¼ k3LN (k3 þ k4)NL, (4:6) (4:6) and where ki, k1, k−1, k2, k−2, k3, k−3 and k4 are the rate constants, Lair and L are the odorant concentrations in the air and in the sensil- lum lymph, respectively, R, RL and R are the concentrations of the receptors in the free, receptor–ligand complexed and acti- vated states, respectively, N and NL are the deactivating enzyme concentrations in the free and complexed states, where ki, k1, k−1, k2, k−2, k3, k−3 and k4 are the rate constants, Lair and L are the odorant concentrations in the air and in the sensil- lum lymph, respectively, R, RL and R* are the concentrations of the receptors in the free, receptor–ligand complexed and acti- vated states, respectively, N and NL are the deactivating enzyme concentrations in the free and complexed states, homogen. model heterogen. in g heterogen. in (D, t) heterogen. in (g, D, t) 0 0.2 0.4 0.6 coefﬁcient of determination (median ± inter-quartile range) 0 0.2 0.4 0.6 0.8 1.0 1.2 0 0.5 1.0 1.5 2.0 D (mV) t (s) (b) (a) Figure 5. Heterogeneity in ORN model parameters. (a) Prediction performance of the model with all parameters fixed (homogeneous model) and three models with heterogeneous parameters (heterogeneity in γ, heterogeneity in (Δ, τ) and heterogeneity in (γ, Δ, τ)). (b) Scatter plot of the threshold parameters (Δ and τ) adjusted to individual neurons. The red dot represents the parameters fitted to the average ORN response (table 2). (Online version in colour.) homogen. model heterogen. in g heterogen. in (D, t) heterogen. (2) If the voltage reaches the threshold at time tsp, V(tsp) ≥θ(tsp), the threshold increases by a step Δ/τ, therefore (2) If the voltage reaches the threshold at time tsp, V(tsp) ≥θ(tsp), the threshold increases by a step Δ/τ, therefore respectively. The total amounts of receptors Rtot and the deacti- vating enzyme Ntot do not change over time. Using RL ¼ Rtot R R (4:7) and NL ¼ Ntot N, (4:8) u(tþ sp) ¼ u(t sp) þ D=t, (4:16) (4:16) and where Δ represents the strength of adaptation due to a single spike. the system of equations (4.1)–(4.6) can be reduced to Equations (4.9)–(4.12), (4.13), (4.14) and (4.16) were solved numerically using the forward Euler integration method with a time step of 0.01 ms. The initial conditions were R(0) = Rtot, N(0) = Ntot, V(0) = EL and θ(0) = θ0, that is, all of the receptors and the degrading enzymes were in the free state, the voltage was at the resting value and the threshold was at the asymptotic level. The simulation code was written in R [61]. dL dt ¼ kiLair n(k1Ln þ k1)R nk1R (k3L þ k3)N þ nk1Rtot þ k3Ntot, (4:9) dR dt ¼ (k1Ln þ k1)R k1R þ k1Rtot, (4:10) dR dt ¼ k2R (k2 þ k2)R þ k2Rtot (4:11) and dN dt ¼ (k3L þ k3 þ k4)N þ (k3 þ k4)Ntot: (4:12) 4.1.1. Receptor activation in (g, D, t) 0 0.2 0.4 0.6 coefﬁcient of determination (median ± inter-quartile range) (a) 0 0.2 0.4 0.6 0.8 1.0 1.2 0 0.5 1.0 1.5 2.0 D (mV) t (s) (b) royalsocietypublishing.org/journal/rsif J. R. Soc. Interface 16: 20190246 7 Figure 5. Heterogeneity in ORN model parameters. (a) Prediction performance of the model with all parameters fixed (homogeneous model) and three models with heterogeneous parameters (heterogeneity in γ, heterogeneity in (Δ, τ) and heterogeneity in (γ, Δ, τ)). (b) Scatter plot of the threshold parameters (Δ and τ) adjusted to individual neurons. The red dot represents the parameters fitted to the average ORN response (table 2). (Online version in colour.) (2) If the voltage reaches the threshold at time tsp, V(tsp) ≥θ(tsp), the threshold increases by a step Δ/τ, therefore 4.1.2. Spike generation Time was divided into bins of a fixed duration (50 or 100 ms). In each bin, the probability of the (1) When the neuron does not generate spikes, the threshold θ(t) decays exponentially to its asymptotic level θ0, (1) When the neuron does not generate spikes, the threshold θ(t) decays exponentially to its asymptotic level θ0, t du dt ¼ (u u0): (4:14) (4:14) This implies that u(t) ¼ u0 þ h u(tþ f ) u0 i exp t t f t , for t f t, (4:15) where tf is the time of the last spike and t+ represents the limit from above. stimulation 0 50 100 firing rate (Hz) cell 1 observed model 0 50 100 firing rate (Hz) cell 2 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 0 50 100 time (s) firing rate (Hz) mean trajectory 1st–3rd quartile (b) (a) Figure 6. Fit of the model with an adaptive spike threshold to individual ORN responses. (a) Top: Time course of the pheromone stimulus. The stimulus was switching between ON and OFF states. In the ON state, the pheromone dose was 100 pg. Bottom: Firing rate time courses of two neurons (cells 1 and 2) obtained from experiments (black) and those of the model with individually tuned threshold parameters (red). (b) The distribution of firing rates of the model neurons whose threshold parameters were derived from 84 ORNs. The dark blue line represents the mean trajectory and the light blue area represents the range between the first and the third quartile. The individual trajectories vary only in the amplitude of the fluctuations, not in the temporal pattern. (Online version in colour.) stimulation 0 50 100 firing rate (Hz) cell 1 observed model 0 50 100 firing rate (Hz) cell 2 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 (a) Figure 6. Fit of the model with an adaptive spike threshold to individual ORN responses. (a) Top: Time course of the pheromone stimulus. The stimulus was switching between ON and OFF states. In the ON state, the pheromone dose was 100 pg. 4.1.2. Spike generation Bottom: Firing rate time courses of two neurons (cells 1 and 2) obtained from experiments (black) and those of the model with individually tuned threshold parameters (red). (b) The distribution of firing rates of the model neurons whose threshold parameters were derived from 84 ORNs. The dark blue line represents the mean trajectory and the light blue area represents the range between the first and the third quartile. The individual trajectories vary only in the amplitude of the fluctuations, not in the temporal pattern. (Online version in colour.) valve being open was 0.5. Unique sequences of puffs and pauses were generated for each ORN. The dose of pheromone was constant throughout one recording session. where fd(t\|Lair) is the average firing rate for the experimental data, fm(t\|Lair) is the firing rate of the model and the summation was conducted across all concentrations of Lair. The minimization was performed using the Nelder–Mead algorithm [65]. In total, recordings of 84 moth ORNs were obtained: 41 recordings with a 50 ms minimum puff/pause duration, 43 recordings with a 100 ms minimum puff/pause duration. Each combination of pheromone dose and minimum puff duration was tested on six or more ORNs. The first 100 s of each recording was discarded because the ORN activity was not stationary. Subsequently, we fitted threshold parameters (Δ and τ) to the recording from each neuron. These parameters were tuned by minimizing the integrated square error in the 10 s training period e2 ind ¼ ð (fd(t) fm(t))2 dt, (4:18) (4:18) where fd(t) is the firing rate of the recorded neuron and fm(t) is the firing rate of the model neuron. The model simulation was initiated 1 s before the start of the training period to reduce the influence of the initial conditions. Finally, the model performance was evalu- ated by the coefficient of determination in the subsequent 10 s prediction period. 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The firing rate of the model neuron was obtained similarly by assuming a 0.5 s stimulation with the odorant concentration Lair equal to 0.1, 1, 10 and 100 pM that corresponds to the pheromone doses 1 pg, 10 pg, 100 pg, 1000 pg, respectively. The firing rate of the model was also calculated by convolving the spike train with a Gaussian kernel function (standard deviation 0.03 s). R2 ¼ 1 Ð (fd(t) fm(t))2 dt Ð (fd(t) hfdi)2 dt , (4:19) (4:19) where 〈fd〉is the average firing rate of the experimental data. Data accessibility. Data and R code are available from GitHub at: https:// github.com/MarieLevakova/Adaptive-integrate-and-fire-model.git. Competing interests. We declare we have no competing interests. Data accessibility. Data and R code are available from GitHub at: https:// github.com/MarieLevakova/Adaptive-integrate-and-fire-model.git. Competing interests. We declare we have no competing interests. Funding. This work was supported by the Institute of Physiology RVO:67985823, by the Czech Science Foundation project no. 17-06943S, by Agence Nationale de la Recherche grant ANR15-CE02-010-01 ‘Odorscape’, by Mobility Project between France and the Czech Republic through grant no. 7AMB17FR059, by JSPS KAKENHI grant nos. JP17H03279, JP18K11560 and JP19H01133, by JST ACT-I grant no. JPMJPR16UC, and by the Okawa Foundation for Information and Tele- communications. We are also grateful for the open collaborative research and MOU grant from the National Institute of Informatics. The parameters n, γ, τ, Δ were tuned by minimizing the integrated square error e2 ave ¼ X Lair ð (fd(tjLair) fm(tjLair))2 dt, (4:17) (4:17) Acknowledgements. We thank P. Lansky for helpful discussions and criti- cal reading of the manuscript. References 2010 Equilibrium and response properties of the integrate-and-fire neuron in discrete time. Front. Comput. Neurosci. 3, 29. (doi:10.3389/neuro.10.029. 2009) 39. 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https://openalex.org/W4234171703	https://bmcmedethics.biomedcentral.com/track/pdf/10.1186/s12910-020-00554-7	English	null	Just Say “No”: Can Dentists Refuse Care on the Basis of Finances? A Survey Using an Ethical Vignette in an Iranian Dental School	Research Square (Research Square)	2,019	cc-by	3,516	© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Decision making when patients ask a dentist for fee reduction is a real ethical dilemma at dental set- tings. The aim of this study was to evaluate how dental students and tutors think about their position for, or against fee reduction at dental offices. Method: It was a questionnaire-based survey, which examined the ethical attitudes of students and tutors of an Iranian Dental School. The questionnaire included a vignette about an ethical dilemma at a dental office. Different ethical approaches, i.e. duty-based, virtue-oriented and consequentialist arguments, for or against fee reduction at dental office were suggested. Respondents were asked to rank those ethical options.Data was entered and analyzed in SPSS 16.0. Result: 121 dental students and thirty-six faculty members (dental specialists) participated in this study. It revealed that a majority of dental students and tutors (68%) are in favor of charging patients less, when facing an imagined request at dental office, using either virtue-oriented (54%) or consequentialist (14%) argument for fee reduction. The difference between rankings of four options was statistically significant, while no statistically significant difference exists neither between male and female respondents, nor students and tutors. Conclusion: This case study provides a basis for fruitful discussions in ethics courses for dental students. Our study suggests that financial issues should be considered as a part of ethical training within the dental student’s curriculum. Keywords: Ethics, Dental Education, Financial Management, Questionnaire Just say “no”: Can dentists refuse care on the basis of finances? A survey using an ethical vignette in an Iranian Dental School Ali Kazemian1* , Mahsa Fayyazi2 and Shahrzad Shafiee2 Ali Kazemian1* , Mahsa Fayyazi2 and Shahrzad Shafiee2 Introduction with lower social-economic status to see a dentist may reduce the single most important impediment to regular dental care [6]. For both patients who need dental care, and for the dentists who treat them, financial issues are among the main questions that arise for both parties. Several sur- veys have shown that the high cost of treatment, and the financial limitations faced by many patients are the main barriers to accessing dental care [1–3]. These barriers inevitably have negative effects on oral health outcomes [4, 5]. Policies that promote opportunities for patients Furthermore, the financial discussions that must take place, affect the dentist-patient relationship, and are a routine challenge at dental settings. Offices that have an efficient, thoughtful, and context specific process for talking to their patients about the cost of treatment and the method of payment have higher financial profits, for having a straightforward initial discussion relieves uncer- tainty for both dentists and patients [7, 8]. Many of the treatment plans are rejected simply because the patient cannot afford to pay either at the time, or even over a period of weeks [9]. Because each dentist must confront Correspondence: kazemiana@mums.ac.ir 1 Department of Community Oral Health, School of Dentistry, Mashhad University of Medical Sciences, Vakilabad Blvd, P.O. Box 984, Mashhad, Iran Full list of author information is available at the end of the article Kazemian et al. BMC Med Ethics (2020) 21:109 https://doi.org/10.1186/s12910-020-00554-7 Kazemian et al. BMC Med Ethics (2020) 21:109 https://doi.org/10.1186/s12910-020-00554-7 Correspondence: kazemiana@mums.ac.ir 1 Department of Community Oral Health, School of Dentistry, Mashhad University of Medical Sciences, Vakilabad Blvd, P.O. Box 984, Mashhad, Iran Full list of author information is available at the end of the article Kazemian et al. BMC Med Ethics (2020) 21:109 Kazemian et al. BMC Med Ethics (2020) 21:109 Kazemian et al. BMC Med Ethics (2020) 21:109 Page 2 of 4 know how his or her colleagues behave in such circum- stances, and how they think about this problem. the choice to offer fee reduction or refuse care where the request is made, we argue that it is a significant ethi- cal and practical challenge as each decides, for example, how to react to the request from disadvantaged patients for reduction in their treatment fees. This situation is more likely to happen in oral health systems where out- of-pocket payment is dominant, but also is encountered when eligibility for exemptions or reduced prices, known as disease-specific responses, are adjudicated. Financial inaccessibility and inability to pay are typical system con- straints [10].i Four approaches are then taken by the colleagues of Dr. E. They include: A Consequentialist argument against fee reduction— Dr. A. believes if a dentist began to accept requests for fee reduction, it would gradually raise other patient’s expectations, which may result in unfavora- ble consequences. B Virtue-oriented argument for fee reduction—Dr. B. considers maintaining the virtuous character of the dentist as the most important response to the dilemma. A good dentist could not remain indiffer- ent about the unaffordable tariffs for treatments for his or her patient, and could never deny care. “On average, do graduates benefitting from state tui- tion subsidies charge their patients less?” [11] This is one of the questions Bartolami raised in his reflection on den- tal graduates’ sense of gratitude for the public’s generos- ity. While charging patients less could be seen as a moral obligation of dentists, especially those who benefitted from public funds, or who have a practice with a sig- nificant economic advantages and generally well-paying patients, this obligation might be understood in other, but equally ethical ways. C Duty-based argument against fee reduction— According to Dr. C. making fee reduction is never listed as a dentist’s duty. We have to be careful to adhere to our obligations, not necessarily more. We are not charity. Dentists tend to state that they choose their profes- sion based on a desire to behave altruistically [12], which seems to be supported by prosocial attitude of dental stu- dents [11]. Focusing on professional issues, such as social and economic situations, which are related to patients’ non-medical problems, could be an effective way for dis- cussing ethical issues of dental practice and promoting ethical sensitivity of dental students within the teaching of professional conduct. In this study, we studied how dental students and tutors thought about their position for, or against fee reduction at dental offices and the way they put forward their arguments to justify their position. D Consequentialist argument for fee reduction—Dr. D. believes rejecting patients’ request to make a reduc- tion would damage our reputation and would be harmful for our profession in long-term. Respondents were asked to rate these four approaches according to their own opinion and intuition in such a way that the first rank is the most favorable option for them. Data was entered and analyzed in SPSS 16.0. Statistical analysis of the ordinal data was done using non-paramet- ric tests including Friedman and Mann–Whitney U tests. Resulth The questionnaire was filled out by 157 respondents, including 121 sixth-year dental students and thirty-six tutors of Mashhad Dental School. Ninety-four respond- ents (59.9%) were female.h This study was a questionnaire-based survey, which examined the ethical attitudes of students and tutors of Mashhad Dental School, in Mashhad, Iran. The question- naire was distributed in the Medical Ethics course to two sequential groups of six-year dental students in July and December 2017. Additionally, it was sent via email to the tutors of the School of Dentistry, Mashhad Univer- sity of Medical Sciences, in Mashhad, Iran. Those who did not respond in a week received a reminder. While all the students at the classes completed the question- naire, the response rate of the tutors to our online survey was 40.0%. The study was approved by the Institutional Review Board of Mashhad Dental School.h The second option, which accounted for the virtue-ori- ented argument for fee reduction, was chosen as the first best option by 54 percent of respondents. The option D, accounted for the consequentialist argument against fee reduction, was selected as the least favored option by 47 percent of respondents. Figure 1 shows the descriptive report of proportion of rankings for each option.hf The difference between rankings of four options was statistically significant (p value < 0.001). The most favora- ble option, i.e. virtue-oriented argument for fee reduc- tion, had the mean rank of 1.85. It followed sequentially by duty-based argument against, consequentialist argu- ment for, and consequentialist argument against fee reduction. Table 1 displays the mean rank of four options. The questionnaire included a vignette about an ethical dilemma at a dental office (Additional file 1).The vignette describes a meeting of five dentists. The host, Dr. E. raises an issue regarding his difficulty in decision making when patients ask him or her for fee reduction. Dr. E. wants to Kazemian et al. BMC Med Ethics (2020) 21:109 Page 3 of 4 Fig. 1 Descriptive ranking of the suggested approaches Fig. 1 Descriptive ranking of the suggested approaches This approach scored lowest among the options. Seventy- two percent of respondents regarded the consequential- ist arguments, either for or against fee reduction, as the least justifiable response. There was no significant differ- ence, neither between male and female respondents, nor between faculty members and students. Resulth We suggest that this may be a sign that there is convergent ethical intui- tion of the participants. Table 1 Mean ranks of the options of the vignette Order Option Mean Rank 1 B—virtue-oriented argument for fee reduction 1.85 2 C—duty-based argument against fee reduction 2.32 3 D—consequentialist argument for fee reduction 2.74 4 A—consequentialist argument against fee reduc- tion 3.09 Table 1 Mean ranks of the options of the vignette Similar arguments about other ethical and profes- sional dilemmas arise in previous studies. For example, the same consequentialist approach at option D seems to be taken by Ayn, et al., who puts emphasis on improv- ing communication skills in dentistry education [7], or by Gosden, et al., stating that clinical behavior of patients is affected by the method of payment [14]. The other conse- quentialist argument, put forward in the option A, is also in line with some suggested cost control strategies [15]. No statistically significant difference exists neither between male and female respondents (p value = 0.60), nor students and tutors (p value = 0.22). Discussionh This study examined the ethical response and moral jus- tification of dental students and faculty members when confronted with controversial issue in dentistry—fee reduction. It revealed that the majority of dental stu- dents and tutors in the second biggest city of Iran believe that accepting patients’ request for a fee reduction is the ethically preferable behavior. Almost 68 percent of par- ticipants selected either a virtue-oriented (54%) or a con- sequentialist (14%) argument for fee reduction as their best option.i Yet the majority of our students and tutors acted in accordance with virtue, rejecting consequentialist argu- ments. The virtue-oriented approach has also been the mainstream attitude of many medical ethicists and is taken by many who discussed ethical aspects of den- tal practice [8, 16, 17]. However, it could be reasonably argued that there is a constant interplay between vir- tue, moral duty and consequentialist doctrine during fee reduction. Further investigation could be pursued to establish criteria for determining which patients will receive free or reduced-fee care. It would help individual dentists contribute in helping to overcome barriers in access to dental care. Virtues define how we behave when no one else is watching, and generally are described as the qualities or excellences of character to which humans ought to aspire. As described by Aristotle, and extrapolated by medieval philosophers [13], it refers to a trait or character that is deemed to be morally good and thus is valued as a foun- dation of a principled life. Virtue considerations include reflection on how each act shapes the moral agent. In our survey a virtue ethics approach was the most favored one among the options offered. The consequentialist approach judges an action based on its consequences. The main limitation of our study was related to using the plain short vignette, which for the sake of encourag- ing compliance, did not involve the complex factors inter- playing in the real clinical settings. There are a bunch of different patient- and dentist-factors that could affect the communication between patient and dentist during Kazemian et al. BMC Med Ethics (2020) 21:109 Kazemian et al. BMC Med Ethics (2020) 21:109 Kazemian et al. BMC Med Ethics (2020) 21:109 Page 4 of 4 fee reduction. Issues such as the alternative treatment planning by dentists, bargaining attitude of patients, and the extent of the fee reduction could be incorporated in developing the vignettes in next studies. Conclusion y 3. kenwilo D, . A difference-in-differences analysis of the effect of free dental check-ups in Scotland. Soc Sci Med. 2013;83:10–8. We argue that using this case study provides a basis for useful discussions in ethics courses for dental students. It is also suggested that such financial issues be considered as a part of the professionalization and ethical training within the dental student’s curriculum. 4. Yuen HK, Shotwell MS, Magruder KM, et al. Factors associated with oral problems among adults with spinal cord injury. J Spinal Cord Med. 2009;32:408–15. 5. Locker D, Maggirias J, Quiñonez C. Income, dental insurance coverage and financial barriers to dental care among Canadian adults. J Public Health Dent. 2011;71(4):327–34. 6. Grembowski D, Andersen RM, Chen M-S. A public health model of the dental care process. Med Care Rev. 1989;46:439–96. References 1. Aguirre-Zero O, Westerhold C, Goldsworthy R, et al. Identification of barriers and beliefs influencing engagement by adult and teen Mexican-Americans in oral health behaviors. Community Dent Health. 2016;33(1):44–7. 1. Aguirre-Zero O, Westerhold C, Goldsworthy R, et al. Identification of barriers and beliefs influencing engagement by adult and teen Mexican-Americans in oral health behaviors. Community Dent Health. 2016;33(1):44–7. 2. Thompson B, Cooney P, Lawrence H, et al. The potential oral health impact of cost barriers to dental care: findings from a Canadian population-based study. BMC Oral Health. 2014;25(14):78. Funding There has been no significant financial support for this work that could have influenced its outcome. 16. Nayak PP, Raju VK, Nanjundaiah V, et al. Ethical conventions: a study on dental practitioner’s knowledge and practice of ethics in their line of work in Bangalore, India. J Clin Diagn Res. 2016;10(8):ZC84-7. 16. Nayak PP, Raju VK, Nanjundaiah V, et al. Ethical conventions: a study on dental practitioner’s knowledge and practice of ethics in their line of work in Bangalore, India. J Clin Diagn Res. 2016;10(8):ZC84-7. Received: 5 June 2020 Accepted: 26 October 2020 Received: 5 June 2020 Accepted: 26 October 2020 Competing interests Another limitation of our study was the potential dif- ference in training about ethical schools of thought for different populations of students. There was also no com- parison between senior and junior students. The authors declare that there is no conflict of interest regarding the publication of this article. Abbreviation IRB: Institutional Review Board. 11. Bertolami CN, Berne R. Access to care: leveraging dental education. J Dent Educ. 2014;78(12):1481–8. 11. Bertolami CN, Berne R. Access to care: leveraging dental education. J Dent Educ. 2014;78(12):1481–8. Discussionh Considering such complexities would be helpful for simulating real life scenarios and making participants careful ethical deliberation. established educational setting was regarded as consent to participate. The par- ticipants had an option to opt-out providing they did not consent to participate in the study. Acknowledgements 12. Galizzi MM, Tammi T, Godager G, et al. Provider altruism in health econom- ics. Washington: National Institute for Health and Welfare; 2015. 12. Galizzi MM, Tammi T, Godager G, et al. Provider altruism in health econom- ics. Washington: National Institute for Health and Welfare; 2015. This paper considerably benefitted from comments made by Professor Laurie Zoloth. 13. DuBois JM, Kraus EM, Mikulec AA, Cruz-Flores S, Bakanas E. A hum- ble task: restoring virtue in an age of conflicted interests. Acad Med. 2013;88(7):924–8. Availability of data and materials 17. Broida JH, Lerner M. Knowledge of patient’s method of payment by physi- cians in a group practice. Public Health Rep. 1975;90(2):113–8. The dataset supporting the results of this article are available from the cor- responding author on reasonable request. The dataset supporting the results of this article are available from the cor- responding author on reasonable request. Consent for publication We declare that the manuscript has been read and approved by all authors, that the requirements for authorship have been met, and that we believe that the manuscript represents honest work. Authors’ contributions A.K.: Designed and performed experiments, supervised the research and co-wrote the paper. M.F. and S.Sh.: Performed analyses, co-wrote the paper. All authors read and approved the final manuscript. 14. Gosden T, Forland F, Kristiansen IS, et al. Capitation, salary, fee-for-service and mixed systems of payment: effects on the behaviour of primary care physi- cians. Cochrane Database Syst Rev. 2000;3:CD002215. 14. Gosden T, Forland F, Kristiansen IS, et al. Capitation, salary, fee-for-service and mixed systems of payment: effects on the behaviour of primary care physi- cians. Cochrane Database Syst Rev. 2000;3:CD002215. y 15. Barer ML, Evans RG, Labelle RJ. Fee controls as cost control: tales from the frozen North. Milbank Q. 1988;66(1):1–64. 15. Barer ML, Evans RG, Labelle RJ. Fee controls as cost control: tales from the frozen North. Milbank Q. 1988;66(1):1–64. Supplementary information 7. Ayn C, Robinson L, Nason A, et al. Determining recommendations for improvement of communication skills training in dental education: a scop- ing review. J Dent Educ. 2017;81(4):479–88. Supplementary information accompanies this paper at https://doi. org/10.1186/s12910-020-00554-7. Additional file 1. The vignette used as the questionnaire of the study. Abbreviation IRB: Institutional Review Board. Acknowledgements This paper considerably benefitted from comments made by Professor Laurie Zoloth. Authors’ contributions A.K.: Designed and performed experiments, supervised the research and co-wrote the paper. M.F. and S.Sh.: Performed analyses, co-wrote the paper. All authors read and approved the final manuscript. Funding There has been no significant financial support for this work that could have influenced its outcome. Availability of data and materials The dataset supporting the results of this article are available from the cor- responding author on reasonable request. Ethics approval and consent to participate The study was considered to be exempt from review by the IRB of Mashhad Dental School, since the research was conducted in established educational set- ting. It complies with national guidelines. The letter of exempt from IRB review is submitted separately. According to national regulations and the IRB of Mashhad Dental School, the participation of students in the study that was conducted in Supplementary information accompanies this paper at https://doi. org/10.1186/s12910-020-00554-7. Supplementary information accompanies this paper at https://doi. org/10.1186/s12910-020-00554-7. 8. Nash DA. Ethics in dentistry: review and critique of Principles of Ethics and Code of Professional Conduct. J Am Dent Assoc. 1984;109(4):597–603. Additional file 1. The vignette used as the questionnaire of the study. 9. Larijani B, Aramesh K. Fee splitting among general practitioner sectional study in Iran. Arch Iran Med. 2016;19(12):861–5. y 10. Travis P, Bennett S, Haines A, et al. Overcoming health-systems constraints to achieve the Millennium Development Goals. Lancet. 2004;364(9437):900–6. y 10. Travis P, Bennett S, Haines A, et al. Overcoming health-systems constraints to achieve the Millennium Development Goals. Lancet. 2004;364(9437):900–6. Abbreviation IRB: Institutional Review Board. Author details 1 1 Department of Community Oral Health, School of Dentistry, Mashhad Uni- versity of Medical Sciences, Vakilabad Blvd, P.O. Box 984, Mashhad, Iran. 2 School of Dentistry, Mashhad University of Medical Sciences, Mashhad, Iran. Creating imaginative ways of thinking about common, yet important dilemmas, such as fee reduction is impor- tant. More study will need to be done to explore whether reducing the fee for dental work will reduce the value of the work among patients; whether asking for a fee reduc- tion will become a habit; whether dentists will begin to expect this from their patients, and how any these ques- tions, by focusing on the materialistic view of the profes- sion of dentistry diminishes its nobility as a calling. Publisher’s Note The study was considered to be exempt from review by the IRB of Mashhad Dental School, since the research was conducted in established educational set- ting. It complies with national guidelines. The letter of exempt from IRB review is submitted separately. According to national regulations and the IRB of Mashhad Dental School, the participation of students in the study that was conducted in Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W3009411980	https://europepmc.org/articles/pmc7253201?pdf=render	English	null	Relative Efficiencies of Simple and Complex Substitution Models in Estimating Divergence Times in Phylogenomics	Molecular biology and evolution	2,020	cc-by	13,281	Relative Efficiencies of Simple and Complex Substitution Models in Estimating Divergence Times in Phylogenomics Qiqing Tao ,1,2 Jose Barba-Montoya,1,2 Louise A. Huuki,1 Mary Kathleen Durnan,1,2 a Sudhir Kumar ,1,2,3 1Institute for Genomics and Evolutionary Medicine, Temple University, Philadelphia, PA 2Department of Biology, Temple University, Philadelphia, PA 3Center for Excellence in Genome Medicine and Research, King Abdulaziz University, Jeddah, Saudi Arabia Corresponding author: E-mail: s.kumar@temple.edu. Associate editor: Jeffrey Thorne Qiqing Tao ,1,2 Jose Barba-Montoya,1,2 Louise A. Huuki,1 Mary Kathleen Durnan,1,2 and Sudhir Kumar ,1,2,3 1Institute for Genomics and Evolutionary Medicine, Temple University, Philadelphia, PA 2Department of Biology, Temple University, Philadelphia, PA 3Center for Excellence in Genome Medicine and Research, King Abdulaziz University, Jeddah, Saudi Arabia Corresponding author: E-mail: s.kumar@temple.edu. Associate editor: Jeffrey Thorne Tao ,1,2 Jose Barba-Montoya,1,2 Louise A. Huuki,1 Mary Kathleen Durnan,1,2 and Kumar ,1,2,3 Center for Excellence in Genome Medicine and Research, King Abdulaziz University, Jeddah, Saudi Arabia Corresponding author: E-mail: s.kumar@temple.edu. Associate editor: Jeffrey Thorne Abstract The conventional wisdom in molecular evolution is to apply parameter-rich models of nucleotide and amino acid substitutions for estimating divergence times. However, the actual extent of the difference between time estimates produced by highly complex models compared with those from simple models is yet to be quantiﬁed for contemporary data sets that frequently contain sequences from many species and genes. In a reanalysis of many large multispecies alignments from diverse groups of taxa, we found that the use of the simplest models can produce divergence time estimates and credibility intervals similar to those obtained from the complex models applied in the original studies. This result is surprising because the use of simple models underestimates sequence divergence for all the data sets analyzed. We found three fundamental reasons for the observed robustness of time estimates to model complexity in many practical data sets. First, the estimates of branch lengths and node-to-tip distances under the simplest model show an approximately linear relationship with those produced by using the most complex models applied on data sets with many sequences. Second, relaxed clock methods automatically adjust rates on branches that experience considerable underestimation of sequence divergences, resulting in time estimates that are similar to those from complex models. And, third, the inclusion of even a few good calibrations in an analysis can reduce the difference in time estimates from simple and complex models. The robustness of time estimates to model complexity in these empirical data analyses is encouraging, because all phylogenomics studies use statistical models that are oversimpliﬁed descriptions of actual evolutionary substitution processes. Key words: molecular dating, substitution model, relaxed clock, phylogenomics, RelTime. Do the time estimates from the most complex models differ significantly from those produced using a relatively sim- ple model when analyzing large data sets that contain many species, genes, and calibrations? The answer to this question is of high practical significance. If it is affirmative, time estimates may be vulnerable to both incorrect model specification and the overall limitations of current substitution models. On the other hand, if time estimates are generally similar for simple and complex substitution models, then currently available models could be deemed sufficient for molecular dating, be- cause even the most complex model is a simplification of actual evolutionary processes that are much more complex due to differences in regional mutation patterns and spatial and temporal selective pressures (Arenas 2015; Abadi et al. 2019). The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Open A The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access Mol. Biol. Evol. 37(6):1819–1831 doi:10.1093/molbev/msaa049 Advance Access publication March 2, 2020 1819 A Plant Data Set Analysis We first present results from a reanalysis of a large-sequence alignment containing 103 plant species (Morris et al. 2018) (“Plants,” table 1). Pairwise sequence distances ranged from 0.01 to 3.18 (median ¼ 0.83) nucleotide substitutions per site (fig. 1a). The GTR model with rate variability among sites (þC) was used in the original analyses and is presented here as the complex model (Morris et al. 2018). For comparison, we ana- lyzed this data set using the JC model as our simple model. The difference in the maximum likelihood (ML) values for the GTRþ C and JC models was very large (DlnL ¼ 14757.9) and highly significant (P< 1016). Conventionally, the JC model would be a poor choice for molecular dating analysis. Indeed, the use of the JC model led to the underestimation of pairwise evolutionary distances by as much as 73%, and there was severe substitution saturation resulting in a classic curvi- linear trend (fig. 1a, the gray area). We focus on models of the general time-reversible (GTR) class because they are employed in all current empirical dat- ing analyses. Although many new substitution models have been proposed to relax the assumptions of stationarity, re- versibility, and homogeneity of base substitution patterns, as well as to incorporate complex structural constraints and epistatic fitness landscapes, these advanced models are not yet available in popular phylogenetic software packages due to their implicit complexity and onerous computational requirements (Jayaswal et al. 2014; Arenas 2015; Arenas et al. 2015; Usmanova et al. 2015). Therefore, in our reanalysis, we used the models selected as the best-fit models in the source phylogenomic studies as “complex models” (table 1). The “simple models” used were the Jukes–Cantor (JC) model for nucleotide substitutions (Jukes and Cantor 1969) and the Poisson model for amino acid substitutions (Nei and Kumar 2000). Both assume that all substitution types are equally likely at a given site, an assumption that is always violated in reality. To ensure the most powerful contrast, we applied these simplest models without partitioning the sequence alignment by genes, genomic features (e.g., codon positions), or sets of positions with similar substitution patterns (see Materials and Methods). Surprisingly, Bayesian estimates of divergence times obtained using the JC model were very similar to those obtained using the GTR þ C model when the same se- quence alignment, topology, and calibrations were used (fig. 1b). Introduction Article Models of nucleotide and amino acid substitution are of fundamental importance in molecular phylogenetic analy- ses (Nei and Kumar 2000; Yang 2006; Arenas 2015). Many sophisticated substitution models have been developed, and the complexity of models developed for use in phylo- genomic studies continues to increase (Arenas 2015; Abadi et al. 2019). Indeed, complex models can provide a more complete description of nucleotide and amino acid substi- tution processes that involve transition/transversion rate differences, biased base compositions, inequality of evolu- tionary rates among sites, and substitution pattern hetero- geneity among genomic regions and sequence partitions (Sumner et al. 2012; Arenas 2015). Because the difference between the estimated and actual numbers of substitutions grows quickly and nonlinearly over time (Nei and Kumar 2000; Yang 2006), researchers often select the most com- plex model available to improve the accuracy of divergence time estimates (Arbogast et al. 2002; Sumner et al. 2012; Arenas 2015; Abadi et al. 2019). Although no studies have directly examined the impact of model complexity on time estimation in phylogenomic inves- tigations, there have been reports that simple substitution models often perform similarly to or only slightly worse than complex substitution models in some types of phyloge- netic inferences (Tamura et al. 2004; Yoshida and Nei 2016; The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access Mol. Biol. Evol. 37(6):1819–1831 doi:10.1093/molbev/msaa049 Advance Access publication March 2, 2020 1819 The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Open Open Access Biol. Evol. 37(6):1819–1831 doi:10.1093/molbev/msaa049 Advance Access publication March 2, 2020 1819 MBE Tao et al. . doi:10.1093/molbev/msaa049 Table 1. Detailed Information about the Empirical Data Analyzed. Detailed Information about the Empirical Data Analyzed Table 1. Detailed Information about the Empirical Data Analyzed. Spielman and Kosakovsky Pond 2018; Abadi et al. 2019; Dornburg et al 2019; Spielman 2019) However in molecular practice in phylogenomic studies to use the phylogeny reli- ably inferred by using a sophisticated tree-building method as a fixed topology for molecular dating (e.g., Li et al. 2019; Oliveros et al. 2019). In addition, the use of the same pub- lished tree topologies ensured consistent placement of cali- brations and eliminated the confounding effects of using alternative phylogenies and calibrations. g p ) dating, it is intuitively assumed that underestimation of se- quence divergences caused by the use of simple models will result in significantly distorted time estimates. Here, we used diverse large data sets to test the conventional wisdom that the use of simple models will result in poor estimates of divergence times (table 1). To detect potential benefits of- fered by the use of complex models, we compared Bayesian estimates of divergence times obtained when using extremely simple models with those obtained when using complex models that contained many biological attributes and param- eters (table 1). Introduction Taxonomic Group Data Type Sequence Count Sequence Lengtha Tree Depthb Ratec Substitution Modeld Partition Count Rate Modele Calibration Count DlnLf References Mammals (A) M 274 7,370 185 2.28 HKY1C 1 ABR 36 96,320.5 dos Reis et al. (2012) Mammals (B) A 162 11,010 187 1.53 JTT1C 26 ARB 64 110,854.0 Meredith et al. (2011) Birds N 51 722,202 102 0.21 HKY1C NA ABR 18 3,794.2 Jarvis et al. (2014) Fishes N 118 85,363 140 0.51 HKY1C 4 IBR 13 13,048.1 Alfaro et al. (2018) Metazoans A 54 38,577 757 0.41 LG1C1F 1 IBR 33 68,405.1 dos Reis et al. (2015) Spiders A 43 55,447 561 0.54 WAG1C 1 IBR 8 37,088.5 Bond et al. (2014) Plants N 103 856,439 798 0.87 GTR1C 1 IBR 37 14,757.9 Morris et al. (2018) Eukaryotes & prokaryotes A 102 9,874 4,511 0.29 LG1C 29 IBR 11 183,666.0 Betts et al. (2018) N, nuclear DNA; M, mitochondrial DNA; A, amino acid; ABR, autocorrelated branch rate model; IBR, independent branch rate model. aSequence length is the number of sites in the alignment used in the original study. We randomly selected 10,000 sites in our analyses if sequences are longer than 10,000 sites. For the “Plants” data set, we used an alignment of 2,217 sites. bTree depth is the root age obtained from the original published study. Times are in millions of years. cThe evolutionary rate is calculated by dividing the sum of the maximum likelihood (ML) branch lengths obtained using the original complex model over the sum of published times elapsed. The unit is substitutions per site per billion years. dThe substitution model used for the majority of partitions in the original study for estimating divergence times. eThe branch rate model used in the original study for estimating divergence times. fDifference between the log-likelihoods of the original complex model and the simple model. A positive value means that the complex model provides a better ﬁt to the data. prokaryotes N, nuclear DNA; M, mitochondrial DNA; A, amino acid; ABR, autocorrelated branch rate model; IBR, independent branch rate model. aSequence length is the number of sites in the alignment used in the original study. We randomly selected 10,000 sites in our analyses if sequences are longer than 10,000 sites. For the “Plants” data set, we used an alignment of 2,217 sites. bTree depth is the root age obtained from the original published study. Introduction Times are in millions of years. cThe evolutionary rate is calculated by dividing the sum of the maximum likelihood (ML) branch lengths obtained using the original complex model over the sum of published times elapsed. The unit is substitutions per site per billion years. dThe substitution model used for the majority of partitions in the original study for estimating divergence times. eThe branch rate model used in the original study for estimating divergence times. fDifference between the log-likelihoods of the original complex model and the simple model. A positive value means that the complex model provides a better ﬁt to the data. A Plant Data Set Analysis (b) Similar divergence time estimates are produced by using the JC and GTR þ C models when all calibrations are used in Bayesian analyses. Times are in millions of years. Gray-dashed line represents 1:1 line. The slope and coefﬁcient of determination (R2) for the linear regression through the origin are shown. Arrows mark three nodes that showdifferent time estimates. (c) Relationship betweenthe complexity of models andthe slope of divergence timesinferred using theGTR þ C and other models. JC, K2, HKY, TN, andGTR represents Jukes–Cantor, Kimura 2-parameter, Hasegawa–Kishino–Yano, Tamura–Nei, and general time-reversible models, respectively. The number of model parameters is shown in the parentheses. Circles indicate whether a gamma distribution (þ C) for incorporating rate variation across sites is used (open circle) or is not used (closed circle) with the substitution model. The Bayesian method produces similar time estimates between the JC and GTR þ C models when (d) all internal calibrations are excluded, and (e) one internal calibration and a diffused root calibration are used. Times are in millions of years. (f) The RelTime method produces similar divergence times between the JC and GTR þ C models. Times are normalized to the sum of node ages. (g) Comparison of 95% Bayesian credibility intervals generated under the JC (dark red) and GTR þ C (cadet blue) models. Dots are point estimates of divergence times. Distributions of posterior time estimates for three nodes pointed in panel (b) are shown (inset). arrows in fig. 1b). However, these node times generated using the JC model fell within the 95% credibility intervals (CrIs) obtained using the GTR þ C model (fig. 1g). CrIs from the JC and GTR þ C models overlapped for every node (fig. 1g), suggesting that estimates of divergence times and CrIs from the simplest model will be as useful as those obtained via complex substitution models in downstream biological anal- yses and hypothesis testing. simple JC model. So, we compared times obtained using the GTRþ C and JC models after eliminating all the internal calibrations but retaining the original root calibration. The linear pattern persisted (slope ¼ 0.95, R2 ¼ 0.99, fig. 1d). We then tested the possibility that a well-constrained root calibration caused the observed linear relationship of dates from simple and complex models, even though the root cal- ibration was expected to only dictate the overall time span, rather than the patterns of individual node times. A Plant Data Set Analysis The Ba method produces similar time estimates between the JC and GTR þ C models when (d) all internal calibrations are excluded, and (e) one i calibration and a diffused root calibration are used. Times are in millions of years. (f) The RelTime method produces similar divergence between the JC and GTR þ C models. Times are normalized to the sum of node ages. (g) Comparison of 95% Bayesian credibility in generated under the JC (dark red) and GTR þ C (cadet blue) models. Dots are point estimates of divergence times. Distributions of posteri estimates for three nodes pointed in panel (b) are shown (inset) Pairwise distances Divergence mes (all calibraons) Divergence mes (no internal calibraons) Divergence mes (diﬀused root, one internal calibraon) RelTime (no calibraons) Slope = 0.97 R2 = 0.99 Slope = 0.95 R2 = 0.99 Slope = 0.95 R2 = 0.99 Slope = 0.99 R2 = 0.99 Saturaon Node 1 Nod Time Node 1 Node 2 Node 2 Time Node 2 Node 3 Node 3 Time ( sla v r e t ni ytilibid e r C Myr) JC JC JC JC GTR + Γ GTR + Γ GTR + Γ GTR + Γ GTR + Γ Nodes Without gamma With gamma (+ Γ) (a) (c) (d) (g) (b) (e) (f) GTR + Γ JC FIG. 1. Plant data analyses. (a) Severe underestimation of pairwise distances via the JC model. The gray-dashed line represents equality b time estimates, and the gray area represents the underestimation resulting from using the JC model. (b) Similar divergence time estima Pairwise distances Divergence mes (all calibraons) Divergence mes (no internal calibraons) Divergence mes (diﬀused root, one internal calibraon) RelTime (no calibraons) Slope = 0.97 R2 = 0.99 Slope = 0.95 R2 = 0.99 Slope = 0.95 R2 = 0.99 Slope = 0.99 R2 = 0.99 Saturaon Node 1 Node 2 Node 3 JC JC JC JC GTR + Γ GTR + Γ GTR + Γ GTR + Γ GTR + Γ Without gamma With gamma (+ Γ) (a) (c) (d) (g) (b) (e) (f) (g) Node 1 Nod Time Node 2 Node 2 Time Node 3 Time Nodes Γ Nodes FIG. 1. Plant data analyses. (a) Severe underestimation of pairwise distances via the JC model. The gray-dashed line represents equality between time estimates, and the gray area represents the underestimation resulting from using the JC model. A Plant Data Set Analysis This trend was also observed for divergence time estimates obtained via substitution models of intermediate complexity (fig. 1c). The linear regression slope between time estimates under the JC and GTR þ C models was 0.97, with low dispersion (R2 ¼ 0.99). Although time estimates gener- ated using the GTRþ C and JC models showed high overall similarity, a few node times showed local discrepancies (e.g., We also used the same tree topology as in the source publications in our comparisons, because it is a common 1820 MBE Dating with Simple versus Complex Models . doi:10.1093/molbev/msaa049 Pairwise distances Divergence mes (all calibraons) Divergence mes (no internal calibraons) Divergence mes (diﬀused root, one internal calibraon) RelTime (no calibraons) Slope = 0.97 R2 = 0.99 Slope = 0.95 R2 = 0.99 Slope = 0.95 R2 = 0.99 Slope = 0.99 R2 = 0.99 Saturaon Node 1 Nod Time Node 1 Node 2 Node 2 Time Node 2 Node 3 Node 3 Time ( sla v r e t ni ytilibid e r C Myr) JC JC JC JC GTR + Γ GTR + Γ GTR + Γ GTR + Γ GTR + Γ Nodes Without gamma With gamma (+ Γ) (a) (c) (d) (g) (b) (e) (f) GTR + Γ JC FIG. 1. Plant data analyses. (a) Severe underestimation of pairwise distances via the JC model. The gray-dashed line represents equality be time estimates, and the gray area represents the underestimation resulting from using the JC model. (b) Similar divergence time estima produced by using the JC and GTR þ C models when all calibrations are used in Bayesian analyses. Times are in millions of years. Gray-dash represents 1:1 line. The slope and coefﬁcient of determination (R2) for the linear regression through the origin are shown. Arrows mark three that showdifferent time estimates. (c) Relationship betweenthe complexity of models andthe slope of divergence timesinferred using theG C and other models. JC, K2, HKY, TN, andGTR represents Jukes–Cantor, Kimura 2-parameter, Hasegawa–Kishino–Yano, Tamura–Nei, and g time-reversible models, respectively. The number of model parameters is shown in the parentheses. Circles indicate whether a gamma distri (þ C) for incorporating rate variation across sites is used (open circle) or is not used (closed circle) with the substitution model. A Plant Data Set Analysis So, we reanalyzed the phylogeny, making the probability density dis- tribution of the root calibration diffused and adding a ran- domly selected internal calibration in the Bayesian analysis We hypothesized that the inclusion of 37 calibration points, and their associated probability densities, in the “Plants” data set constrained the node time estimates and eliminated the bias anticipated to be caused by the use of the 1821 MBE Tao et al. . doi:10.1093/molbev/msaa049 (a) Normalized pairwise distances (complex model) s e c n a tsid e si w ria p d e zila m r o N (simple model) 0.0 1.0 0.0 1.0 Mammals (A) Mammals (B) Spiders Eukaryotes & Prokaryotes Birds Fishes Metazoans Plants Average diﬀerence in pairwise distances (%) 0 20 40 60 Data set (b) FIG. 2. (a) Curvilinear relationships of pairwise distances. Pairwise distances are normalized to the maximum pairwise distance obtained using the complex model for a given empirical data set to enable comparisons across empirical data sets. The gray-dashed line repre- sents equality between distance estimates. (b) Average percent differ- ences between pairwise distances obtained using simple and complex models. The error bar shows 1 SD. Data sets for “Mammals (A),” “Mammals (B),” “Birds,” “Fishes,” “Metazoans,” “Spiders,” “Plants,” and “Eukaryotes & Prokaryotes” are from dos Reis et al. (2012), Meredith et al. (2011), Jarvis et al. (2014), Alfaro and Holder (2006), dos Reis et al. (2015), Bond et al. (2014), Morris et al. (2018), and Betts et al. (2018), respectively. (a) s e c n a tsid e si w ria p d e zila m r o N ( i l d l) (b (see Materials and Methods). The resulting divergence times from using the simple JC model were still very similar to those from the more complex GTR þ C model (slope ¼ 0.95, R2 ¼ 0.99, fig. 1e). Therefore, dates from simple and complex mod- els show good linear relationships in Bayesian analyses with even a few calibrations. The Complexity of the Substitution Model Has Limited Impact on Time Inference for Many Data Sets We examined the similarity of times estimated via simple and complex models for many other data sets, which contained small or large numbers of species (43–274), had varying evo- lutionary time depths (102–4,511 My), evolved with slow or fast rates (0.21–2.28 substitution per site per billion years), or employed small or large numbers of calibration points (8–64) (table 1). For all these data sets, the use of simple models underestimated pairwise evolutionary distances and showed curvilinear relationships with distances estimated using com- plex models (fig. 2). The curvilinear relationship was partic- ularly dramatic for more ancient divergences due to substitutional saturation (fig. 2a). FIG. 2. (a) Curvilinear relationships of pairwise distances. Pairwise distances are normalized to the maximum pairwise distance obtained using the complex model for a given empirical data set to enable comparisons across empirical data sets. The gray-dashed line repre- sents equality between distance estimates. (b) Average percent differ- ences between pairwise distances obtained using simple and complex models. The error bar shows 1 SD. Data sets for “Mammals (A),” “Mammals (B),” “Birds,” “Fishes,” “Metazoans,” “Spiders,” “Plants,” and “Eukaryotes & Prokaryotes” are from dos Reis et al. (2012), Meredith et al. (2011), Jarvis et al. (2014), Alfaro and Holder (2006), dos Reis et al. (2015), Bond et al. (2014), Morris et al. (2018), and Betts et al. (2018), respectively. Despite the curvilinear relationship of pairwise distances estimated using simple and complex models, their divergence time estimates showed strong linear relationships for nuclear nucleotide, mitochondrial nucleotide, and amino acid se- quence alignments (fig. 3a). The linear regression slopes ranged from 0.92 to 1.01 (fig. 3a). The linear relationships persisted in all analyses even when internal calibrations were removed (only the root calibration was kept); slopes ranged from 0.96 to 1.11 (fig. 3b). The use of a random internal calibration and a diffused root calibration produced a similar range of slopes (0.94–1.01) (supplementary fig. S1, Supplementary Material online). As with the “Plants” data set, RelTime dating analysis also produced excellent linear relationships of node times es- timated via simple and complex models (supplementary fig. S2, Supplementary Material online). “Spiders” data set, divergence estimates from the “Eukaryotes & Prokaryotes” data set showed larger differences between simple and complex models when all the internal calibrations were removed. A Plant Data Set Analysis (a) (a) s e c n a tsid e si w ria p d e zila m r o N (simple model) 0.0 1.0 0.0 1.0 Mammals (A) Mammals (B) Spiders Eukaryotes & Prokaryotes Birds Fishes Metazoans Plants To eliminate the effect of calibrations in mediating the similarity of times obtained using simple and complex mod- els, we estimated divergence times using the RelTime method in which no calibrations and no branch rate model are re- quired. For this data set, divergence times under the JC and GTR þ C models were very similar (fig. 1f), and the confi- dence intervals also showed broad overlap. This result sug- gested that the specification of a branch rate model and calibrations used in the Bayesian analysis for the data set analyzed are unlikely to explain the high similarity in diver- gence times between the JC and GTR þ C model. Overall, substitution model complexity appears to have a limited im- pact on divergence time estimates for the “Plants” data set. Normalized pairwise distances (complex model) Normalized pairwise distances (complex model) 0.0 1.0 Average diﬀerence in pairwise distances (%) 0 20 40 60 Data set (b) Normalized pairwise distances (complex model) (b) Dating with Simple versus Complex Models . doi:10.1093/molbev/msaa049 Divergence mes (complex model) Mammals (A) Mammals (B) Birds Fishes Metazoans Spiders Plants Eukaryotes & Prokaryotes Slope = 0.96 R2 = 0.99 Slope = 0.97 R2 = 1.00 Slope= 0.98 R2 = 1.00 Slope = 0.98 R2 = 1.00 Slope = 0.96 R2 = 0.99 Slope = 1.02 R2 = 0.99 Slope = 0.95 R2 = 0.99 Slope = 1.11 R2 = 0.97 Divergence mes (complex model) )le d o m elp m is( s e m it e c n e g r e vi D (b) No internal calibraons FIG. 3. Comparisons of Bayesian divergence times obtained via simple and complex models. Similar divergence time estimates are produced w (a) all calibrations are used and (b) all internal calibrations are excluded in Bayesian analyses. The time unit is millions of years. The gray-dashed marks equal time estimates. The slope and coefﬁcient of determination (R2) for the linear regression through the origin are shown. Sou publications for data sets are listed in ﬁgure 2. Mammals (A) Mammals (B) Slope = 0.96 R2 = 0.99 Slope = 0.97 R2 = 1.00 )le d o m elp m is( s e (b) No internal calibraons (b) No internal calibraons Birds Slope= 0.98 R2 = 1.00 Fishes Slope = 0.98 R2 = 1.00 Metazoans Slope = 0.96 R2 = 0.99 e m it e c n e g r e vi D Plants Slope = 0.95 R2 = 0.99 Plants Eukaryotes & Prokaryotes Slope = 0.95 R2 = 0.99 Slope = 1.11 R2 = 0.97 es (complex model) Spiders Slope = 1.02 R2 = 0.99 Divergence mes (complex model) FIG. 3. Comparisons of Bayesian divergence times obtained via simple and complex models. Similar divergence time estimates are produced when (a) all calibrations are used and (b) all internal calibrations are excluded in Bayesian analyses. The time unit is millions of years. The gray-dashed line marks equal time estimates. The slope and coefﬁcient of determination (R2) for the linear regression through the origin are shown. Source publications for data sets are listed in ﬁgure 2. (88–90.6 My) models rejected the evolutionary model in which the last common ancestor of placental mammals appeared after the Cretaceous–Paleogene (K–Pg) event, con- sistent with conclusions in some previous studies (Hedges et al. 1996; Kumar and Hedges 1998; dos Reis et al. 2012). observation and to establish its generality. Dating with Simple versus Complex Models . doi:10.1093/molbev/msaa049 We hypothesized that the branch length estimates obtained via simple and complex models were linearly related (even though not 1:1) for the data sets analyzed. This linearity would result in similar relative branch lengths, and thus divergence times. This hy- pothesis arose from our observation that the RelTime method produced similar time estimates under simple and complex models (supplementary fig. S2, Supplementary Material online). RelTime is based on a relative rate frame- work in which the relationship between branch lengths and time estimates is established algebraically (Tamura et al. 2012, The Complexity of the Substitution Model Has However, Bayesian CrIs obtained using simple models often contained the point estimates obtained using complex models, and vice versa (fig. 4 and supplementary fig. S3, Supplementary Material online). CrIs overlapped for >97% of the nodes across all the analyses between simple and complex models (fig. 4), and the over- lapping region was significant for the majority of the nodes (supplementary fig. S4, Supplementary Material online). Therefore, simple and complex models seem to offer similar statistical power for biological hypothesis testing. For exam- ple, in the analysis of “Mammals (A)” data set, divergence estimates from both simple (88.6–91.3 My) and complex The mean of the relative absolute difference between times estimated by simple and complex models was small (<6.2%), but some node times deviated considerably from the 1:1 linear trend for these two models. For example, three nodes in the “Spiders” data set showed 15–23% difference between time estimates obtained from simple and complex models (fig. 3a). Interestingly, these differences disappeared (<1%) when all the internal calibrations were excluded (fig. 3b). In contrast to the patterns observed for the 1822 MBE Dating with Simple versus Complex Models . doi:10.1093/molbev/msaa049 Dating with Simple versus Complex Models . doi:10.1093/molbev/msaa049 Mammals (A) Mammals (B) Birds Fishes Metazoans Spiders Plants Eukaryotes & Prokaryotes Slope = 0.99 R2 = 1.00 Slope = 1.00 R2 = 1.00 Slope= 0.99 R2 = 0.99 Slope = 1.01 R2 = 1.00 Slope = 1.00 R2 = 1.00 Slope = 0.92 R2 = 0.97 Slope = 0.97 R2 = 0.99 Slope = 0.98 R2 = 0.99 Divergence mes (complex model) )le d o m elp m is( s e m it e c n e g r e vi D (a) All calibraons 88–90.6 My) models rejected the evolutionary model in observation and to establish its generality. We hypothesiz Mammals (A) Mammals (B) Birds Fishes Metazoans Spiders Plants Eukaryotes & Prokaryotes Slope = 0.99 R2 = 1.00 Slope = 1.00 R2 = 1.00 Slope= 0.99 R2 = 0.99 Slope = 1.01 R2 = 1.00 Slope = 1.00 R2 = 1.00 Slope = 0.92 R2 = 0.97 Slope = 0.97 R2 = 0.99 Slope = 0.98 R2 = 0.99 Divergence mes (complex model) )le d o m elp m is( s e m it e c n e g r e vi D Mammals (A) Mammals (B) Birds Fishes Metazoans Spiders Plants Eukaryotes & Prokaryotes Slope = 0.96 R2 = 0.99 Slope = 0.97 R2 = 1.00 Slope= 0.98 R2 = 1.00 Slope = 0.98 R2 = 1.00 Slope = 0.96 R2 = 0.99 Slope = 1.02 R2 = 0.99 Slope = 0.95 R2 = 0.99 Slope = 1.11 R2 = 0.97 Divergence mes (complex model) )le d o m elp m is( s e m it e c n e g r e vi D (a) All calibraons (b) No internal calibraons FIG. 3. Comparisons of Bayesian divergence times obtained via simple and complex models. Similar divergence time estimates are produced wh a) all calibrations are used and (b) all internal calibrations are excluded in Bayesian analyses. The time unit is millions of years. The gray-dashed l marks equal time estimates. The slope and coefﬁcient of determination (R2) for the linear regression through the origin are shown. Sou publications for data sets are listed in ﬁgure 2. Fundamental Factors Enabling Robustness of Inferred Times to Model Complexity Comparisons of Bayesian credibility intervals (CrIs) inferred by using simple and complex models. Shown are the proportions of node times for which simple and complex models produce overlapping CrIs (solid), CrIs produced by complex models contain point time estimates produced by simple models (open), and CrIs produced by simple models include point time estimates produced by complex models (hatch) when (a) all calibrations are used and (b) no internal calibrations are used. See also supplementary ﬁgure S3, Supplementary Material online, for more detailed information about (a). However, this adjustment may not be possible for phylog- enies with few species or sparse taxon sampling within some clades, producing isolated long branches and causing time estimates from simple models to differ from complex models. For example, many very long branches may appear in an unbalanced phylogeny (supplementary fig. S6a, Supplementary Material online). Interestingly, we found that the slope of times estimated using simple and complex models was close to 1 when the rates were similar to those observed in empirical data sets (1 category in supplemen- tary fig. S6b and c, Supplementary Material online), which is consistent with results from our empirical analyses. For data sets where evolutionary rates are extremely fast (e.g., 10), or sequence divergences are very large (see Materials and Methods), we found that the use of a single, very shallow calibration often produced overly young estimates (blue dots, 2–10 in supplementary fig. S6c, Supplementary Material online). It is because the underestimation of lengths for long branches is more severe when using a simple model, and the rate adjustment offered by relaxed clock methods is not as effective. However, the use of good calibrations at deeper nodes alleviated the discrepancy and produced less biased time estimates for the simple model (green and pink dots, 2–10 in supplementary fig. S6c, Supplementary Material online). Because reliable alignment of sequences showing large divergences is very challenging (Edwards et al. 1995), researchers will (and should) generally be apprehensive of using highly divergent genes (e.g., 10). 2018). Divergence times are the ratios of the linear combina- tions of branch length estimates (and of node-to-tip distan- ces), which allows us to predict that the relative branch lengths between simple and complex models will be similar. Indeed, linear models (through the origin) described the relationship between branch lengths from simple and com- plex models for all empirical data sets we examined (fig. 5). Fundamental Factors Enabling Robustness of Inferred Times to Model Complexity Elucidation of causal factors mediating the similarity of time estimates produced under simple and complex substitution models is needed to reveal the fundamental basis of our 1823 MBE Tao et al. . doi:10.1093/molbev/msaa049 Nodes (%) (a) All calibraons (b) No internal calibraons CrIs from simple and complex models overlap CrIs from complex models contain mes from simple models CrIs from simple models contain mes from complex models FIG. 4. Comparisons of Bayesian credibility intervals (CrIs) inferred by using simple and complex models. Shown are the proportions of node times for which simple and complex models produce overlapping CrIs (solid), CrIs produced by complex models contain point time estimates produced by simple models (open), and CrIs produced by simple models include point time estimates produced by complex models (hatch) when (a) all calibrations are used and (b) no internal calibrations are used. See also supplementary ﬁgure S3, Supplementary Material online, for more detailed information about (a). Nodes (%) (a) All calibraons (b) No internal calibraons CrIs from simple and complex models overlap CrIs from complex models contain mes from simple models CrIs from simple models contain mes from complex models FIG. 4. Comparisons of Bayesian credibility intervals (CrIs) inferred by using simple and complex models. Shown are the proportions of node times or which simple and complex models produce overlapping CrIs (solid), CrIs produced by complex models contain point time estimates produced by simple models (open), and CrIs produced by simple models include point time estimates produced by complex models (hatch) when (a) all alibrations are used and (b) no internal calibrations are used. See also supplementary ﬁgure S3, Supplementary Material online, for more detailed nformation about (a). CrIs from simple and complex models overlap CrIs from complex models contain mes from simple models CrIs from simple models contain mes from complex models FIG. 4. Comparisons of Bayesian credibility intervals (CrIs) inferred by using simple and complex models. Shown are the proportions of node times for which simple and complex models produce overlapping CrIs (solid), CrIs produced by complex models contain point time estimates produced by simple models (open), and CrIs produced by simple models include point time estimates produced by complex models (hatch) when (a) all calibrations are used and (b) no internal calibrations are used. See also supplementary ﬁgure S3, Supplementary Material online, for more detailed information about (a). FIG. 4. Fundamental Factors Enabling Robustness of Inferred Times to Model Complexity This trend is dramatically different from that observed for pairwise distances, where a curvilinear relationship was ob- served in every case (fig. 2). Linear regression slopes of branch lengths are all <1 because simple models underestimate se- quence divergences. However, branch lengths were uniformly underestimated via simple models for the empirical data sets tested, resulting in similar relative branch lengths between simple and complex model scenarios (fig. 5) as well as similar relative node-to-tip distances (supplementary fig. S5, Supplementary Material online). Because divergence times are a function of the ratios of the linear combinations of branch lengths, and the ratios of node-to-tip distances, they become comparable under simple and complex models. p p p Despite the linear patterns described earlier, we expected a greater magnitude of underestimation for longer branches and deeper sequence divergences when using simple models (see fig. 2a), which could distort time estimates. This effect is indeed observed for the longest branches in most of the data sets analyzed, as they show significant deviation from the linear trend (fig. 5). We confirmed this pattern in a systematic anal- ysis of short, long, and intermediate branch lengths (fig. 6a); and shallow, deep, and intermediate node-to-tip distances (fig. 6c). However, divergence time estimates on deep nodes and branch times on long branches were often not very dif- ferent between simple and complex models (fig. 6b and d). These results suggest that relaxed clock methods automatically adjust evolutionary rates within a phylogeny to produce robust time estimates for the empirical data sets analyzed. Increasing Numbers of Sequences Makes Time Estimates from Simple and Complex Models More Similar Increasing Numbers of Sequences Makes Time Estimates from Simple and Complex Models More Similar We investigated the effect of the number of sequences in a data set on the similarity of estimated dates from simple and complex models, because a data set with sparse taxon 1824 MBE Dating with Simple versus Complex Models . doi:10.1093/molbev/msaa049 Mammals (A) Mammals (B) Birds Fishes Metazoans Spiders Plants Eukaryotes & Prokaryotes Slope = 0.57 R2 = 0.95 Slope = 0.94 R2 = 0.99 Slope= 0.67 R2 = 1.00 Slope = 0.58 R2 = 0.98 Slope = 0.59 R2 = 0.95 Slope = 0.66 R2 = 0.96 Slope = 0.68 R2 = 0.96 Slope = 0.65 R2 = 0.94 Branch lengths (complex model) )le d o m elp m is( s h t g n el h c n a r B FIG. 5. Relationship between ML branch lengths obtained by using simple and complex models. The gray-dashed line represents the best-ﬁt linear regression through the origin. The slope and coefﬁcient of determination (R2) are shown. Birds Slope= 0.67 R2 = 1.00 Spiders Slope = 0.66 R2 = 0.96 B h l th Plants Slope = 0.68 R2 = 0.96 ( l d l) Plants Eukaryotes & Prokaryotes Slope = 0.68 R2 = 0.96 Slope = 0.65 R2 = 0.94 s (complex model) Branch lengths (complex model) FIG. 5. Relationship between ML branch lengths obtained by using simple and complex models. The gray-dashed line represents the best-ﬁt linear regression through the origin. The slope and coefﬁcient of determination (R2) are shown. (a) (b) Slopes of branch lengths Slopes of branch mes Short branches Intermediate branches Long branches (c) (d) Slopes of node-to-p distances Slopes of divergence mes Shallow divergence Intermediate divergence Deep divergence (a) (b) Slopes of branch lengths Slopes of branch mes Short branches Intermediate branches Long branches FIG. 6. Linear regression slopes of (a) branch lengths and (b) branch times estimated using simple and complex models for short (solid), intermediate (open), and long (hatch) branches. Linear regression slopes of (c) node-to-tip distances and (d) divergence times estimated using simple and complex models for shallow (solid), intermediate (open), and deep (hatch) locations in the phylogeny. A slope of 1 represents equality between estimates from simple and complex models, which is marked by a gray-dashed line. Smaller slope values represent more considerable underestimation when using simple models. Discussion Divergence times estimated using simple and complex sub- stitution models were remarkably similar across a range of phylogenomic data sets, a pattern that we observed for both Bayesian and RelTime methods. More surprisingly, similar estimates were observed even when a small number of cali- brations (e.g., only the root calibration) was used in Bayesian and non-Bayesian analyses. We found that three fundamental reasons can explain the observed robustness of time esti- mates to model complexity in many phylogenomic data sets. First, the estimates of branch lengths and node-to-tip distances under simple and complex models show strong linear relationships, especially for data sets with many sequen- ces. Second, relaxed clock methods are able to automatically adjust evolutionary rates on branches that experience under- estimation of sequence divergences, resulting in time esti- mates that are similar to those from complex models. Third, the use of calibrations (especially deep calibrations) further narrows differences in time estimates from simple and complex models, as calibrations often offer strong con- straints on node ages. Number of sequences FIG. 7. Relationships between the number of sequences and the dis- persion around the linear trends of branch lengths from simple and complex models. Boxes show the variation of the coefﬁcient of de- termination of the linear regression (through the origin, R2) between branch lengths obtained using the GTR þ C and the corresponding models (JC, K2, HKY, TN, and GTR) based on an analysis of 20 repli- cates. A narrower box indicates a more stable linear relationship of branch lengths. Model abbreviations are as those in ﬁgure 1. The number of model parameters is shown in the parentheses. sampling may show a greater discrepancy. We evaluated the linearity of the relationship of branch lengths generated via simple and complex models for data subsets with increasingly larger numbers of sequences subsampled from the “Plants” data set. We found that when the number of sequences sampled was small (e.g., 10), the linear relationships between branch lengths were weak for some subsets (lower linear co- efficient, R2) and strong for others (higher R2) (fig. 7). With an increasing number of sequences, the dispersion of R2 values became smaller, resulting in a more robust linear relationship between branch lengths (fig. 7). In the empirical data sets analyzed, the dispersion of R2 became very small for data sets that contain as few as 40 sequences. Discussion Therefore, a stronger linear relationship of branch length estimates between simple and complex models exists for data sets with many species, resulting in similar divergence time estimates. Our results may explain the inconsistent patterns reported in previous studies (Yang 1996; Schenk and Hufford 2010), which analyzed rela- tively small data sets (5–25 sequences). Although divergence time estimates derived via simple and complex models show remarkable overall similarity, use of different substitution models may produce very different point estimates for some nodes (e.g., >20% difference for node 3 in fig. 1g). It is mainly because the distributions of posterior times under simple and complex models differ for those nodes. Even for phylogenomic data sets, divergence time estimates are generally associated with large estimation errors due to the variance of branch length estimates, the degree of evolutionary rate heterogeneity, and the uncer- tainty related to clock calibration (Zhu et al. 2015; Tao et al. 2020). Therefore, CrIs, which represent the uncertainty surrounding divergence time estimates, are more useful than point estimates in biological hypothesis testing (Warnock et al. 2017). CrIs produced by simple and complex models largely overlapped (fig. 4 and supplementary fig. S4, Supplementary Material online), and the widths of CrIs around time estimates were also very similar when the same set of calibrations were used (fig. 1g and supplementary fig. S3, Supplementary Material online). Increasing Numbers of Sequences Makes Time Estimates from Simple and Complex Models More Similar (b) Short branches Intermediate branches Long branches Slopes of branch lengths (c) ( Slopes of node-to-p distances (d) s Slopes of divergence mes Shallow divergence Intermediate divergence Deep divergence (d) (c) Slopes of divergence mes Slopes of node-to-p distances FIG. 6. Linear regression slopes of (a) branch lengths and (b) branch times estimated using simple and complex models for short (solid), intermediate (open), and long (hatch) branches. Linear regression slopes of (c) node-to-tip distances and (d) divergence times estimated using simple and complex models for shallow (solid), intermediate (open), and deep (hatch) locations in the phylogeny. A slope of 1 represents equality between estimates from simple and complex models, which is marked by a gray-dashed line. Smaller slope values represent more considerable underestimation when using simple models. 1825 MBE Tao et al. . doi:10.1093/molbev/msaa049 Number of sequences Linear coeﬃcient R2 10 20 30 60 40 50 70 80 90 JC (1) K2 (2) HKY (5) TN (6) GTR (9) FIG. 7. Relationships between the number of sequences and the dis- persion around the linear trends of branch lengths from simple and complex models. Boxes show the variation of the coefﬁcient of de- termination of the linear regression (through the origin, R2) between branch lengths obtained using the GTR þ C and the corresponding models (JC, K2, HKY, TN, and GTR) based on an analysis of 20 repli- cates. A narrower box indicates a more stable linear relationship of branch lengths. Model abbreviations are as those in ﬁgure 1. The number of model parameters is shown in the parentheses. and non-GTR models showed strong linear relationships (fig. 8c and d). Simple and complex models generated com- parable time estimates and, hence, may provide equivalent statistical power for hypothesis testing within the range of evolutionary conditions observed in empirical data sets ana- lyzed for this study. Number of sequences Linear coeﬃcient R2 10 20 30 60 40 50 70 80 90 JC (1) K2 (2) HKY (5) TN (6) GTR (9) Effects of Irreversibility and Nonstationarity of Substitution Patterns 8. Relationships of RelTime divergence times estimated with branch lengths obtained using the JC model and models that are (a) non-time- reversible and (b) non-stationary for all nucleotide data sets. Gray solid lines represent 95% conﬁdence intervals. The sum of node ages was used to normalize divergence times and conﬁdence intervals. Relationships of branch lengths obtained using the JC model and models that are (c) non- time-reversible and (d) non-stationary. The gray-dashed line represents the best-ﬁt linear regression through the origin. The slope and coefﬁcient of determination (R2) for the linear regression are shown. FIG. 8. Relationships of RelTime divergence times estimated with branch lengths obtained using the JC model and models that are (a) non-time- reversible and (b) non-stationary for all nucleotide data sets. Gray solid lines represent 95% conﬁdence intervals. The sum of node ages was used to normalize divergence times and conﬁdence intervals. Relationships of branch lengths obtained using the JC model and models that are (c) non- time-reversible and (d) non-stationary. The gray-dashed line represents the best-ﬁt linear regression through the origin. The slope and coefﬁcient of determination (R2) for the linear regression are shown. Oliveros et al. 2019). In the future, we plan to investigate the impact of substitution model complexity on the joint inference of phylogeny and times. required when using an external mutation or substitution rate to set evolutionary distances to times because actual branch lengths are best estimated via a complex model. Therefore, complex models are needed if one is interested in estimating not only divergence times but also absolute and relative evolutionary rates. Complex models may also be re- quired when inferring the phylogeny and divergence times jointly, although use of a predetermined topology is a com- mon practice in phylogenomic studies (e.g., Li et al. 2019; required when using an external mutation or substitution rate to set evolutionary distances to times because actual branch lengths are best estimated via a complex model. Therefore, complex models are needed if one is interested in estimating not only divergence times but also absolute and relative evolutionary rates. Complex models may also be re- quired when inferring the phylogeny and divergence times jointly, although use of a predetermined topology is a com- mon practice in phylogenomic studies (e.g., Li et al. Effects of Irreversibility and Nonstationarity of Substitution Patterns Time mes (non-me-reversible model) Normalized RelTime mes (non-me-reversible model) s e m it d e zila m r o N (JC) Mammals (A) Slope = 0.99 R2 = 0.99 (b) s e m it d e zila m r o N (JC) Mammals (A) Birds Slope = 0.99 R2 = 0.99 Slope= 1.01 R2 = .0.98 (b) (b) Normalized RelTime mes (non-staonary model) s e m it d e zila m r o N (JC) Mammals (A) Birds Fishes Plants Slope = 0.99 R2 = 0.99 Slope= 1.01 R2 = .0.98 Slope = 1.00 R2 = 1.00 Slope = 1.02 R2 = 0.99 (b) mes (non-staonary model) Fishes Plants Slope = 1.00 R2 = 1.00 Slope = 1.02 R2 = 0.99 Plants Slope = 1.02 R2 = 0.99 Birds Slope= 1.01 R2 = .0.98 Fishes Slope = 1.00 R2 = 1.00 Normalized RelTime mes (non-staonary model) Normalized RelTime mes (non-staonary model) s h t g n el h c n a r B (JC) Mammals (A) Slope = 0.44 R2 = 0.91 (c) s h t g n el h c n a r B (JC) Mammals (A) Birds Slope = 0.44 R2 = 0.91 Slope= 0.68 R2 = 1.00 (c) ( y ) Branch lengths (non-me-reversible model) s h t g n el h c n a r B (JC) Mammals (A) Birds Fishes Plants Slope = 0.44 R2 = 0.91 Slope= 0.68 R2 = 1.00 Slope = 0.57 R2 = 0.98 Slope = 0.60 R2 = 0.90 (c) ibl d l) Fishes Plants Slope = 0.57 R2 = 0.98 Slope = 0.60 R2 = 0.90 (c) Fishes Slope = 0.57 R2 = 0.98 Birds Slope= 0.68 R2 = 1.00 Branch lengths (non-me-reversible model) s h t g n el h c n a r B (JC) Mammals (A) Slope = 0.56 R2 = 0.94 (d) g ( ) Branch lengths (non-staonary model) s h t g n el h c n a r B (JC) Mammals (A) Birds Fishes Plants Slope = 0.56 R2 = 0.94 Slope= 0.67 R2 = 1.00 Slope = 0.57 R2 = 0.98 Slope = 0.60 R2 = 0.92 (d) Branch lengths (n Birds Slope= 0.67 R2 = 1.00 non-staonary model) Fishes Plants Slope = 0.57 R2 = 0.98 Slope = 0.60 R2 = 0.92 (d) Plants Slope = 0.60 R2 = 0.92 Fishes Slope = 0.57 R2 = 0.98 Branch lengths (non-staonary model) FIG. Effects of Irreversibility and Nonstationarity of Substitution Patterns As mentioned earlier, our primary focus is on complex mod- els used in practical analyses, which are from the GTR class. However, it is important to consider whether a violation of the assumption of model stationarity and time-reversibility has biased time estimates significantly. Because non-GTR sub- stitution models are not available for use in the Bayesian dating software, we used the RelTime method to infer diver- gence times directly for phylogenies in which branch lengths were estimated under an unrestricted model (in which the time-reversibility in substitution models is not assumed) and a model in which the stationarity of the substitution patterns is not assumed (see Materials and Methods). We found that the JC model and non-GTR models produced similar diver- gence time estimates and confidence intervals (fig. 8a and b). It is because branch length estimates obtained using the JC Even though we found that simple models produced results comparable to those from complex models across many empirical data sets, we anticipate that there will be situations in which complex models are best suited for mo- lecular dating. This includes the analysis of data sets in which the number of sequences is small, substitution patterns have shifted substantially in some groups, sequences divergences are large, or taxon sampling in some clades is so sparse as to create many long branches. Complex models are also 1826 MBE Dating with Simple versus Complex Models . Effects of Irreversibility and Nonstationarity of Substitution Patterns doi:10.1093/molbev/msaa049 Normalized RelTime mes ) C J( s e m it d e zila m r o N Mammals (A) Birds Slope = 0.98 R2 = 0.99 Slope= 1.01 R2 = 0.99 (a) Normalized RelTime mes (non-me-reversible model) ) C J( s e m it d e zila m r o N Mammals (A) Birds Fishes Plants Slope = 0.98 R2 = 0.99 Slope= 1.01 R2 = 0.99 Slope = 1.01 R2 = 1.00 Slope = 0.99 R2 = 0.99 (a) ) C J( s e m it d e zila m r o N Mammals (A) Slope = 0.98 R2 = 0.99 (a) s (non-me-reversible model) Fishes Slope = 1.01 R2 = 1.00 s (non-me-reversible model) Fishes Plants Slope = 1.01 R2 = 1.00 Slope = 0.99 R2 = 0.99 (a) ) Plants Slope = 0.99 R2 = 0.99 Normalized RelTime mes (non-me-reversible model) ) C J( s e m it d e zila m r o N Mammals (A) Birds Fishes Plants Slope = 0.98 R2 = 0.99 Slope= 1.01 R2 = 0.99 Slope = 1.01 R2 = 1.00 Slope = 0.99 R2 = 0.99 (a) Normalized RelTime mes (non-staonary model) s e m it d e zila m r o N (JC) Mammals (A) Birds Fishes Plants Slope = 0.99 R2 = 0.99 Slope= 1.01 R2 = .0.98 Slope = 1.00 R2 = 1.00 Slope = 1.02 R2 = 0.99 (b) Branch lengths (non-me-reversible model) s h t g n el h c n a r B (JC) Mammals (A) Birds Fishes Plants Slope = 0.44 R2 = 0.91 Slope= 0.68 R2 = 1.00 Slope = 0.57 R2 = 0.98 Slope = 0.60 R2 = 0.90 (c) Branch lengths (non-staonary model) s h t g n el h c n a r B (JC) Mammals (A) Birds Fishes Plants Slope = 0.56 R2 = 0.94 Slope= 0.67 R2 = 1.00 Slope = 0.57 R2 = 0.98 Slope = 0.60 R2 = 0.92 (d) FIG. 8. Relationships of RelTime divergence times estimated with branch lengths obtained using the JC model and models that are (a) non-time reversible and (b) non-stationary for all nucleotide data sets. Gray solid lines represent 95% conﬁdence intervals. The sum of node ages was used to normalize divergence times and conﬁdence intervals. Relationships of branch lengths obtained using the JC model and models that are (c) non time-reversible and (d) non-stationary. Effects of Irreversibility and Nonstationarity of Substitution Patterns The gray-dashed line represents the best-ﬁt linear regression through the origin. The slope and coefﬁcien of determination (R2) for the linear regression are shown. Effects of Irreversibility and Nonstationarity of Substitution Patterns 2019; A majority of phylogenomic analyses make a simplifying assumption that the same substitution model applies across all the sites in a concatenated data set or within each data partition. However, evolutionary dynamics and processes dif- fer regionally (and even positionally) in mutation patterns, sequence contexts, and selective pressures (Yang et al. 1994; 1827 MBE Tao et al. . doi:10.1093/molbev/msaa049 Yang and Swanson 2002; Shapiro et al. 2006; Kosakovsky Pond et al. 2008; Bordner and Mittelmann 2014; Jayaswal et al. 2014; Arenas 2015). Therefore, one may imagine that even a seemingly complex substitution model (e.g., GTR þ C) is relatively simple when compared with the actual reality. The observation that the simplest models produce time esti- mates similar to those obtained using much more complex models may be used to suggest that the current model com- plexity is appropriate for estimating divergence times. But, extensive analyses of simulated data are required to fully ex- plore the sufficiency of current substitution models and un- derstand whether the difference between simple and complex models can be interpreted as a problem with simple models, which is beyond the scope of this article and an exciting future direction of research. the approximate likelihood calculation. The substitution models used in the original studies were employed as the complex models (table 1). JC and Poisson models without the assumption of rate variation across sites under the gamma distribution were used as the simple model for nu- cleotide and amino acid sequences, respectively. For the “Plants” data, we also estimated divergence times using Kimura 2-parameter (K2) (Kimura 1980), Hasegawa– Kishino–Yano (HKY) (Hasegawa et al. 1985), and Tamura– Nei (TN) (Tamura and Nei 1993) models with and without a gamma parameter for accounting for rate variation across sites, and the GTR model without a gamma parameter. We used a single partition in all simple and complex model anal- yses, although multiple partitions might be used in the orig- inal studies. However, we used 29 partitions for the “Eukaryotes & Prokaryotes” data analysis since the original research (Betts et al. 2018) showed a strong influence of partitioning on time estimation. We used the same rate mod- els, prior settings (e.g., tree prior and overall rate prior), and calibration constraints and densities as published in the orig- inal studies for all analyses. Empirical Data Acquisition Empirical Data Acquisition We selected eight large-scale empirical data sets distributed across the tree of life. Species groups, data types, sequence lengths, sequence counts, calibration counts, branch rate model, the number of partitions, and the original substitution models used in the majority of partitions are summarized in table 1. These studies used complex substitution models (e.g., GTRþ C) along with one or multiple partitions, and none of the studies selected the JC or Poisson models as the best model for any partitions. All empirical data were analyzed initially in MCMCTree (Yang 2007) to estimate the divergence times, except the “Spiders” data that were analyzed in RelTime and then reanalyzed in MCMCTree by Mello et al. (2017). In all analyses, we used the published topologies. We obtained the published divergence times and CrIs from the original studies, except for “Mammals A” and “Eukaryotes & Prokaryotes” data sets. Because the original studies of these two data sets did not provide CrIs of Bayesian time estimates, we reproduced time- trees for these two data sets with the same settings as used in the original studies with MCMCTree (v4.9h). g p We first examined whether the use of a single partition and subsampled alignments would significantly impact the time estimates generated by the complex model analysis. Therefore, we compared the published times obtained using the full data sets and multiple partitions with times estimated using the subsampled alignments and a single partition. Concordant time estimates were found in all empirical data sets (supplementary fig. S7, Supplementary Material online). Thus, we considered the effect of data subsampling and par- titioning to be small for the empirical data sets analyzed. Our observations are consistent with studies showing that site subsampling has a limited impact on the accuracy and pre- cision of time estimates (dos Reis and Yang 2013; Zhu et al. 2015). Therefore, we used the divergence times, and CrIs obtained using the subsampled alignments, the original com- plex substitution model, and a single partition as the infer- ences from complex models when a reanalysis was needed. We then compared them with the results obtained using the subsampled alignments, simple models, and a single partition to eliminate any site-subsampling bias, and observed good linear relationships (fig. 3a). Effects of Irreversibility and Nonstationarity of Substitution Patterns Two independent runs were con- ducted to ensure convergence and that ESS values were >200 after removing 10% burn-in samples for each run. Empirical Data Acquisition We also found similar linear trends in direct comparisons between time estimates from simple models and published times for all data sets (results not shown), indicating that the length of sequences has lim- ited impact on the robustness of time estimates to substitu- tion model complexity. We also computed the mean of relative absolute error between times estimated under simple 1 P n t t Because analyses of long sequences can require long com- putational times, mainly in ML branch length calculations, we used the original alignments when they were shorter than 10,000 sites (see table 1). Otherwise, we randomly selected 10,000 sites from the original alignments (10K data sets) for all phylogenetic analyses. The exception was the “Plants” data, for which the original study (Morris et al. 2018) showed that similar time estimates were obtained by using the full align- ment (856,439 sites) and a trimmed subsample with high site coverage (2,217 sites). We, therefore, used the 2,217 sites data for the “Plants” data set. The subsampled alignments were used in all following analyses of simple and complex models. All empirical data sets are available at https://doi.org/10.6084/ m9.figshare.11873874, last accessed March 9, 2020. and complex models for each data set by using 1 n P i tsitci tci %, Relationship between Bayesian Time Estimates Using Simple and Complex Models where tc_i and ts_i is the node time estimated under the complex and simple model for node i, respectively, and n is the number of nodes. Although we only used MCMCTree for inferring Bayesian divergence times, we expect results to be similar when using other Bayesian dating software, for We estimated divergence times in MCMCTree (v4.9h) using simple models with topologies and calibrations from the orig- inal studies. All MCMCTree analyses were conducted using 1828 MBE Dating with Simple versus Complex Models . doi:10.1093/molbev/msaa049 Testing the Relationship of Branch Lengths and Node- to-Tip Distances Estimated Using Simple and Complex Models example, BEAST2 (Bouckaert et al. 2014), because previous studies have shown that different Bayesian dating software packages tend to generate similar time estimates when priors and calibration constraints are consistent (Warnock et al. 2012, 2015). For each data set, we estimated ML branch lengths using both simple and complex models and the published topology in MEGA X. A single partition was used in all ML analyses. Empirical Data Acquisition We compared the branch lengths estimated using simple and complex models to obtain the relationship. We then calcu- lated the node-to-tip distances using the resulting ML tree. For each node, the node-to-tip distance is the sum of the lengths of all paths from this node to all descendent tips divided by the total number of descendant tips. Influence of Calibrations on the Relationship of Bayesian Times between Simple and Complex Models We re-estimated divergence times in MCMCTree (v4.9h) us- ing both simple and complex models without any internal calibrations to examine whether the use of many calibrations constrained the final time estimates and concealed bias caused by the use of simple models. In this case, only the age of root was constrained. A single partition and the sub- sampled alignments were used in the analysis. All other priors, including the root calibration, were the same as those used in the original studies. We also investigated whether the use of root calibration caused a linear relationship between time estimates under simple and complex models. We performed Bayesian analysis for each data set using a single internal cal- ibration that was randomly selected from all the internal calibrations used in the original study and only the maximum constraint (tmax) for the root. The use of only a maximum constraint for the root resulted in a diffused uniform density [0, tmax]. Testing Relationships of Branch Lengths and Node-to- Tip Distances for Different Branches and Sequence For each data set, we compared branch lengths and branch times estimated using simple complex models for short, in- termediate, and long branches. Branch length categories were assigned by comparing individual branch length to the mean branch length across a given tree. Long branches were longer than 1 SD from the mean value of all branches. Short branches were those with lengths shorter than the mean value. The remaining branches were classified as intermediate branches. We also compared node-to-tip distances from sim- ple and complex models for shallow, intermediate, and deep nodes based on the timetree inferred using the complex model and no internal calibrations. The shallow region is the period spanning from 0 My to 30% of the root age. The deep region for all data sets, except for the “Eukaryotes & Prokaryotes” data, is the period spanning from 70% of the root age to the root age. Empirical Data Acquisition For the “Eukaryotes & Prokaryotes” data, the deep region is the period spanning from 50% of the root age to the root age because all internal nodes are youn- ger than 70% of the root age. The remaining timespan belongs to the intermediate region for all data sets. We com- puted the slopes of node-to-tip distances and of divergence times for nodes that were located in shallow, intermediate, and deep divergences regions. Testing the Need for Multiple-Hits Correction in Pairwise Distance Estimation We tested whether the concordance of times between simple and complex models arose because minimal multiple-hits correction was needed, which was often true for data sets that contained recently diverged or slow-evolving taxa (Nei and Kumar 2000). We estimated pairwise sequence distances using simple and complex models in MEGA X (Kumar et al. 2018). We used TN and Jones–Taylor–Thornton (Jones et al. 1992) models with a gamma parameter estimated by the ML method as the complex model for nucleotide and amino acid data sets, respectively. Testing the Linearity of Relationships among Substitution Model Complexity, Number of Ingroup Sequences, and the Dispersion of Branch Lengths We first randomly sampled ten ingroup sequences from the full “Plant” data set (99 ingroup þ 4 outgroup sequences). We then used “expanded sampling” to generate data sets with 20, 30, 40, 50, 60, 70, 80, and 90 ingroup sequences, so that the larger data sets always contained the sequences in the smaller data sets. For example, we kept ten sampled sequences and sampled another ten sequences to generate a data set with 20 ingroup sequences. We repeated this procedure 20 times, so we had 20 replicates for each number of ingroup sequences. We estimated branch lengths for each replicate using JC, K2, HKY, TN, GTR, and the model used in the original study, GTR þ C. We compared the branch lengths estimated using the Testing the Relationship of Divergence Times Estimated Using Simple and Complex Models for a Non-Bayesian Approach Testing the Relationship of Divergence Times Simulation To assess the impact of sparse taxon sampling and long branches on divergence time estimation between simple and complex models, we conducted a computer simulation. We used a completely unbalanced tree with 16 tips as the model timetree (root age ¼ 3 time units, supplementary fig. S6a, Supplementary Material online) and simulated sequen- ces under the strict clock with different mean rates. All sequences were simulated using SeqGen (Grassly et al. 1997) under the GTR þ C (a ¼ 0.25) model with 5,000 bp and a biased base composition (T¼ 0.25, C¼ 0.33, A¼ 0.31, and G¼ 0.11). We set the mean evolutionary rate of 0.1 substitutions per site per time unit as the baseline case (1) to make the simulated distribution of pairwise sequence distances (estimated under the TN þ C model) to be similar to that produced from the analysis of all eight empirical data sets analyzed (1 in supplementary fig. S6b, Supplementary Material online). Then we accelerated the mean rate to be 2-, 4-, 6-, 8-, and 10 times faster. For the fastest rate simulated (10), the median of pairwise sequence distances was 4.7 substitutions per site, which is much larger than the empirical value (supplementary fig. S6b, Supplementary Material online). Data Availability Statement All empirical and simulated data sets are deposited to Figshare: https://doi.org/10.6084/m9.figshare.11873874. Supplementary Material To examine whether the linear relationships of divergence times and branch lengths between simple and complex mod- els are unique phenomena for models in GTR class, we com- pared the ML branch lengths estimated using the simple model (JC) and models that are non-time-reversible or non-stationary for all nucleotide data sets. We obtained the branch lengths under non-time-reversible (i.e., unrestricted) model (model ¼ 10) (Yang 1994) and non-stationary model (nhomo ¼ 3) with a single partition in baseml (v4.9h) (Yang 2007). Because the direct usage of non-time-reversible and nonstationary models is not allowed in MCMCTree for esti- mating divergence times, we obtained time estimates using the RelTime method with the ML trees produced by baseml and without calibrations. We then normalized time estimates produced by RelTime to the sum of node ages to obtain the relationship. Supplementary data are available at Molecular Biology and Evolution online. Acknowledgments We thank Drs Sergei Pond, Heather Rowe, Maria Pacheco, Ananias Escalante, Koichiro Tamura, Antonia Chroni, Jeffrey Thorne, Mario dos Reis, and two anonymous reviewers for critical comments and editorial suggestions. We also thank Mario dos Reis for suggesting the phylogeny, calibration strat- egies, and simulation parameters in supplementary figure S6, Supplementary Material online. This research was supported in part by grants from the National Institutes of Health (NIH GM0126567-03), National Science Foundation (NSF 1661218), and National Aeronautics and Space Administration (NASA NNX16AJ30G) to S.K. Non-Bayesian Approach We estimated divergence times using the RelTime method (Tamura et al. 2012, 2018) in MEGA X. A single partition was used in these analyses. No calibrations are required for RelTime analyses. To directly compare the dates estimated by RelTime when no calibrations are used, we normalized time estimates to the sum of node ages. This normalization is simply a post hoc scaling of relative times and is not the same as assigning calibrations in the Bayesian approaches in which the tree prior interacts with calibration probability densities. Outgroups were removed in the time comparisons because RelTime does not produce time estimates for sequences in the outgroup. 1829 MBE Tao et al. . doi:10.1093/molbev/msaa049 simpler models with those estimated using the GTR þ C model and computed the coefficient of determination of linear regression through the origin (R2). Therefore, for each subsampled category, we had 20 R2 of branch lengths com- pared between analyses using the GTR þ C and a corre- sponding simpler model. shallow node (o–p divergence in supplementary fig. S6a, Supplementary Material online) and a diffused root calibra- tion; a precise calibration at a middle node (j–p divergence) and a diffused root calibration; and a precise root calibration. All priors (e.g., mean evolutionary rate) were set to be as the true values in all Bayesian analyses. Simulated data sets and prior settings are available at https://doi.org/10.6084/m9.fig- share.11873874, last accessed March 9, 2020. 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https://openalex.org/W1970805620	https://www.scielo.br/j/isz/a/SXk8cMrz5qxGc5S7gJkkSSs/?lang=pt&format=pdf	Portuguese	null	Baetidae (Insecta, Ephemeroptera) em córregos do cerrado matogrossense sob diferentes níveis de preservação ambiental	Iheringia. Série zoologia/Iheringia. Série Zoologia	2,011	cc-by	7,459	KEYWORDS. Abundance, richness, similarity, Index of Habitat Integrity, indicator species. RESUMO. O efeito dos diferentes níveis de preservação ambiental de córregos de 1ª a 4ª ordens sobre a riqueza, abundância, similaridade na composição de Baetidae (Ephemeroptera) e o potencial das espécies como bioindicadoras foram investigados em uma região de cerrado matogrossense. Um total de vinte espécies/morfoespécies foi catalogado dentre as 1752 ninfas amostradas, sendo reportado dois novos registros de espécies para o país. Os valores do Índice de Integridade de Hábitat (HII) obtidos foram categorizados para ambientes degradados, alterados e conservados. A riqueza de espécies foi diferente entre os locais, sendo maior em ambientes alterados. A abundância foi maior entre ambientes conservados e alterados, sendo significantemente diferente dos locais degradados. A análise NMDS indicou que locais conservados e alterados apresentam similaridade de composição de espécies, diferindo dos locais degradados. Quatro espécies demonstraram relação positiva com o aumento dos valores do HII. Baetidae apresentou espécies indicadoras de ambientes com diferentes níveis de preservação, sendo Zelusia principalis Lugo-Ortiz & McCafferty, 1998 e Baetodes sp. indicadoras de ambientes conservados e Aturbina nigra Salles, Boldrini & Shimano, 2011, Callibaetis sp. 2, Camelobaetidius aff. janae Dominique & Thomas, 2000, Paracloeodes binodulus Lugo-Ortiz & McCafferty, 1996, Waltzoyphius roberti Thomas & Peru, 2002 como indicadoras de ambientes alterados. O conhecimento da ecologia das espécies avança no sentido de fornecer subsídios ao biomonitoramento da bacia e uso de espécies indicadoras. PALAVRAS-CHAVE. Abundância, riqueza, similaridade, Índice de Integridade de Hábitat, espécies indicadoras. Macroinvertebrados aquáticos são o grupo de organismos mais utilizado em programas de biomonitoramento, especialmente insetos (Rosemberg & Resh, 1993). Dentre esses, Ephemeroptera se destaca, compreendendo um dos grupos dominantes em ecossistemas lóticos; são importantes nas cadeias tróficas, no processamento da matéria orgânica, bem como na transferência de energia (Galdean et al., 2000; Salles et al., 2004b). Possuem curto período de vida adulta (efêmera), longa duração de seu período ninfal, alta abundância e capacidade de ocupar todos os habitats aquáticos disponíveis (Bispo & Crisci-Bispo, 2006). Alterações provocadas pelas ações antrópicas nos ecossistemas aquáticos geralmente levam a uma diminuição ou completa perda das características ambientais do sistema (Bauernfeind & Moog, 2000). Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 Baetidae (Insecta, Ephemeroptera) em córregos do cerrado matogrossense sob diferentes níveis de preservação ambiental Hilton M. de L. Souza 1, Helena S. R. Cabette 2 & Leandro Juen 3 o em Ciências Ambientais, Universidade do Estado de Mato Grosso, Av. Santos Dumont, Bairro Santos Dumont, Cidade Universitária, 78200-000, Cáceres MT, t il ) Programa de Pós-Graduação em Ciências Ambientais, Universidade do Estado de Mato Grosso, Av. Santos Dumont, Bairro Santos Dumont, Cidade Universitária, 78 Brasil (hilton_marcelo@hotmail.com) 2. Departamento de Ciências Biológicas, Universidade do Estado de Mato Grosso, BR 158, Km 148, Caixa Postal 08, 78690-000, Nova Xavantina MT, Brasil (hcabette@uol.com.br) 3. Instituto de Ciências Biológicas, Universidade Federal do Pará, Rua Augusto Correia, no1, Bairro Guama, 66075-110, Belém, Pará, Brasil (leandrojuen@yahoo.com.br) ABSTRACT. Baetidae (Insecta, Ephemeroptera) of Cerrado streams in the state of Mato Grosso, Brazil, under different levels of environmental preservation. The effect of different stream environmental preservation levels, in 1 st to 4 th orders, over the richness, abundance, similarity in the composition and the potential of Baetidae (Ephemeroptera) species as bioindicators were investigated in a Cerrado region located in the state of Mato Grosso, Brazil. A total of twenty species/morphospecies were catalogued from the 1752 samples of nymphs, resulting in a new record for two species in Brazil. Values of Habitat Integrity Index (HII) were categorized among damaged, modified, and preserved environments. Species richness was different between sites, being greater in disturbed habitats. Total abundance was higher among preserved and altered environments, and differed significantly from the degraded areas. The NMDS analysis indicates that, in preserved and modified sites, the similarity in the composition of species differs from degraded areas. Four species showed a positive relationship with increasing values of HII. Baetidae showed environmental indicator species with different levels of preservation, with Zelusia principalis Lugo-Ortiz & McCafferty, 1998 and Baetodes sp. being indicators of preserved environments and Aturbina nigra Salles, Boldrini & Shimano, 2011, Callibaetis sp. 2, Camelobaetidius aff. janae, Paracloeodes binodulus Lugo-Ortiz & McCafferty, 1996 and Waltzoyphius roberti Thomas & Peru, 2002 as indicators of modified environments. The knowledge on the ecology of species advances in terms of providing subsidies for the biomonitoring of basins and the use of indicator species. EYWORDS. Abundance, richness, similarity, Index of Habitat Integrity, indicator species. Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 KEYWORDS. Abundance, richness, similarity, Index of Habitat Integrity, indicator species. Baetidae apresenta algumas vantagens como indicadores da integridade de riachos; (1) a taxonomia é baseada na forma imatura, não sendo necessária a associação com a forma adulta; (2) são facilmente encontrados em diversos tipos de substratos, tais como seixos, folhiços, entre outros (Buss et al., 2004); (3) a evolução na taxonomia deste grupo é evidente nos últimos 10 anos na região neotropical (Buss & Salles, 2007), com descrição de novos gêneros e espécies (Gillies, 2001; Lugo-Ortiz et al., 2002; Salles & Lugo-Ortiz, 2003; Salles et al., 2003, 2004a,c, 2005; Francischetti et al., 2004; Salles & Francischetti, 2004; Salles & Serrão, 2005; Domínguez et al., 2006; Nieto & Salles, 2006; Salles, 2007; Salles & Polegatto, 2008). No total foram realizadas 64 coletas, sendo três etapas nos córregos CRC, CRM, CRP e CRT (período chuvoso, seca e início das chuvas de 2005) e quatro etapas no CRCV (estação seca, início das chuvas, auge da estação chuvosa e na vazante em 2007/2008); cada etapa em cada córrego foi tratada como uma réplica diferente, exceto na ordenação. A amostragem das ninfas foi feita pelo método de varredura, com áreas fixas em transectos de 100 m e subdivisões a cada 5 m, perfazendo 20 segmentos amostrais por local de estudo (adaptado de De Marco Jr., 1998; Ferreira-Peruquetti & De Marco Jr., 2002; Ferreira- Peruquetti & Fonseca-Gessner, 2003), com auxílio de uma peneira de 18 cm de diâmetro e malha de 0,50 mm. Em cada local foram anotados os aspectos físico-químicos da água. Este método vem sendo usado com eficiência em outros estudos em Mato Grosso (Cabette et al., 2010). i Estudos ecológicos com esta família ainda são restritos, entretanto, Buss et al. (2002) indicam os baetídeos como pouco sensíveis às alterações ambientais, encontrados em locais com níveis intermediários de degradação. Outras contribuições relatam variação nas espécies quanto às exigências por locais com integridades ambientais mais elevadas e tipos de substratos específicos associados a esses ambientes (Domínguez et al., 2006; Buss & Salles, 2007). As espécies/morfoespécies foram identificadas através de chaves taxonômicas (Salles et al., 2004c; Domínguez et al., 2006) e por comparação com as descrições de Waltz & McCafferty (1987), Lugo-Ortiz & McCafferty (1996a,b), Gillies (2001), Francischetti et al. (2003), Salles & Serrão (2005), Salles & Polegatto (2008). O material foi preservado em álcool a 85% e está depositado na Coleção Zoobotânica “James A. KEYWORDS. Abundance, richness, similarity, Index of Habitat Integrity, indicator species. Tem-se observado expressiva queda da qualidade da água e redução da integridade ambiental em corpos d’água no Brasil, levando a modificações na dinâmica natural das comunidades biológicas, culminando na perda de biodiversidade aquática (Galdean et al., 2000; Callisto et al., 2001; Dodds, 2002).i As principais modificações estruturais em ambientes lóticos são consequência da retirada da vegetação ciliar que deixam as margens expostas facilitando o processo de assoreamento, resultando na homogeneização do leito e na diminuição da diversidade de habitats (microhabitats) (Delong & Brusven, 1998). Em consequência disso, os organismos que dependem de condições ambientais específicas para sua coexistência são os primeiros a serem afetados (Cortezzi et al., 2009) e sua distribuição reflete diretamente os impactos provocados no ecossistema (Rios & Bailey, 2006; Crisci-Bispo et al., 2007). Segundo Bauernfeind & Moog (2000), a estrutura da comunidade de efemerópteros reflete eficientemente a situação ambiental dos corpos d’água, em virtude da intrínseca relação com a disponibilidade de habitats, podendo ser usada como ferramenta na avaliação da integridade ecológica e monitoramento ambiental. Sob condições naturais, sua diversidade é diretamente correlacionada com a variedade de habitat e, portanto, uma modificação na estrutura física dos córregos deve 182 Souza et. al. refletir efetivamente o estado da integridade ecológica da área de amostragem (Kothe, 1962; Dinakaram & Anbalagan, 2007). Köppen, com duas estações climáticas bem definidas: um período seco de cinco meses (de maio a setembro) e outro período chuvoso (dezembro a março), com temperaturas médias em torno de 20ºC a 25ºC, sendo o período de agosto a outubro o mais quente. As médias de precipitação anual variam de 1.200 a 1.600mm (Brasil, 1981). Dentre as famílias de Ephemeroptera registradas no Brasil, Baetidae é a mais abundante (Romero & Fernández, 2001; Francischetti et al., 2004; Goulart & Callisto, 2005; Buss & Salles, 2007) e a que possui maior número de espécies registradas (69), além de ser o grupo mais amplamente distribuído entre as diversas regiões geográficas do mundo (Lugo-Ortiz & McCafferty, 1999; Barber-James et al., 2008; Salles, 2011). As áreas de estudo correspondem a pontos da rede de drenagem da sub-bacia do rio Corrente nos córregos Taquaral (CRT) e Papagaio (CRP) e, porção superior e média da sub-bacia do rio Pindaíba nos córregos Cachoeirinha (CRC), da Mata (CRM) e Caveira (CRCV). Foram selecionados 20 locais de estudo em afluentes de 1 a a 4 a ordens (Strahler, 1957) (Fig. 1). KEYWORDS. Abundance, richness, similarity, Index of Habitat Integrity, indicator species. Ratter”, Universidade do Estado de Mato Grosso, campus de Nova Xavantina (CZNX). Diante deste cenário, este trabalho tem como objetivo avaliar o efeito da integridade ambiental sobre a abundância, riqueza e similaridade na composição das espécies de Baetidae em córregos do cerrado matogrossense, procurando indicar se existem espécies com especificidade/fidelidade a determinado nível de conservação. Para tanto, foi testada a seguinte hipótese: Baetidae apresenta maior riqueza e abundância de espécies em locais com níveis mais elevados de integridade e ambientes com menor integridade apresentarão redução na riqueza e composição de espécies mais homogêneas do que nos locais mais íntegros. Assim, se de fato a integridade ambiental é importante para a distribuição, as espécies apresentarão baixa tolerância às variações de integridade e poderão ser usadas como espécies indicadoras. A integridade ambiental dos locais foi avaliada através do Índice de Integridade de Habitat (Habitat Integrity Index - HII) (Nessimian et al., 2008), que analisa diversos aspectos ambientais, tais como padrão do uso da terra, largura da vegetação ripária, estado de preservação da mata ciliar, dispositivos de retenção, sedimentos no canal, estrutura do barranco, leito do rio, corredeiras, vegetação aquática e detritos. O índice é expresso numericamente de 0 a 1 - valores mais próximos de 1 representam ambientes com os níveis mais elevados de integridade. Os dados obtidos a partir do HII foram categorizados de acordo com o critério de Scott (1979) para a formação dos grupos, estabelecendo três categorias de conservação: ambientes degradados, alterados e conservados. Os ambientes considerados como degradados apresentaram valores de HII=0,52 a ┤0,67, alterados com HII=├ 0,68 a ┤ 0,82 e conservados com HII=├ 0,83 a 0,97. Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 MATERIAL E MÉTODOS A bacia do Rio Pindaíba, com área de 10.029 km 2, localiza-se em região de cerrado ao leste do Estado de Mato Grosso (14°59’ - 15°39’S / 52°03’ - 52°28’O), onde a atividade econômica predominante é a agropecuária extensiva. O clima regional é do tipo Cwa conforme Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 Baetidae (Insecta, Ephemeroptera) em córregos do cerrado... 183 Fig. 1. Áreas de amostragens na Bacia do Rio Pindaíba (CRC - Córrego Cachoeirinha; CRCV - Córrego Caveira; CRM - Córrego da Mata; CRP - Córrego Papagaio; CRT - Córrego Taquaral; [1(1ª ordem), 2(2ªordem), 3(3ª ordem),4(4ª ordem]). Fig. 1. Áreas de amostragens na Bacia do Rio Pindaíba (CRC - Córrego Cachoeirinha; CRCV - Córrego Caveira; CRM - Córrego da Mata; CRP - Córrego Papagaio; CRT - Córrego Taquaral; [1(1ª ordem), 2(2ªordem), 3(3ª ordem),4(4ª ordem]). Fig. 1. Áreas de amostragens na Bacia do Rio Pindaíba (CRC - Córrego Cachoeirinha; CRCV - Córrego Caveira; CRM - Córrego da Mata; CRP - Córrego Papagaio; CRT - Córrego Taquaral; [1(1ª ordem), 2(2ªordem), 3(3ª ordem),4(4ª ordem]). Análise de dados. Para testar se houve diferenças na abundância e na riqueza de espécies entre as categorias de integridade ambiental, foi aplicada a análise de variância (ANOVA unifatorial) (Zar, 1999). Os pressupostos de normalidade e de homocedasticidade (teste de Levene) foram calculados e testados. O teste a posteriori de Tukey foi aplicado quando a ANOVA foi significativa, para identificar quais categorias de integridade ambiental apresentaram médias diferentes. Monte Carlo aplicando 10.000 permutações, para os três níveis de integridade (conservado, alterado e degradado). Todas as análises foram realizadas no programa R (R Development Core Team, 2009). Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 RESULTADOS Foram coletados 1.752 indivíduos da família Baetidae, distribuídos em 11 gêneros e 20 espécies/ morfoespécies (Tab. I), o que representa aproximadamente 50% dos gêneros e 40% das espécies registradas para o Brasil. Os gêneros mais abundandes foram Zelusia (641 exemplares), Waltzoyphius (213), Aturbina (210) e Americabaetis (207), ocorrendo em todos os córregos, exceto Zelusia que não ocorreu no CRCV. Para sumarizar os dados da estrutura e composição da assembleia, utilizamos a análise de escalonamento multidimensional não-métrico (non- metric multidimentional scaling – NMDS) (Delaney et al., 2000) ordenando os 20 locais estudados com base na similaridade de composição a partir da presença e ausência das espécies. Para testar a diferença na composição de espécies entre as áreas degradadas, alteradas e conservadas, aplicou-se a Análise de Similaridade Bi- fatorial (ANOSIN), método usado para detectar diferenças na estrutura da composição (Melo & Hepp, 2008). As espécies mais abundantes foram Zelusia principalis Lugo-Ortiz & McCafferty, 1998 (n=641), Americabaetis alphus Lugo-Ortiz & McCafferty, 1996 (n=207), Aturbina georgei Lugo-Ortiz & McCafferty, 1996 (n=170), Paracloeodes sp. (n=143) e Cloeodes redactus Nieto & Richard, 2008 (n=126). Para indicar a relação de cada espécie aos níveis de integridade, foi utilizada regressão linear simples (Zar, 1999) entre os valores do HII e abundância das espécies que apresentaram ocorrência mínima de 1,5 indivíduos por local amostrado. A escolha desse valor foi devido às espécies com valores inferiores apresentarem grande heterogeneidade de variância. O potencial de bioindicação das espécies foi avaliado através do Teste de Espécies Indicadoras (IndVal) (Dufrêne & Legendre, 1997), submetidos ao teste de No CRT houve maior frequência de C. redactus (69,8%), Z. principalis (39%) e Paracloeodes sp. (37%), no CRM foram Z. principalis (32,8%) e A. georgei (29,4%). Americabaetis alphus apresentou maior frequência para o CRP (39,6%) e A. georgei para o CRC (35%). Americabaetis alphus, A. georgei e Callibaetis sp., com ampla distribuição, ocorreram em todos os locais de coleta. Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 Souza et. al. 184 Tab. I. Abundância das espécies de Baetidae (Insecta, Ephemeroptera) coletados em córregos da bacia do rio Pindaíba, Mato Grosso, em 2005 e 2007/2008 (CRC, córrego Cachoeirinha; CRCV, córrego Caveira; CRM, córrego da Mata; CRP, córrego Papagaio; CRT, córrego Taquaral). Tab. I. RESULTADOS Abundância das espécies de Baetidae (Insecta, Ephemeroptera) coletados em córregos da bacia do rio Pindaíba, Mato Grosso, em 2005 e 2007/2008 (CRC, córrego Cachoeirinha; CRCV, córrego Caveira; CRM, córrego da Mata; CRP, córrego Papagaio; CRT, córrego Taquaral). Tab. I. Abundância das espécies de Baetidae (Insecta, Ephemeroptera) coletados em córregos da bacia do rio Pindaíba, Mato Grosso, em 2005 e 2007/2008 (CRC, córrego Cachoeirinha; CRCV, córrego Caveira; CRM, córrego da Mata; CRP, córrego Papagaio; CRT, córrego Taquaral). Localização geográfica e categorização ambiental dos pontos amostrais de coleta em córregos da bacia do rio Pindaíba, período de 2005, eográfica e categorização ambiental dos pontos amostrais de coleta em córregos da bacia do rio Pindaíba, período de 2005, rego Cachoeirinha; CRCV, córrego Caveira; CRM, córrego da Mata; CRP, córrego Papagaio; CRT, córrego Taquaral). Tab. II. Localização geográfica e categorização ambiental dos pontos amostrais de coleta em córregos da bacia do rio Pind 2007/2008 (CRC, córrego Cachoeirinha; CRCV, córrego Caveira; CRM, córrego da Mata; CRP, córrego Papagaio; CRT, c ç g gi g ç p g , C, córrego Cachoeirinha; CRCV, córrego Caveira; CRM, córrego da Mata; CRP, córrego Papagaio; CRT, córreg Baetidae (Insecta, Ephemeroptera) em córregos do cerrado... 185 Fig. 2. Riqueza estimada (A) e abundância (B) de Baetidae (Ephemeroptera) entre ambientes com diferentes níveis de integridade na bacia do rio Pindaíba, MT, 2005 e 2007/08. (A) (F(2,17); p=0,015); (B) (F(2,17); p=0,003). Fig. 2. Riqueza estimada (A) e abundância (B) de Baetidae (Ephemeroptera) entre ambientes com diferentes níveis de integridade na bacia do rio Pindaíba, MT, 2005 e 2007/08. (A) (F(2,17); p=0,015); (B) (F(2,17); p=0,003). imada (A) e abundância (B) de Baetidae (Ephemeroptera) entre ambientes com diferentes níveis de integridade na bacia do ri 5 e 2007/08. (A) (F(2,17); p=0,015); (B) (F(2,17); p=0,003). O HII apresentou valores que variaram de 0,52 a 0,96. Seguindo as categorias estabelecidas, oito locais foram classificados como degradados (CRC 1ª e 4ª ordens; CRCV 1ª a 4ª ordens; CRP 2ª ordem; CRT 4ª ordem), seis como alterados (CRC 2ª e 3ª ordens; CRM 3ª ordem; CRP 3ª e 4ª ordens; CRT 3ª ordem) e seis como conservados (CRM 1ª, 2ª e 4ª ordens; CRP 1ª ordem; CRT 1ª e 2ª ordens) (Tab. II). pastagens a locais com mata ciliar preservada) e o CRT com HII de 0,62 a 0,96 (locais com influência de pastagem a locais com vegetação ciliar preservada). RESULTADOS A riqueza de espécies foi diferente entre os locais com distintos níveis de integridade (F(2,17)=8,486; p=0,003). Em média, locais alterados apresentaram oito espécies a mais do que degradados (p=0,003) e sete a mais que ambientes conservados (p=0,015); os ambientes conservados tiveram riqueza de espécies similares aos degradados (Fig. 2A). A abundância das espécies também diferiu entre locais (F(2,17)=5,418; p=0,015). Ambientes conservados apresentaram, em média, 75 indivíduos a mais do que os degradados (Tukey p=0,022), enquanto que locais alterados, 65 indivíduos a mais do que os locais degradados (Tukey p=0,050) (Fig. 2B). Houve alteração do valor desse índice entre locais (ordens) de um mesmo córrego; assim, o CRC variou entre 0,61 a 0,73 (nascente intermitente e outros locais com agricultura e pecuária), o CRCV entre 0,52 a 0,65 (barragens no curso e locais de agricultura e pastagem), CRM entre 0,82 a 0,96 (locais com alguma influência de pastagens a outros com vegetação ciliar preservada), CRP entre 0,65 a 0,84 (mata ciliar circundada por Fig. 3. Ordenação dos escores derivados da análise de escalonamento multidimensional não-métrico (NMDS), realizado com matriz binária da composição de Baetidae entre trechos e época do ano em cinco córregos da Bacia do Rio Pindaíba/MT. [CRC - Córrego Cachoeirinha; CRCV - Córrego Caveira; CRM - Córrego da Mata; CRP - Córrego Papagaio; CRT - Córrego Taquaral; 1 a 4 – ordens dos córregos]. Fig. 3. Ordenação dos escores derivados da análise de escalonamento multidimensional não-métrico (NMDS), realizado com matriz binária da composição de Baetidae entre trechos e época do ano em cinco córregos da Bacia do Rio Pindaíba/MT. [CRC - Córrego Cachoeirinha; CRCV - Córrego Caveira; CRM - Córrego da Mata; CRP - Córrego Papagaio; CRT - Córrego Taquaral; 1 a 4 – ordens dos córregos]. Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 Souza et. al. 186 Fig. 4. Relação entre abundância de Americabaetis alphus (A), Baetodes sp. (B), Cloeodes redactus (C) e Zelusia principalis (D) (Ephemeroptera: Baetidae) com o Índice de Integridade de Hábitat (HII) em córregos da Bacia do Rio Pindaíba, MT, 2005 e 2007/08. Fig. 4. Relação entre abundância de Americabaetis alphus (A), Baetodes sp. (B), Cloeodes redactus (C) e Zelusia principalis (D) (Ephemeroptera: Baetidae) com o Índice de Integridade de Hábitat (HII) em córregos da Bacia do Rio Pindaíba, MT, 2005 e 2007/08. Tab. III. Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 DISCUSSÃO O número de espécies de Baetidae na bacia do rio Pindaíba (n=20), pode ser considerado elevado se comparado com outros estudos no Brasil, fato que pode estar associado à diversidade deste grupo, ainda pouco conhecida na região Centro-Oeste. Salles et al. (2003) reportou dez gêneros e 13 espécies em córregos de baixa ordem no Estado de São Paulo e Buss & Salles (2007) registraram nove espécies de Baetidae para o Estado do Rio de Janeiro. As várias morfoespécies identificadas, tais como Aturbina sp. nov., Baetodes sp., Callibaetis sp., Callibaetis sp. 1, Callibaetis sp. 2, Camelobaetidius sp. e Paracloeodes sp. reforça a necessidade de intensificar estudos com focos taxonômicos e ecológicos, visando conhecer e descrever as espécies da região, bem como verificar suas exigências ecológicas. Waltzoyphius roberti e C. redactus foram registradas pela primeira vez para o Brasil. Os locais conservados apresentaram baixa riqueza. De acordo com Connell (1978), locais sem distúrbio tendem a apresentar maior exclusão competitiva de espécies, uma vez que poderá haver predominância daquelas mais hábeis na colonização. Segundo Resh et al. (1988), quando há ausência de distúrbio em uma comunidade, o resultado é a competição, onde competidores superiores eliminaram os inferiores, reduzindo a riqueza de espécies do sistema e dando espaço a dominância de algumas espécies. Assim, faz-se necessário a compreensão de outros aspectos ecológicos do sistema, tais como a preferência das espécies a determinados tipos de substratos.i Os córregos classificados como degradados apresentaram baixa riqueza de Baetidae, devido à maior intensidade das pressões antrópicas (retilinização e atividades agropastoris) sobre os locais. Segundo Townsend et al. (1997), locais que apresentam maior intensidade de distúrbio mostram baixa riqueza porque poucas espécies são hábeis para colonizar ambientes que apresentam homogeneização do sistema devido à perda de microhábitat. A riqueza foi alta em ambientes alterados, refutando nossa hipótese de que ambientes mais íntegros apresentariam maior número de espécies. Em outras pesquisas abordando comunidades de macroinvertebrados, vários autores têm verificado uma relação entre a riqueza de espécies com locais íntegros. Para Rios & Bailey (2006) há uma forte relação entre a vegetação ciliar, uso do solo e estrutura de macroinvertebrados. Desta forma, riqueza e diversidade de táxons - principalmente de EPT (Ephemeroptera, Plecoptera e Trichoptera) - aumentam com maior quantidade de cobertura vegetal da zona ripária. Couceiro et al. RESULTADOS Espécies de Baetidae (Insecta, Ephemeroptera) indicadoras da Integridade de Hábitat (HII) na bacia do rio Pindaíba, Mato Grosso, em 2005 e 2007/2008 [degradados (0,52 a ┤0,67), alterados (├ 0,68 a ┤ 0,82) e conservados (├ 0,83 a 0,97)]; Valor de Indicador das espécies (%). cies de Baetidae (Insecta, Ephemeroptera) indicadoras da Integridade de Hábitat (HII) na bacia do rio Pindaíba, Mato Gros 2008 [degradados (0,52 a ┤0,67), alterados (├ 0,68 a ┤ 0,82) e conservados (├ 0,83 a 0,97)]; Valor de Indicador das espécie Tab. III. Espécies de Baetidae (Insecta, Ephemeroptera) indicadoras da Integridade de Hábitat (HII) na bacia do rio Pindaíba, Mato Grosso, em 2005 e 2007/2008 [degradados (0,52 a ┤0,67), alterados (├ 0,68 a ┤ 0,82) e conservados (├ 0,83 a 0,97)]; *Valor de Indicador das espécies (%). Ao analisar a ordenação (NMDS) dos locais estudados, foi possível observar um gradiente no agrupamento indicando que os locais conservados apresentaram composição similar aos de ambientes alterados e ambos diferiram dos locais degradados (Fig. 3). Os resultados da ANOSIN também indicam diferenças na composição de espécies entre os dois grupos de ordenação; conservados/alterados e degradados (R=0,337, p=0,001). riqueza de espécies (r 2=0,034; p=0,437). Porém, quando avaliamos esse efeito sobre a abundância de espécimes, houve um efeito positivo; neste sentido, a redução da integridade reflete na abundância, e essa relação não pode ser explicada pelo acaso (r 2=0,428; p=0,001). A integridade ambiental também apresentou efeito positivo sobre a abundância de A. alphus (r 2=0,229; p=0,032), C. redactus (r 2=0,243; p=0,026), Z. principalis (r 2=0,364; p=0,004) e Baetodes sp. (r 2=0,276; p=0,017), sendo mais abundantes em locais com maior nível de conservação (Fig. 4). A integridade ambiental não mostrou efeito sobre a Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 Baetidae (Insecta, Ephemeroptera) em córregos do cerrado... 187 raramente está em um estado de equilíbrio, sendo alta a riqueza quando os distúrbios são intermediários em escala de frequência e intensidade. Isso ocorre por haver coexistência de diferentes grupos de macroinvertebrados, levando a uma colonização favorável destes organismos, que por sua vez, aumentam, assim, a riqueza de espécies do local. Outro aspecto a ser considerado é a existência de uma maior variedade de guildas alimentares em Baetidae. As comunidades estudadas apresentam alguns representantes da guilda dos raspadores (e.g. Baetodes sp., Camelobaetidius sp. e C. aff. RESULTADOS janae), que encontram mais alimento, especialmente o perifíton, em locais que tem maior abertura de dossel, como ocorre em ambientes alterados pela retirada de vegetação ciliar (Svitok, 2006). Ao avaliar o potencial de indicação das espécies, de acordo com os critérios de fidelidade e especificidade estabelecidos pela análise de IndVall, Z. principalis e Baetodes sp. indicaram preferência por locais conservados; por outro lado, W. roberti, Camelobaetidius aff. jane, Aturbina nigra, Paracloeodes binodulus Lugo-Ortiz & McCafferty, 1996 e Callibaetis sp. 2 podem ser usadas para indicar ambientes que sofreram alterações em sua integridade (Tab. III). Nenhuma espécie apresentou especificidade e fidelidade ambiental suficiente para serem usadas como indicadoras de ambientes degradados. Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 DISCUSSÃO (2007) observaram redução na riqueza de insetos aquáticos com a retirada da vegetação ciliar resultante do desmatamento, sendo este um dos principais fatores que afetam a composição das comunidades. A abundância de Baetidae foi alta em ambientes conservados e alterados, refutando parcialmente a hipótese de que o grupo apresentaria maior abundância em locais com níveis elevados de integridade, corroborando com Buss et al. (2002) que observaram alta abundância do grupo em locais com níveis intermediários de impacto. Segundo Merrit & Cummins (1996), essa família é formada basicamente por organismos coletores (representados aqui por Z. principalis, W. roberti, A. georgei, Paracloeodes sp., dentre os mais abundantes) e raspadores (e.g., C. aff. janae). Baptista et al. (2006) indicaram que espécies de Americabaetis, Baetodes, Cloeodes e Camelobaetidius podem ser pastadoras (“grazers”), manipuladoras de detritos vegetais e raspadoras de substratos rochosos. Galdean et al. (2000) anotaram espécies raspadoras em locais com maior entrada de luz e, consequentemente, aumento da produtividade primária no sistema, fato que pode explicar a alta abundância de baetídeos em ambientes com algum grau de impacto. O fato da maior riqueza de Baetidae ter ocorrido em trechos alterados pode ser explicado com base na hipótese do distúrbio intermediário de origem antrópica, que estes locais vêm sofrendo pela retirada parcial da vegetação ciliar para implantação de áreas de pastagens. Siegloch et al. (2008) também relataram a influência do distúrbio intermediário sobre a riqueza de ninfas de efemerópteros no Sudeste do Brasil. Para Connell (1978), a composição das espécies de uma comunidade Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 Souza et. al. 188 Bauernfeind & Moog (2000) e Chakona et al. (2008) relataram que a distribuição e abundância da comunidade de efemerópteros é fortemente dependente da composição do habitat, estrutura de micro e mesohabitats. Ambientes alterados podem apresentar menor disponibilidade de habitats, com menos entrada de material alóctone no sistema e predomínio de matéria orgânica particulada fina, favorecendo a prevalência de organismos coletores. Para Delong & Brusven (1998), os impactos na bacia de drenagem, como erosão e remoção da vegetação ripária, resultam no aumento da sedimentação, entrada de luz e diminuição de matéria orgânica, levando a uma diminuição de macroinvertebrados que tem sua distribuição fortemente associada a tipos específicos de microhabitats. variação das condições ambientais, vivendo em áreas com baixo fluxo de água, frequentemente associada a folhiços de origem alóctone. DISCUSSÃO Para Goulart & Callisto (2005), Cloeodes foi registrado associado com o biofilme de algas (perifíton) ou substratos rochosos, enquanto que Domínguez et al. (2006) observaram que ninfas habitam ampla diversidade de habitats, encontradas tanto em riachos bem oxigenados quanto em locais pobremente oxigenados. Os aspectos ecológicos citados demonstram que estes gêneros em questão podem estar em ambientes conservados, mas não demonstram preferência por este tipo de categoria ambiental.ii Já a especificidade e fidelidade de Z. principalis para ambientes conservados (IndVal) corrobora com Buss & Salles (2007), que a classificaram como muito sensível a alterações ambientais e Lugo-Ortiz et al. (2002) que a consideraram intolerante a tais modificações. Esta espécie foi cerca de três a quatro vezes mais abundante que A. alphus e A. georgei, respectivamente; a distribuição foi mais restrita, devido sua ausência no CRCV, reforçando sua preferência ecológica. Baetodes sp., ao apresentar-se como indicadora ecológica de ambientes com elevada integridade, diverge do conjunto do gênero, que foi classificado como pouco sensível por Buss & Salles (2007), mesmo que Domínguez et al. (2006) tenham registrado espécies em locais bem oxigenados e associados ao substrato rochoso. Wantzen & Pinto-Silva (2006) relataram que os desmatamentos, construção de estradas e agricultura não-sustentável são as principais causas de erosões em riachos no Mato Grosso. Aliados a isso, apontaram que locais assoreados tendem a ter menor abundância de macroinvertebrados, possivelmente devido à baixa colonização e falta de substratos. A baixa abundância de baetídeos em locais categorizados como degradados pode estar diretamente associada a estes aspectos. Segundo Brittain (1982), outros fatores devem ser considerados, tais como inundações, alimentação e competição e que também podem influenciar a abundância e distribuição. Os agrupamentos dos locais de coleta demonstram diferenças na composição de Baetidae entre ambientes degradados com os conservados/alterados. Locais degradados apresentaram maior similaridade de fauna, corroborando nossa hipótese. Esse resultado já era esperado, uma vez que a uniformidade ambiental dos locais degradados afeta significantemente a diversidade e abundância de efemerópteros (Bauernfeind & Moog, 2000). Em diversas pesquisas com macroinvertebrados, as análises de agrupamentos têm mostrado a separação dos indivíduos quanto à preferência a um determinado tipo ambiental. Buss & Salles (2007) e Barbosa et al. (2001) relataram que baetídeos foram predominantes em locais sob níveis intermediários de impacto, onde as populações são capazes de se adaptar às novas condições, refletindo na resiliência do ecossistema. Agradecimentos. À Fundação de Amparo a Pesquisa de Mato Grosso (FAPEMAT, Processo n°098/04), pelo apoio financeiro para execução do projeto. A Universidade do Estado de Mato Grosso (UNEMAT), Departamento de Ciências Biológicas e aos membros Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 REFERÊNCIAS BIBLIOGRÁFICAS Dinakaran, S. & Anbalagan, S. 2007. Anthropogenic impacts on aquatic insects in six streams of South Western Ghats. Journal of Insect Science 7(37):1-9. Baptista, D. F.; Buss, D. F.; Dias, L. G.; Nessimian, J. L.; Da Silva, E. R.; De Morais Neto, A. H. A.; De Carvalho, S. N.; De Oliveira, M. A. & Andrade, L. R. 2006. Functional feeding groups of Brazilian Ephemeroptera nymphs: ultrastructure mouthparts. Annales de Limnologie – Internacional Journal of Limnology 42(2):87-96. Dodds, W. K. 2002. Freshwater ecology: concepts and environmental applications. San Diego, London, Academic Press. 569p. Domínguez, E.; Molineri, C.; Pescador, M. L.; Hubbard, M. D. & Nieto, C. 2006. Ephemeroptera of South America. In: Adis, J.; Arias, J. R.; Rueda-Delgado, G. & Wantzen, K. M. eds. Aquatic Biodiversity in Latin America (ABLA). Sofia-Moscow, Pensoft. v. 2, 644p. ( ) Barber-James, H. M.; Gattolliat, J-L.; Sartori, M. & Hubbard, M. D. 2008. Global diversity of mayflies (Ephemeroptera, Insecta) in freshwater. Hydrobiologia 595:339-350. ( )i p Dufrène, M. & Legendre, P. 1997. Species assemblages and indicator species: the need for a flexible asymmetrical approach. Ecological Monographs 67:345-366. Barbosa, F. A. R.; Callisto, M. & Galdean, N. 2001. Diversity macroinvertebrates as an indicator of water quality and ecosystem health: a case study for Brazil. Aquatic Ecosystem Health and Management Society 4:51-59.l Ferreira-Peruquetti, P. & De Marco Jr., P. 2002. Efeito da alteração ambiental sobre comunidades de Odonata em riachos de Mata Atlântica de Minas Gerais, Brasil. Revista Brasileira de Zoologia 19(2):317-327. y Management Society 4:51-59. Bauernfeind, E. & Moog, O. 2000. Mayflies (Insecta: Ephemeroptera) and the assessment of ecological integrity: a methodological approach. Hydrobiologia 422:71-83. Ferreira-Peruquetti, P. S. & Fonseca-Gessner, A. A. 2003. Comunidade de Odonata (Insecta) em áreas naturais de Cerrado e monocultura no nordeste do Estado de São Paulo, Brasil: relação entre o uso do solo e a riqueza faunística. Revista Brasileira de Zoologia 20(2):219-224. Bispo, P. da C. & Crisci-Bispo, V. L. 2006. Ephemeroptera. In: Costa, C.; Ide S. & Simonka, E. org. Insetos imaturos: metamorfose e identificação. Ribeirão Preto, Holos. p. 55-59. Francischetti, C. N.; Salles, F. F.; Lugo-Ortiz, C. R. & Da-Silva, E. R. 2003. First report of Americabaetis Kluge (Ephemeroptera: Baetidae) from Rio de Janeiro, Brazil. Entomotropica 18(1):69-71. i p Brasil. 1981. Projeto RADAMBRASIL, Folha SD 22. Goiás: geologia, geomorfologia, pedologia, vegetação, uso potencial da terra. Ministério das Minas e Energia/ Divisão de Publicação. 636p.l Francischetti, C. REFERÊNCIAS BIBLIOGRÁFICAS P. 1996a. Taxonomy of the Neotropical Genus Americabaetis, new status (Insecta: Ephemeroptera: Baetidae). Studies on Neotropical Fauna and E i 31 156 169 , p Callisto, M.; Moretti, M. & Goulart, M. 2001. Macroinvertebrados bentônicos como ferramenta para avaliar a saúde de riachos. Revista Brasileira de Recursos Hídricos 6(1):71-82. Environment 31:156-169. _____. 1996b. The genus Paracloeodes (Insecta: Ephemeroptera: Baetidae) and its presence in South America. Annales Limnologie 32(3):161-169. ( ) Connell, J. H. 1978. Diversity in tropical rain forest and coral reefs. Science California 199(4335):1302-1310. ( ) _____. 1999. Global biodiversity of the family Baetidae (Ephemeroptera): a generic perspective. Trends in Entomology 2(1):45-54. Cortezzi, S. S.; Bispo, P. da C.; Paciência, G. de P. & Leite, R. C. 2009. Influência da ação antrópica sobre a fauna de macroinvertebrados aquáticos em riachos de uma região do cerrado do sudoeste do Estado de São Paulo. Iheringia, Série Zoologia, 99(1):36-43. ( ) Lugo-Ortiz, C. R.; Salles, F. F. & Furieri, K. S. 2002. First record of small minnow mayflies (Ephemeroptera: Baetidae) from the state of Espírito Santo southeastern Brazil. Lundiana 3(1):79-80. Chakona, A.; Phiri, C.; Magagadza, C. H. D. & Brendonck, L. 2008. The influence of habitat structure and flow permanence on macroinvertebrate assemblages in temporary rivers in northwestern Ziwbabwe. Hydrobiologia 607:199-209. Melo, A. S. & Hepp, L. U. 2008. Ferramentas estatísticas para análises de dados provenientes de biomonitoramento. Oecologia Brasiliensis 12(3):463-486. Couceiro, S. R. M.; Hamada, N.; Luz, S. L. B.; Forsberg, B. R. & Pimentel, T. P. 2007. Deforestation and sewage effects on aquatic macroinvertebrates in urban stream in Manaus, Amazonas, Brazil. Hydrobiologia 575:271-284. ( ) Merrit, R. W. & Cummis, K. W. 1996. An introduction to the aquatic insects of North America. 3 ed. Dubuque, Kendall/Hunt Publishing Company. 862p. Nessimian, J. L.; Venticinque, E. M.; Zuanon, J.; De-Marco-Jr., P.; Gordo, M.; Fidelis, L.; Batista, J. D. & Juen, L. 2008. Land use, habitat integrity and aquatic insect assemblages in Central Amazonian streams. Hydrobiologia 614(1):117-131. y g Crisci-Bispo, V. L.; Bispo, P. C. & Froehlich, C. G. 2007. Ephemeroptera, Plecoptera e Trichoptera assemblages in two Atlantic rainforest streams, Southeastern Brazil. Revista Brasileira de Zoologia 24(2):312-318. Nieto, C. & Salles, F. F. 2006. Revision of the Genus Paracloeodes (Ephemeroptera: Baetidae) in South America. Zootaxa 1303:1-33. Delaney, C.; Reed, D. & Clarke, M. 2000. Describing patient problems & nursing treatment patterns using nursing Minimum data sets (NMDS & NMMDS) & UHDDS repositories. DISCUSSÃO Assim, pode-se verificar que Baetidae vem sendo citado como um grupo que apresenta preferência por trechos de integridade intermediária, o que corrobora em parte com os dados ecológicos para esta família até então registrados. Waltzoyphius roberti, C. aff. janae, Aturbina nigra, P. binodulus, Callibaetis sp. 2, não responderam significantemente em relação ao aumento dos valores do HII, sendo mais abundantes em locais alterados e indicadoras de ambientes com algum grau de alteração na integridade (IndVal). Estas espécies demonstraram preferência a ambientes que possam apresentar perda de microhabitat, menor sombreamento, pedregosos, com maior entrada de luz no sistema e assoreamento, ocorridos principalmente pela devastação de mata ciliar. Ambientes com estas características são habitados por organismos generalistas ou que se privilegiam por terem estratégias alimentares diferentes daqueles organismos encontrados em ambientes conservados (Domínguez et al., 2006; Baptista et al. 2006). A hipótese de que baetídeos podem ser usados como organismos bioindicadores de alterações ambientais não foi corroborada; no entanto, os resultados apresentam espécies sensíveis (Baetodes sp. e Z. principalis) e outras cinco que se beneficiam com pequenas alterações ambientais (W. roberti; C. aff. janae, Aturbina nigra, P. binodulus e Calibaetis sp. 2), indicadoras do início de alterações antrópicas no sistemas aquáticos da região. Assim, os resultados revelam que a comunidade de Baetidae apresenta ampla resposta em meio a diferentes níveis de integridade ambiental. Algumas espécies apresentaram relação com o aumento da integridade. Entre as onze espécies analisadas, A. alphus, Baetodes sp., C. redactus e Z. principalis revalaram relação positiva da abundância com os valores de HII. Entretanto, A. alphus e C. redactus não demonstraram fidelidade aos ambientes conservados. Segundo Siegloch et al. (2008), Americabaetis tem ampla distribuição, ocorrendo em vários tipos de habitats, incluindo locais com algum grau de impacto. Buss & Salles (2007) descreveram que A. alphus é tolerante à Agradecimentos. À Fundação de Amparo a Pesquisa de Mato Grosso (FAPEMAT, Processo n°098/04), pelo apoio financeiro para execução do projeto. A Universidade do Estado de Mato Grosso (UNEMAT), Departamento de Ciências Biológicas e aos membros Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 Baetidae (Insecta, Ephemeroptera) em córregos do cerrado... 189 do Laboratório de Entomologia pelo suporte logístico e de parcerias no trabalho. Ao Dr. Frederico Falcão Salles (UFES) e Yulie Shimano Feitosa (UNEMAT), pela orientação taxonômica e revisão da identificação das espécies. L. Juen é bolsista da Capes. Delong, M. D. & Brusven, M. A. 1998. DISCUSSÃO Macroinvertebrate community structure along the longitudinal gradient of an agriculturally impacted stream. Environmental Management 22(3):445-457. l p g ( ) De Marco Jr., P. 1998. The Amazonian Campina dragonfly assemblage: patterns in microhabitat use and behavior in a foraging habitat. Odonatologica 27:239-248. REFERÊNCIAS BIBLIOGRÁFICAS N.; Da Silva, E. R.; Salles, F. F. & Nessimian, J. L. 2004. A efemeropterofauna (Insecta: Ephemeroptera) do trecho ritral inferior do Rio Campo Belo, Itatiaia, RJ: composição e mesodistribuição. Lundiana 5(1):33-39. g ç p Brittain, J. E. 1982. Biology of mayflies. Annual Review of Entomology 27:119-147. gy Buss, D. F. & Salles, F. F. 2007. Using Baetidae species as biological indicators of environmental degradation in a Brazilian River Basin. Environmental Monitoring Assessment 130:365-372. Galdean, N.; Callisto, M. & Barbosa, F. A. R. 2000. Lotic ecosystems of Serra do Cipó, southeast Brazil: water quality and a tentative classification based on the benthic macroinvertebrate community. Aquatic Ecosystem Health & Management 3(4):545-552. Buss, D. F.; Baptista, D. F.; Nessimian, J. L. & Egler, M. 2004. Substrate specificity, environmental degradation and disturbance structuring macroinvertebrate assemblages in neotropical streams. Hydrobiologia 518:179-188. q y g ( ) Goulart, M. & Callisto, M. 2005. Mayfly diversity in the Brazilian tropical headwaters of Serra do Cipó. Brazilian Archives of Biology and Technology 48(6):983-996. y g Buss, D. F.; Baptista, D. F.; Silveira, M. P.; Nessimian, J. L. & Dorvillé, L. F. M. 2002. Influence of water chemistry and environmental degradation on macroinvertebrate assemblages in a river basin in south-east Brazil. Hydrobiologia 481:125-136. Gillies, M. T. 2001. A new species of Aturbina (Ephemeroptera: Baetidae) Lugo-Ortiz and McCafferty from Uruguay. In: Domínguez, E. ed. Trends in Research in Ephemeroptera & Plecoptera. New York, Kluwer Academic, Plenum Publishers. p. 331-335. river basin in south-east Brazil. Hydrobiologia 481:125-136. Cabette, H. S. R.; Giehl, N. F. S.; Dias-Silva, K; Juen, L. & Batista, J. B. 2010. Distribuição de Nepomorpha e Gerromorpha (Insecta: Heteroptera) da Bacia Hidrográfica do Rio Suiá-Miçu, MT: riqueza relacionada à qualidade da água e do hábitat. In: Santos, J. E.; Galbiati, C. & Moschini, L. E. orgs. Gestão e educação ambiental: água, biodiversidade e cultura. São Carlos, RiMa. v. 2, p.113-137. Kothe, P. 1962. Der Artenfehlbetrag, ein einfaches Güterkriterium und seine Anwenung bei biologischen Vorflutersuntersuchungen. Deutsche Gewässerkundliche Mitteilungen 6:60-65. Deutsche Gewässerkundliche Mitteilungen 6:60-65. g Lugo-Ortiz, C. R. & McCafferty, W. P. 1996a. Taxonomy of the Neotropical Genus Americabaetis, new status (Insecta: Ephemeroptera: Baetidae). Studies on Neotropical Fauna and Environment 31:156-169. _____. 1996b. The genus Paracloeodes (Insecta: Ephemeroptera: Baetidae) and its presence in South America. Annales Limnologie 32(3):161-169. _____. 1999. Global biodiversity of the family Baetidae (Ephemeroptera): a generic perspective. Trends in Entomology 2(1):45-54. Lugo-Ortiz, C. R. & McCafferty, W. Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 REFERÊNCIAS BIBLIOGRÁFICAS Procedings of the American Medical Informatics Association Symposium. p.176-179. R Development Core Team. 2009. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Disponível em: <http://www.rproject.org>. Acesso em: 23.06.2009. Iheringia, Série Zoologia, Porto Alegre, 101(3): 181-190, 30 de setembro de 2011 190 Souza et. al. Resh, V. H.; Brown, A.; Covich, A. P.; Gurtz, M. E.; Li, H. W.; Minshall, G. W.; Reice, S. R.; Sheldon, A. L.; Wallace, J. B. & Wismar, R. C. 1988. The role of the disturbance in stream ecology. Journal of the North American Benthological Society 7(4):433-455. Salles, F. F.; Da-Silva, E. R.; Hubbard, M. D.; Serrão, J. E. 2004b. 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https://openalex.org/W2129100605	https://bmcimmunol.biomedcentral.com/counter/pdf/10.1186/1471-2172-2-11	English	null	Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state.	BMC immunology	2,001	cc-by	11,956	Research article Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state David A Hume1, David M Underhill2, Matthew J Sweet1, Ad i O O i k2 F Y Li 3 d Al Ad 2 Address: 1Institute for Molecular Bioscience, University of Queensland, Brisbane, Q4072, Australia, 2Institute for Systems Biology, Seattle, USA and 3Department of Immunology, University of Glasgow, Scotland, UK me - D.Hume@imb.ug.edu.au; David M Underhill - Dunderhill@systemsbiology.org; E-mail: David A Hume* - D.Hume@imb.ug.edu.au; David M Underhill - Dunderhill@systemsbiology.org; Matthew J Sweet - M.Sweet@imb.ug.edu.au; Adrian O Ozinsky - Aozynski@systemsbiology.com; Foo Y Liew Alan Aderem - aaderem@systemsbiology.com E-mail: David A Hume* - D.Hume@imb.ug.edu.au; David M Underhill - Dunderhill@systemsbiology.org; Matthew J Sweet - M.Sweet@imb.ug.edu.au; Adrian O Ozinsky - Aozynski@systemsbiology.com; Foo Y Liew - fyl1h@clinmed.gla.ac.uk; Alan Aderem - aaderem@systemsbiology.com C di th E mail: David A Hume D.Hume@imb.ug.edu.au; David M Underhill Dunderhill@systemsbiology.org; Matthew J Sweet - M.Sweet@imb.ug.edu.au; Adrian O Ozinsky - Aozynski@systemsbiology.com; Foo Y Liew - fyl1h@clinmed.gla.ac.uk; Alan Aderem - aaderem@systemsbiology.com Received: 20 August 2001 Accepted: 12 October 2001 Received: 20 August 2001 Accepted: 12 October 2001 Published: 12 October 2001 BMC Immunology 2001, 2:11 This article is available from: http://www.biomedcentral.com/1471-2172/2/11 © 2001 Hume et al; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any non-com- mercial purpose, provided this notice is preserved along with the article's original URL. For commercial use, contact info@biomedcentral.com © 2001 Hume et al; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any mercial purpose, provided this notice is preserved along with the article's original URL. For commercial use, contact info BioMed Central BioMed Central BioMed Central Abstract Background: Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive. Results: RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1β promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6. Conclusions: These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a microbial challenge. crobial products with induction of genes required for host defence. Amongst these genes is a suite of inducible crobial products with induction of genes required for host defence. Amongst these genes is a suite of inducible Background M li Background li Activation of TLR4 on the cell surface which requires coooperation with at least two other surface proteins, CD14 and MD2 [5] leads to recruitment of adaptor proteins (MyD88, IRAK, TRAF6) that ultimately couple the recognition of the microbial product to activation of the transcription factor complex NF-κB [Reviewed in [6]]. Translocation of this transcription factor complex in turn contributes to activation of the promoters of many inducible genes in macrophages [1]. Another member of the TLR family, TLR2, is absolutely required for recognition of a range of surface components of gram-positive organisms includ- ing bacterial lipopeptides [7]. Apart from bacterial cell wall components, macrophages are able to recognize and respond to bacterial DNA or CpG-containing immunos- timulatory oligonculeotides [8,9]. A recent paper indi- cates that yet another member of Toll-like receptor family, TLR9, is required for optimal recognition of im- munostimulatory DNA [10]. The TLR2, TLR4 and TLR9 pathways apparently converge at the level of MyD88, since macrophages from mice with a mutation in this gene are defective in activation by either gram-negative or gram-positive organisms or bacterial DNA [11,12]. active state is allowed to completely decay. In such a model, the new steady state could have an intrinsic half- life or it could be maintained by continued stimulation. Explanations for tolerance in macrophages in vitro gen- erally involve selective modulation or repression of some component of the signalling cascade, ranging from the putative LPS receptor TLR4, through IRAK and various regulators of NF-κB [[5,17] and references therein]. Many of the proposed mechanisms of tolerance would imply cross-desensitisation of responses to other bacte- rial products presumed to share the same pathway, a phenomenon that has been reported between TLR2 and TLR4 [25,26]. It is self-evident that if genes such as TNF-α and c-fos are induced transiently by LPS, there is a state that might be called "tolerance" or "repression" in that the mRNA and protein declines despite the continued presence of the stimulus. The transitory nature of TNF-α induction is probably a consequence of induction of nucleases that specifically degrade the mRNA [27]. Such a feedback mechanism would clearly interfere with subsequent in- duction by any stimulus for as long as the nuclease activ- ity was retained. Background li By contrast, where mRNAs continue to increase and there is no evidence of feedback control, there is no reason to expect any restriction on additive signaling by different TLR agonists unless there is an in- trinsic limit to the amount of mRNA that can be made or common pathway components are limiting. There is no obvious reason why it would be desirable for macrophag- es to restrict their ability to recognise more than one TLR-related challenge simultaneously, especially in mixed infections. Based upon these considerations, we hypothesised that genes that are induced in a sustained manner by TLR agonists would not display any kind of "tolerance" and that there would be significant advan- tage to macrophages in being able to integrate multiple signals that induce such genes. One clinically-important feature of the mammalian re- sponse to LPS is the induction of tolerance. An animal exposed to a sub-lethal dose of LPS, or to a non-toxic LPS, enters a refractory period during which it can resist lethal LPS exposure. Several groups have attempted to model the phenomenon of endotoxin tolerance in vitro by studying the ability of macrophages exposed to LPS to recognise a secondary stimulus [5,13–22]. In these mod- els, some responses that are measured (e.g. activation of transcription factor NF-κB, TNF-α mRNA production) occur transiently after LPS addition. Whereas TNF-α mRNA is induced transiently in response to LPS and then rapidly degraded [23] and the protein product is produced and secreted in a bolus from the stimulated cells [24], other mRNAs are elevated for as long as LPS is present, up to 48 hrs after LPS addition [23]. Such genes could be induced by autocrine stimuli produced in the in- itial phase of activation. Alternatively, LPS may act to cause a change of "steady state", and TNF-α induction and other early response genes could be a feature of a transition state that cannot be reactivated until the fully To address this hypothesis, we have developed novel re- porter gene systems. The firefly luciferase gene product is very unstable in the macrophage cell line RAW264, and stably-transfected cells have provided a sensitive in- dicator of transient activation of κB-dependent tran- scription in these cells [28]. We examined the regulation of promoters of the IL-1β, IL-6 and IL-12p40 genes as well as the widely-used κB-responsive gene, ELAM (E- selectin) in stably-transfected RAW264 cells. Background li Background Mammalian macrophages respond to a wide range of mi- http://www.biomedcentral.com/1471-2172/2/11 BMC Immunology 2001, 2:11 cytokines, including IL-1, TNF-α, IL-6 and IL-12, which are required for protective innate and acquired immuni- ty but also mediate much of the pathology of disseminat- ed infections including toxic shock [1]. The archetypal macrophage activating bacterial product is lipopolysac- charide (LPS) or endotoxin. An understanding of the mechanism of action of LPS was greatly expedited by the identification of the Toll-like receptor [TLR] family and the parallels between recognition of bacterial stimuli in mammals and drosophila. Mice with mutations in the TLR4 gene are hypo-sensitive to LPS [2–4]. Activation of TLR4 on the cell surface which requires coooperation with at least two other surface proteins, CD14 and MD2 [5] leads to recruitment of adaptor proteins (MyD88, IRAK, TRAF6) that ultimately couple the recognition of the microbial product to activation of the transcription factor complex NF-κB [Reviewed in [6]]. Translocation of this transcription factor complex in turn contributes to activation of the promoters of many inducible genes in macrophages [1]. Another member of the TLR family, TLR2, is absolutely required for recognition of a range of surface components of gram-positive organisms includ- ing bacterial lipopeptides [7]. Apart from bacterial cell wall components, macrophages are able to recognize and respond to bacterial DNA or CpG-containing immunos- timulatory oligonculeotides [8,9]. A recent paper indi- cates that yet another member of Toll-like receptor family, TLR9, is required for optimal recognition of im- munostimulatory DNA [10]. The TLR2, TLR4 and TLR9 pathways apparently converge at the level of MyD88, since macrophages from mice with a mutation in this gene are defective in activation by either gram-negative or gram-positive organisms or bacterial DNA [11,12]. cytokines, including IL-1, TNF-α, IL-6 and IL-12, which are required for protective innate and acquired immuni- ty but also mediate much of the pathology of disseminat- ed infections including toxic shock [1]. The archetypal macrophage activating bacterial product is lipopolysac- charide (LPS) or endotoxin. An understanding of the mechanism of action of LPS was greatly expedited by the identification of the Toll-like receptor [TLR] family and the parallels between recognition of bacterial stimuli in mammals and drosophila. Mice with mutations in the TLR4 gene are hypo-sensitive to LPS [2–4]. Results G Comparative time and dose response curve analysis were performed for each of the pooled transfectant lines with different combinations of promoters. Each promoter controlling firefly luciferase behaved in a unique man- ner, regardless of the presence of the IL-1 promoter-re- nilla luciferase gene in all of the lines, whereas the IL-1 promoter regulation was remarkably consistent in inde- pendent lines with different combinations of firefly luci- ferase reporters. Generation and characterisation of stably transfected RAW264 cell lines with two integrated luciferase reporters Previous studies examined a stable transfectant of the cell line RAW264, in which the firefly luciferase gene di- rected by the NF-κB-dependent HIV-1-LTR was inte- grated into the genome. This line provided a sensitive indicator of response to LPS and to CpG DNA [8,28]. In- duction of luciferase activity was transient, reaching a peak after around 2 hrs and then declining rapidly to control levels. The time course of luciferase activation was consistent with transient induction of nuclear NF- κB activity [8,28] and TNF-α mRNA [8,28] demonstrat- ed previously using RAW264 cells cultured in similar conditions. Although NF-κB is strongly implicated in regulated gene expression in macrophages, numerous other transcription factors (i.e. PU.1, Ets-2, Sp1, Stat-1, C/EBPβ, γ or δ, IRF-1 etc [1,29]) are regulated or induced in LPS-stimulated cells, so we decided to examine more complex promoters that are not solely dependent upon NF-κB. The ELAM-1 promoter, used commonly as an indicator of κB-dependent transcription [33]. displayed the high- est basal activity and inducibility. In our studies of the HIV-1-LTR, we found that adherence to tissue culture plastic was stimulatory [unpublished], so the transfected RAW264 cells were plated in the evening and stimulated the next morning after the cells were fully adherent. We considered the possibility that overnight culture could lead to accumulation of endogenous stimulatory or in- hibitory cytokines, so we examined the effect of replacing the medium. The outcome depended upon the reporter gene. When cells were exposed to fresh medium, ELAM- luciferase activity decreased transiently, then increased continuously over a 12 hr incubation (this is not obvious in Fig 1A, because of the much larger effect of added LPS). Following addition of 100 ng/ml LPS to unwashed cells, ELAM-luciferase was detectably elevated 2-fold af- ter 30 mins, and continued to increase relative to un- stimulated expression for up to 6–8 hrs, after which it declined (Fig 1A). Background li We dem- onstrate that sustained activation of the promoters of these genes requires the continued presence of microbial agonist and that signaling by one such agonist does not preclude additional activation through a distinct Toll- like receptor pathway. In the second part of the study, we use constitutively active forms of TLRs to generate the primary activation of reporter gene expression, and http://www.biomedcentral.com/1471-2172/2/11 BMC Immunology 2001, 2:11 again show that their actions are additive with those of agents that act through other TLRs. Finally, we show that primary macrophages can, indeed, respond to multiple TLR stimuli with increased cytokine secretion. In over- view, we conclude that macrophages sense microbial challenges continuously and can respond to more than one stimulus simultaneously. inducible by LPS or other agonists [32]. The expression of luciferase and responsiveness to microbial challenge has been stable in the pools of transfectants for at least 2 months in continuous culture. To our knowledge, this is the first example of the use of multiple reporter genes in a stably-transfected macrophage line. http://www.biomedcentral.com/1471-2172/2/11 BMC Immunology 2001, 2:11 Figure 1 Time course of activation of integrated reporter genes in RAW264 cells. Pooled stable transfectants of RAW264 cells, with either the ELAM or IL-12 promoter driving firefly luci- ferase, cotransfected with the IL-1β promoter driving renilla luciferase, were cultured overnight as described in Materials and Methods. Where the cells were washed, the medium was aspirated, and replaced immediately with warm medium at time zero. A typical experiment of three is shown. Results for ELAM and IL12 promoters are the average of duplicates that differ by less than 10% from the mean. In the case of the IL-1β data, the results are the average of 4 datapoints obtained with the two separate pooled transfectant lines in the same experiment. 0 500000 1000000 1500000 2000000 2500000 RLU 0 5 1 0 1 5 Hours Washed-LPS Washed LPS Control 0 10000 20000 30000 40000 50000 RLU 0 5 1 0 1 5 Hours WASHED-LPS WASHED LPS Control A:ELAM C:IL-1 0 50000 100000 150000 200000 RLU 0 5 1 0 1 5 Hours Washed +LPS Washed LPS Control B:IL-12 ficult to interpret, we chose a standard assay procedure in which the cells were plated late in the afternoon and stimulated the next day for 8 hrs without changing the medium. The sensitivity of the assay allowed us to obvi- ate the effect of accumulated inhibitors following over- night culture via the use of relatively low starting cell density (2 × 105/ml). 0 500000 1000000 1500000 2000000 2500000 RLU 0 5 1 0 1 5 Hours A:ELAM 0 500000 1000000 1500000 2000000 2500000 RLU 0 5 1 0 1 5 Hours Washed-LPS Washed LPS Control A:ELAM RLU Figure 1 was also increased marginally by washing and continued to increase when the control activation had peaked. Sim- ilar results were obtained with a population of cells transfected with an IL-6 firefly luciferase reporter to- gether with the IL-1 renilla luciferase reporter (data not shown). These findings suggest that the RAW264 cells produce a feedback suppressor of LPS response that has some selectivity for the target promoter being studied. Because the introduction of fresh medium, or use of freshly plated-cells creates a rising baseline which is dif- was also increased marginally by washing and continued to increase when the control activation had peaked. Sim- ilar results were obtained with a population of cells transfected with an IL-6 firefly luciferase reporter to- gether with the IL-1 renilla luciferase reporter (data not shown). These findings suggest that the RAW264 cells produce a feedback suppressor of LPS response that has some selectivity for the target promoter being studied. Because the introduction of fresh medium, or use of freshly plated-cells creates a rising baseline which is dif- Reporter gene lines respond to a wide range of microbial agonists with distinct dose response curves Hours Hours Washed +LPS Washed LPS Control 0 50000 100000 150000 200000 RLU 0 5 1 0 1 5 Hours B:IL-12 The different indicator cells lines each responded to a wide diversity of different agonists of bacterial origin in addition to LPS, including the synthetic bacterial li- popeptide, PAM3-CSK4 [34], peptidoglycan and bacteri- al DNA (bDNA) or CpG-containing oligonucleotides. Figure 2 shows an example using the IL-6 promoter in combination with the IL-1β promoter in the same cells. This study makes several additional points: RLU WASHED-LPS WASHED LPS Control 1) Activation of expression of both reporter genes was de- tectable in response to LPS at Ing/ml. 0 10000 20000 30000 40000 50000 RLU 0 5 1 0 1 5 Hours WA WA LPS Con C:IL-1 RLU 2) The response of both promoters to low doses (1 or 10 ng/ml) of LPS was transient and declined after 10–15 hrs, where the response to higher LPS concentration (100 ng/ml) was sustained for longer and was still almost maximal after 24 hrs. Hence, the duration, rather than the peak magnitude, of the response was most sensitive to LPS concentration. Hours Figure 1 3) The different agonists have different relative activities on the two promoters. In keeping with the previous ob- servation that IL-1β is weakly induced by bDNA [8], bDNA was as effective as LPS at inducing IL-6 promoter but only half as effective on the IL-1 promoter. g Time course of activation of integrated reporter genes in RAW264 cells. Pooled stable transfectants of RAW264 cells, with either the ELAM or IL-12 promoter driving firefly luci- ferase, cotransfected with the IL-1β promoter driving renilla luciferase, were cultured overnight as described in Materials and Methods. Where the cells were washed, the medium was aspirated, and replaced immediately with warm medium at time zero. A typical experiment of three is shown. Results for ELAM and IL12 promoters are the average of duplicates that differ by less than 10% from the mean. In the case of the IL-1β data, the results are the average of 4 datapoints obtained with the two separate pooled transfectant lines in the same experiment. g Time course of activation of integrated reporter genes in RAW264 cells. Pooled stable transfectants of RAW264 cells, with either the ELAM or IL-12 promoter driving firefly luci- ferase, cotransfected with the IL-1β promoter driving renilla luciferase, were cultured overnight as described in Materials and Methods. Where the cells were washed, the medium was aspirated, and replaced immediately with warm medium at time zero. A typical experiment of three is shown. Results for ELAM and IL12 promoters are the average of duplicates that differ by less than 10% from the mean. In the case of the IL-1β data, the results are the average of 4 datapoints obtained with the two separate pooled transfectant lines in the same experiment. Figure 3 shows dose response curves for LPS, lipopeptide and bacterial DNA for the IL-12, IL-1 and ELAM-1 pro- moters. Activity of both the IL-1 and IL-12 promoters continued to increase up to 500 ng/ml LPS whereas the activation of ELAM luciferase was detectable at 0.1 ng/ ml and maximally at 10 ng/ml. Given that the ELAM-1 promoter is apparently more sensitive to LPS than other promoters tested, the rising baseline seen upon addition of fresh medium (Fig 1) is likely to be due to endotoxin and/or endotoxin-like activity in serum, which cannot be completed avoided and which the cells themselves de- grade with time. Results G In cells provided with fresh medium, the tail-off at 6–8 hrs did not occur, and luciferase activ- ity was still increasing at 12 hrs. Hence, endogenous reg- ulators appear to constrain activation of the NF-κB- dependent promoter. In the same stably-transfected cells, the IL-1β renilla luciferase did not show either the rapid decline upon medium replacement, or the increase in basal activity thereafter (Fig 1C). However, fresh me- dium did accelerate the response to LPS, and permit the response to continue rising up to 12 hrs. To examine genes that are induced at a transcriptional level in RAW264 cells, we made a series of stable RAW264 cell transfectants with cytokine promoters driving luciferase. Stable transfection avoids the compli- cation that derives from the ability of macrophages to recognise and respond to plasmid DNA [8]. The inter- leukin-1β gene is of particular interest, because previous reports indicate that "LPS tolerance" does not prevent re-induction of this gene or of the interleukin 6 gene [30,31]. We cloned the human interleukin-1 β promoter into a renilla luciferase plasmid, which allowed us to pro- duce lines in which the IL-1β induction response could be measured simultaneously with other promoters that may, or may not, exhibit tolerance. RAW264 cells were contransfected by electroporation with two separate reporters and after 2 weeks of selec- tion in G418, several hundred foci of stable transfectants were pooled for further study. Because of the known var- iation between RAW264 subclones in terms of LPS-in- ducible gene expression [23] we chose not to study single cell clones. In a separate study, we have confirmed that all clones derived from the pools used in this study ex- press both the firefly and renilla luciferase genes at low levels, but vary considerably in whether that activity is Fig 1B shows a comparable time course for the IL-12 pro- moter. The data for the IL-1β promoter in this pool of sta- ble transfectants line were indistinguishable from those obtained with the ELAM/IL-1 transfectants, and have been averaged in Fig 1C. Unlike the IL-1 promoter in the same cells, the response of the IL-12 promoter to LPS http://www.biomedcentral.com/1471-2172/2/11 Figure 2 g Time course of activation ofIL-6 and IL-1 promoters in RAW264 cells and the effect of a range of microbial agonists. Pooled stable transfectants with the IL-6 promoter driving firefly luciferase and the IL-6 promoter driving renilla luci- ferase were incubated overnight then stimulated with the agents shown for the time indicated prior to assay of luci- ferase activity. PMA3-CSK was added at 100 ng/ml, peptidog- lycan at 10 µg/ml and E-coli genomic DNA (bDNA) at 10 µg/ ml. A representative experiment of two is shown. Datapoints are the average of duplicate wells. Addition of a different agonist revealed a surprisingly simple pattern of purely additive responses. This is most obvious in the case of lipopeptide-treated cells, where maximal activity was retained despite removal of the pri- mary agonist. Addition of either LPS or bDNA caused an almost additive increase in luciferase activity, whereas addition of peptidoglycan (which probably shares the TLR2-signalling pathway with lipopeptide) was without additional effect (data not shown). of contrasting activation patterns. Each line was incubat- ed overnight (16–18 hrs) with 100 ng/ml LPS, 100 ng/ml lipopeptide or 10 µg/ml of bacterial DNA, washed twice with fresh medium, and then re-exposed to a range of ag- onists. The results are shown In Fig 4. Even after 18 hrs, luciferase activity was still elevated at least 10-fold in all cases. In cells washed to remove LPS, the elevated activ- ity declined by about 70% after 8 hrs, but immediate re- addition of LPS maintained activity of the ELAM-1, IL-12 and IL-1 promoters. By contrast to the pattern observed in LPS-stimulated cells deprived of stimulus, ELAM, IL- 12 or IL-1β promoter luciferase activity stimulated by li- popeptide did not decline greatly after removal of the stimulus. bDNA behaved intermediately for all promot- ers; activity declined 20–60% after removal of the stim- ulus. The effects of pretreatment 1 L P S 1.0 LPS 10 LPS 100 LPS 500 LPS 1 PAM 10 PAM 100 PAM 1ug bDNA 5 ug bDNA 10ug bDNA ELAM IL1 IL12 RLU RLU bDNA Peptidoglycan PAM-CSK LPS100ng/ml LPS10ng/ml LPS1ng/ml 0 5000 10000 15000 20000 25000 30000 RLU 0 5 1 0 1 5 2 0 2 5 HOURS bDNA Peptidoglycan PAM-CSK LPS100ng/ml LPS10ng/ml LPS1ng/ml B:IL1 RLU Figure 3 Comparative dose response curves for activation of IL-12, IL- 1 and ELAM promoters. Pooled stable transfectants of RAW264 cells, with either the ELAM or IL-12 promoter driving firefly luciferase, cotransfected with the IL-1β pro- moter driving renilla luciferase, were cultured overnight as described in Materials and Methods. E. coli genomic DNA (bDNA), LPS or PAM3-CSK lipopeptide (PAM) were added at the concentrations indicated (LPS and PAM are in ng/ml, bDNA in µg/ml). After 8 hrs, the cells were harvested for determination of luciferase activity. Datapoints are the aver- age of duplicate wells. The experiment is representative of two, and reiterates the pattern observed in 2 others with dif- ferences in design. HOURS The effects of pretreatment Pooled stable transfectants of RAW264 cells, with either the ELAM or IL-12 promoter driving firefly luciferase, cotransfected with the IL-1β pro- moter driving renilla luciferase, were cultured overnight as described in Materials and Methods. E. coli genomic DNA (bDNA), LPS or PAM3-CSK lipopeptide (PAM) were added at the concentrations indicated (LPS and PAM are in ng/ml, bDNA in µg/ml). After 8 hrs, the cells were harvested for determination of luciferase activity. Datapoints are the aver- age of duplicate wells. The experiment is representative of two, and reiterates the pattern observed in 2 others with dif- ferences in design. 0 2 5 0 0 0 5 0 0 0 0 7 5 0 0 0 1 0 0 0 0 0 RLU Control 0 . 1 L P S 1.0 LPS 10 LPS 100 LPS 500 LPS 1 PAM 10 PAM 100 PAM 1ug bDNA 5 ug bDNA 10ug bDNA ELAM IL1 IL12 Figure 2 Time course of activation ofIL-6 and IL-1 promoters in RAW264 cells and the effect of a range of microbial agonists. Pooled stable transfectants with the IL-6 promoter driving firefly luciferase and the IL-6 promoter driving renilla luci- ferase were incubated overnight then stimulated with the agents shown for the time indicated prior to assay of luci- ferase activity. PMA3-CSK was added at 100 ng/ml, peptidog- lycan at 10 µg/ml and E-coli genomic DNA (bDNA) at 10 µg/ ml. A representative experiment of two is shown. Datapoints are the average of duplicate wells. 0 50000 100000 150000 RLU 0 5 1 0 1 5 2 0 2 5 bDNA Peptidoglycan PAM-CSK LPS100ng/ml LPS10ng/ml LPS 1ng/ml 0 5000 10000 15000 20000 25000 30000 RLU 0 5 1 0 1 5 2 0 2 5 HOURS bDNA Peptidoglycan PAM-CSK LPS100ng/ml LPS10ng/ml LPS1ng/ml A:IL6 B:IL1 0 50000 100000 150000 RLU 0 5 1 0 1 5 2 0 2 5 bDNA Peptidoglycan PAM-CSK LPS100ng/ml LPS10ng/ml LPS 1ng/ml A:IL6 0 2 5 0 0 0 5 0 0 0 0 7 5 0 0 0 1 0 0 0 0 0 RLU Control 0 . The effects of pretreatment The main purpose of creating these lines of RAW264 cells is to use them as convenient indicators of interac- tions amongst signals generated by different microbe-as- sociated stimuli. We focused upon the IL-12/IL-1 and ELAM-IL-1 lines, which appear to display the spectrum http://www.biomedcentral.com/1471-2172/2/11 BMC Immunology 2001, 2:11 in s. ng i- he i- g- / Figure 3 Comparative dose response curves for activation of IL-12, IL- 1 and ELAM promoters. Pooled stable transfectants of RAW264 cells, with either the ELAM or IL-12 promoter driving firefly luciferase, cotransfected with the IL-1β pro- moter driving renilla luciferase, were cultured overnight as described in Materials and Methods. E. coli genomic DNA (bDNA), LPS or PAM3-CSK lipopeptide (PAM) were added at the concentrations indicated (LPS and PAM are in ng/ml, bDNA in µg/ml). After 8 hrs, the cells were harvested for determination of luciferase activity. Datapoints are the aver- age of duplicate wells. The experiment is representative of two, and reiterates the pattern observed in 2 others with dif- ferences in design. 0 2 5 0 0 0 5 0 0 0 0 7 5 0 0 0 1 0 0 0 0 0 RLU Control 0 . 1 L P S 1.0 LPS 10 LPS 100 LPS 500 LPS 1 PAM 10 PAM 100 PAM 1ug bDNA 5 ug bDNA 10ug bDNA ELAM IL1 IL12 Addition of a different agonist revealed a surprisingly Figure 2 Time course of activation ofIL-6 and IL-1 promoters in RAW264 cells and the effect of a range of microbial agonists. Pooled stable transfectants with the IL-6 promoter driving firefly luciferase and the IL-6 promoter driving renilla luci- ferase were incubated overnight then stimulated with the agents shown for the time indicated prior to assay of luci- ferase activity. PMA3-CSK was added at 100 ng/ml, peptidog- lycan at 10 µg/ml and E-coli genomic DNA (bDNA) at 10 µg/ ml. A representative experiment of two is shown. Datapoints th f d li t ll 0 50000 100000 150000 RLU 0 5 1 0 1 5 2 0 2 5 bDNA Peptidoglycan PAM-CSK LPS100ng/ml LPS10ng/ml LPS 1ng/ml 0 5000 10000 15000 20000 25000 30000 RLU 0 5 1 0 1 5 2 0 2 5 HOURS bDNA Peptidoglycan PAM-CSK LPS100ng/ml LPS10ng/ml LPS1ng/ml A:IL6 B:IL1 Figure 3 Comparative dose response curves for activation of IL-12, IL- 1 and ELAM promoters. Figure 4 Figure 4 Additive effects of different microbial agonists and lack of evi- dence of tolerance in reporter gene activation. Pooled stable transfectants of RAW264 cells, with either the ELAM or IL- 12 promoter driving firefly luciferase, cotransfected with the IL-1β promoter driving renilla luciferase, were cultured over- night for 16–18 hrs with either no addition, LPS (100 ng/ml), PAM3-CSK lipopeptide (PAM, 100 ng/ml) or E. coli genomic DNA (bDNA, 10 µg/ml) as indicated on the vertical legend. The cells were washed by aspirating the medium, replacing it with warm medium, incubating for 5 minutes and replacing the medium a second time. The replacement medium con- tained no additional stimulus, or one of original stimuli as indicated on the legend to the X-axis, so that a 4 × 4 matrix of pretreatment and retreatment was established. After a further 8 hrs stimulation, the cells were harvested for deter- mination of luciferase activity. Each datapoint is the average of duplicate wells. The experiments is representative of two with identical design. The pattern of restimulation has been confirmed for LPS, and for the ELAM/IL-1 line in separate experiments (not shown). Figure 4 Additive effects of different microbial agonists and lack of evi- dence of tolerance in reporter gene activation. Pooled stable transfectants of RAW264 cells, with either the ELAM or IL- 12 promoter driving firefly luciferase, cotransfected with the IL-1β promoter driving renilla luciferase, were cultured over- night for 16–18 hrs with either no addition, LPS (100 ng/ml), PAM3-CSK lipopeptide (PAM, 100 ng/ml) or E. coli genomic DNA (bDNA, 10 µg/ml) as indicated on the vertical legend. The cells were washed by aspirating the medium, replacing it with warm medium, incubating for 5 minutes and replacing the medium a second time. The replacement medium con- tained no additional stimulus, or one of original stimuli as indicated on the legend to the X-axis, so that a 4 × 4 matrix of pretreatment and retreatment was established. After a further 8 hrs stimulation, the cells were harvested for deter- mination of luciferase activity. Each datapoint is the average of duplicate wells. The experiments is representative of two with identical design. The pattern of restimulation has been confirmed for LPS, and for the ELAM/IL-1 line in separate experiments (not shown). RAW264 cells transfected with CD4-TLR plasmids, and the IL-12 promoter, were incubated overnight, washed and treated with microbial agonists. http://www.biomedcentral.com/1471-2172/2/11 BMC Immunology 2001, 2:11 Figure 4 Additive effects of different microbial agonists and lack of evi- dence of tolerance in reporter gene activation. Pooled stable transfectants of RAW264 cells, with either the ELAM or IL- 12 promoter driving firefly luciferase, cotransfected with the IL-1β promoter driving renilla luciferase, were cultured over- night for 16–18 hrs with either no addition, LPS (100 ng/ml), PAM3-CSK lipopeptide (PAM, 100 ng/ml) or E. coli genomic DNA (bDNA, 10 µg/ml) as indicated on the vertical legend. The cells were washed by aspirating the medium, replacing it with warm medium, incubating for 5 minutes and replacing the medium a second time. The replacement medium con- tained no additional stimulus, or one of original stimuli as indicated on the legend to the X-axis, so that a 4 × 4 matrix of pretreatment and retreatment was established. After a further 8 hrs stimulation, the cells were harvested for deter- mination of luciferase activity. Each datapoint is the average of duplicate wells. The experiments is representative of two with identical design. The pattern of restimulation has been confirmed for LPS, and for the ELAM/IL-1 line in separate experiments (not shown). 0 10000 20000 30000 RLU Control L P S PAM bDNA bDNA PAM LPS Control 0 100000 200000 300000 400000 RLU Control L P S PAM bDNA bDNA PAM LPS Control A:IL-12 B:IL-1 0 5 0 0 1000 1500 2000 RLU Control L P S PAM bDNA TREATMENT bDNA PAM LPS Control C:ELAM 0 10000 20000 30000 RLU Control L P S PAM bDNA bDNA PAM LPS Control 0 100000 200000 300000 400000 RLU Control L P S PAM bDNA bDNA PAM LPS Control A:IL-12 B:IL-1 0 5 0 0 1000 1500 2000 RLU Control L P S PAM bDNA TREATMENT bDNA PAM LPS Control C:ELAM pattern of signaling is the same as previously described for activation of TNF-α production [35]. Because of the possible complex effect of stimulatory DNA sequences in the co-transfected plasmids, we confirmed that the re- sponse was due to expression of the protein products by inactivating each signaling domain by mutating a con- served proline in each TIR domain that corresponds to the inactivating mutation in TLR4 in the C3H/HeJ mouse strain, P712H [36,37]. We have previously shown that mutation of the conserved prolines in TLR2 and TLR6 similarly inactivates these receptors [34,35]. Figure 4 The extent of the ac- tivation by CD4-TLR4 was comparable to that induced by addition of a maximal dose of LPS (100 ng/ml) to con- trol cells and LPS did not stimulate further. By contrast, the effects of both lipopeptide and bacterial DNA were approximately additive with CD4-TLR4. The converse experiment gave the expected reciprocal pattern. Li- popeptide addition to cells transfected with CD-4TLR2/ 6 lifted the luciferase activity to the level observed when the agonist was added to control cells, whereas both LPS and bacterial DNA effects were almost perfectly additive. combination with either TLR1 or TLR6. We investigated whether the active TLRs alone could induce IL-12 luci- ferase, and whether their actions were additive with their agonists or, alternatively, produced some kind of toler- ance. http://www.biomedcentral.com/1471-2172/2/11 The ability of CD4-TLR4 to trans-activate IL-12 promoter ac- tivity was abolished by the P-H mutation. In fact, the CD4-TLR4-PH mutant expression plasmid reproducibly reduced the basal IL-12 promoter activity suggesting that the mutant receptor may generate a repressive signal. In the case of TLR2 and TLR6, co-transfection of both P-H mutant expression plasmids together also suppressed the basal activity. However, when wild-type TLR2 or 6 was co-transfected with the reciprocal P-H mutant part- ner, there was a small, but reproducible activation above the control; even more relative to the level observed when both partners were P-H mutants. This complex pattern strongly supports the view that the CD4-TLR2 and CD4-TLR6 heterodimerise in order to signal [35] and also suggests that the P-H mutation reduces, but does not completely abolish, the ability of either partner to contribute some function to an active heterodimer. 0 5 0 0 1000 1500 2000 RLU Control L P S PAM bDNA TREATMENT bDNA PAM LPS Control C:ELAM Interaction of different agonists with the Toll-like receptor pathways In a separate report, we have examined the ability of dominant-positive forms of the Toll-like receptors [TLRs] to mimic actions of the microbial agonists in macrophages [35]. Activation was achieved by fusing the extracellular domain of CD4, which promotes spontane- ous dimerisation, with the intracellular domain of TLRs. CD4-TLR4 alone was able to activate production of the cytokine TNF-α, and ELAM luciferase activity. By con- trast, CD4-TLR2 was active only when transfected in http://www.biomedcentral.com/1471-2172/2/11 Additive effects of different agonists on inducible cytokine production Both the experiments with stable reporter gene lines, and the studies of the dominant-active TLRs, suggest that different Toll-like receptor pathways can operate in- dependently of each other to induce inflammatory cy- tokines. To confirm the biological relevance of this observation, we examined the effect of combined addi- tion of two such agonists, LPS and CpG DNA. Data in Fig 6 show that combined addition of LPS and bacterial DNA or the stimulatory CpG oligonucleotide AO-1 actually produced a more than additive release of TNF-α, IL-6 or When the IL-12 luciferase reporter gene was co-trans- fected into RAW264 cells together with the the CD4- TLR4 expression vector, reporter gene expression was strongly induced (Fig 5). By contrast, CD4-TLR2 or CD4- TLR6 had little effect when added alone. When added in combination, TLR2 plus TLR6 activated IL-12 luciferase activity to the same extent as CD-TLR4 added alone. This http://www.biomedcentral.com/1471-2172/2/11 BMC Immunology 2001, 2:11 Figure 5 Combined effects of constitutively active toll-like receptors and added agonists. RAW264 cells were transiently co-trans- fected with the IL-12 promoter luciferase reporter genes and expression plasmids encoding wild-type and mutated (indi- cated by an asterisk) forms of the CD4/TLR chimaeric recep- tors as indicated either alone for TLR4 or in combination for TLR2 and 6. The experiment is described in detail in Materi- als and Methods. After overnight incubation, the cells were treated with either LPS [100 ng/ml], PAM3-CSK lipopeptide (100 ng/ml) or E-coli genomic DNA (10 µg/ml) as indicated. Each datapoint is the average of duplicate wells. The same pattern was observed in three separate experiments. The major variation between experiments was in the effect of the vector alone on basal reporter gene activity. 0 5 0 0 1000 1500 RLU Vector TLR2 TLR2/6 TLR4 TLR4 TLR2/6 TLR2/6 TLR26 bDNA PAM-CSK L P S Control removed. By contrast, there is a family of transcription factors and target genes that are induced much more slowly by LPS, and which remain elevated at least 48 hrs after ligand addition. They include the transcription fac- tors Ets-2, C/EBP family members and Sp1 [1,38] and the interferon-regulatory factors [29], the serpin plas- minogen activator inhibitor-2 and inducible nitric oxide synthase (iNOS) [23] the cytoskeletal regulators MARCKS and MacMARCKS [39] and many of the induc- ible cytokines such as IL-1, IL-6, IL-10 and IL-12 and other regulators [20,21,31,40–44]. 0 5 0 0 1000 1500 RLU Vector TLR2 TLR2/6 TLR4 TLR4* TLR2/6 TLR2/6 TLR26 bDNA PAM-CSK L P S Control In this study we have used a novel reporter gene system to examine how late-responsive genes are regulated. We show with a number of promoters, including the κB-de- pendent ELAM promoter, that the levels of luciferase ac- tivity increase for at least 8 hrs following addition of microbial agonists, and are maintained thereafter long after early events such as nuclear NF-κB activation and MAP kinase activation and induction of early response genes such as TNF-α has peaked and declined. The ex- amples we have shown, IL-6, IL-12 and ELAM are only a subset of the promoter combinations we have tested suc- cessfully. At least for IL-1β and iNOS, the time course of luciferase activation is consistent with the time course of transcriptional activation of the corresponding gene measured previously using nuclear run-on transcription assays in the same cells [23]. Additive effects of different agonists on inducible cytokine production Amongst these sta- bly-inducible genes is p50NFκB, a repressor of κB-de- pendent transcription, which provides at least one explanation for suppression of transcription of early re- sponse genes such as TNF-α [45]. Figure 5 C b d g Combined effects of constitutively active toll-like receptors and added agonists. RAW264 cells were transiently co-trans- fected with the IL-12 promoter luciferase reporter genes and expression plasmids encoding wild-type and mutated (indi- cated by an asterisk) forms of the CD4/TLR chimaeric recep- tors as indicated either alone for TLR4 or in combination for TLR2 and 6. The experiment is described in detail in Materi- als and Methods. After overnight incubation, the cells were treated with either LPS [100 ng/ml], PAM3-CSK lipopeptide (100 ng/ml) or E-coli genomic DNA (10 µg/ml) as indicated. Each datapoint is the average of duplicate wells. The same pattern was observed in three separate experiments. The major variation between experiments was in the effect of the vector alone on basal reporter gene activity. The key observation in the present paper is that luci- ferase activity declines upon removal of the agonists LPS and bacterial DNA, and this decline can be prevented by immediate re-addition of the agonists. The observation that this is not readily apparent for the agonist PAM3- CSK4 may reflect something fundamentally different about its signaling pathway, or may simply reflect a re- duced ability of the cell to degrade or detoxify residual amounts of this ligand. The implications of the declining signals seen after ligand removal are two-fold. Firstly the cells are not LPS-unresponsive, and secondly mainte- nance of the induced luciferase requires continuous stimulation by LPS (or lipopeptide or bacterial DNA as the case may be). In keeping with this interpretation, the response of the IL-1, IL-6 and IL-12 promoters to sub- maximal doses of LPS peaked and declined earlier (Figs 2,3) presumably because the cells consume the ligand, and the response then declines. A useful analogy can be drawn with the activation of the CSF-1 receptor in mac- rophages. The addition of CSF-1 to cells previously starved of ligand causes a rapid activation of signalling pathways leading to MAP kinase activation, PI-3-kinase, IL-12 protein from RAW264 cells after 24 hrs. The same pattern was evident at 8 hrs (not shown). Controls in this experiment, DNasel-treated DNA and GpC oligonucle- otide (NAO-1) confirm that the response is, indeed, due to stimulatory DNA recognition in combination with LPS. In panel D, the observation is extended to primary bone marrow-derived macrophages, to provide assur- ance that this is not a peculiarity of the RAW264 cell line. Contrasting regulation of late response genes in macro- phages 0 1000 2000 3000 4000 IL-6 (pg/ml) 0 2000 4000 6000 8000 IL-12 (pg/ml) A B 0 1000 2000 3000 4000 5000 CON LPS AO-1 NAO-1 bDNA bDNA DNased LPS+AO-1 LPS+NAO-1 LPS+bDNA LPS+bDNA DNased treatment C TNF (pg/ml) 0 10000 20000 30000 40000 CON LPS AO-1 NAO-1 bDNA b DNA DNased LPS+AO-1 LPS+NAO-1 LPS+bDNA LPS+ bDNA DNased treatment IL-12 (pg/ml) D 0 1000 2000 3000 4000 IL-6 (pg/ml) 0 2000 4000 6000 8000 IL-12 (pg/ml) A B 0 1000 2000 3000 4000 5000 CON LPS AO-1 NAO-1 bDNA bDNA DNased LPS+AO-1 LPS+NAO-1 LPS+bDNA LPS+bDNA DNased treatment C TNF (pg/ml) IL-12 (pg/ml) Fi 6 0 1000 2000 3000 4000 IL-6 (pg/ml) 0 2000 4000 6000 8000 IL-12 (pg/ml) A B 0 1000 2000 3000 4000 5000 CON LPS AO-1 NAO-1 bDNA bDNA DNased LPS+AO-1 LPS+NAO-1 LPS+bDNA LPS+bDNA DNased treatment C TNF (pg/ml) 0 10000 20000 30000 40000 CON LPS AO-1 NAO-1 bDNA b DNA DNased LPS+AO-1 LPS+NAO-1 LPS+bDNA LPS+ bDNA DNased treatment IL-12 (pg/ml) D other response mimicked by LPS; [9]). Yet, uPA mRNA is maintained for as long as CSF-1 is present; removal of CSF-1 leads to rapid degradation of uPA mRNA in a man- ner that is blocked by inhibitors of protein or RNA syn- thesis [47]. The implication is that after the initial signalling pulse, the cells must continue to recognise and respond to ligand in a quite distinct manner. Indeed, al- though the steady level of CSF-1 receptor is low, activity can be discerned from the continued degradation of the ligand by the cells [48]. In the case of LPS, recent availa- bility of TLR4 antibody supports a similar mechanism. The initial recognition of LPS was shown to lead to rapid down-modulation of TLR4 from the cell surface and re- duced expression of TLR4 mRNA [5]. 0 10000 20000 30000 40000 CON LPS AO-1 NAO-1 bDNA b DNA DNased LPS+AO-1 LPS+NAO-1 LPS+bDNA LPS+ bDNA DNased treatment IL-12 (pg/ml) D Although we have questioned the nature of LPS tolerance in macrophages treated in vitro, the data we have ob- tained actually provide a plausible mechanism for LPS tolerance in vivo. The double reporter system enabled us to confirm and extend an earlier finding showing that early LPS responses, such as induction of TNF-α and NF- κB-dependent transcriptional activation (e.g. Contrasting regulation of late response genes in macro- phages part of the ELAM-1 activation and the earlier HIV-1-LTR respons- es) require at least 10-fold lower concentration of LPS than later responses. We suggest that early responses such as TNF-α induction are activated solely in response to an initial bolus of LPS or other agonist interacting with receptor, a response that is truly concentration depend- ent. By contrast, activation of late response genes, which requires the continued presence of the ligand, depends upon the amount of ligand available to each cell. Agonist availability is a function of the concentration of the cells and the rate at which they deplete the medium of agonist. By analogy, that pattern was demonstrated with CSF-1, where the dose response curve for induction of uPA and cell growth is a function of cell concentration [49]. In simple terms, late response genes require more agonist because the cells must receive a continuous supply for the duration of the biological response. When macro- phages respond to local LPS exposure in vivo, where the agonist is rapidly cleared or diluted by body fluids, or they have engulfed a single microorganism, they give a pulse of cytokines and chemokines that activates the acute phase and causes local inflammation. Conversely, continued exposure of the cells to the stimulus is indica- tive of the failure of initial defence, and the need to max- imally activate the host, including the acquired immune system. When LPS is not cleared, the macrophages pro- gressively induce more and more of the cytokine genes in a manner that is a linear function of the duration of ex- posure. Contrasting regulation of late response genes in macro- phages Macrophages that have been treated with a maximal LPS dose in vitro fail to respond to restimulation in most ear- ly response assays including phosphorylation of MAP Ki- nases, activation of NF-κB and AP-1 transcription factors, and induction of a range of cytokine and chem- okine genes [5,17]. Such responses are transient respons- es to LPS, and have declined to "almost" basal levels by the time of restimulation, regardless of whether LPS is http://www.biomedcentral.com/1471-2172/2/11 BMC Immunology 2001, 2:11 Figure 6 Combined effects of LPS and bacterial DNA on pro-inflam- matory cytokine production Panels A-C. RAW264 cells were stimulated for 24 h with medium control, LPS [100 ng/ml], stimulatory CpG oligonucleotide AO-1 (3 µM), non-stimula- tory GpC oligonucleotide NAO-1 (3 µM), E. coli DNA (bDNA, 5 µg/ml), DNase I-treated bDNA (5 µg/ml) or com- binations as indicated. Cell supernatants were harvested and IL-6 (Panel A), IL-12 (Panel B] and TNF-β (Panel C) levels were estimated by ELISA. Data are mean of triplicates ± SD and similar results were obtained in 3 independent experi- ments. Panel D. Bone marrow-derived primary macrophages were stimulated with medium control, LPS (100 ng/ml), AO- 1 (3 µM), NAO-1 (3 µM), E. coli DNA (bDNA, 5 µg/ml), DNase I-treated E. coli DNA (5 µg/ml), or combinations as indicated. After 24 h, cell supernatants were harvested and IL-12 levels in supernatants were estimated by ELISA. Data (mean of triplicates ± SD) are representative of 4 independ- ent experiments. Conclusion h h We have shown that macrophages exposed to LPS or oth- er microbial agonists do not become refractory to stimu- lation. Rather, they have entered a new steady state which requires continued stimulation and in which other agonists can generate a further amplification of the re- sponse. It may be a characteristic of the "activated" steady state that genes activated during the transition phase from the "inactive" state cannot be reactivated. We have presented an analogy with growth factor responses, where macrophage cells do not respond to CSF-1 with re- activation of the Ets/APl pathway unless they have been starved of ligand and maximal cell surface receptor has re-appeared. This is not referred to as CSF-1 tolerance, and the phenomenon that has been studied as LPS toler- ance in vitro is, based upon our findings, equivalent. This state of tolerance is, in reality, a state of sustained and continued activation. Figure 6 g Combined effects of LPS and bacterial DNA on pro-inflam- matory cytokine production Panels A-C. RAW264 cells were stimulated for 24 h with medium control, LPS [100 ng/ml], stimulatory CpG oligonucleotide AO-1 (3 µM), non-stimula- tory GpC oligonucleotide NAO-1 (3 µM), E. coli DNA (bDNA, 5 µg/ml), DNase I-treated bDNA (5 µg/ml) or com- binations as indicated. Cell supernatants were harvested and IL-6 (Panel A), IL-12 (Panel B] and TNF-β (Panel C) levels were estimated by ELISA. Data are mean of triplicates ± SD and similar results were obtained in 3 independent experi- ments. Panel D. Bone marrow-derived primary macrophages were stimulated with medium control, LPS (100 ng/ml), AO- 1 (3 µM), NAO-1 (3 µM), E. coli DNA (bDNA, 5 µg/ml), DNase I-treated E. coli DNA (5 µg/ml), or combinations as indicated. After 24 h, cell supernatants were harvested and IL-12 levels in supernatants were estimated by ELISA. Data (mean of triplicates ± SD) are representative of 4 independ- ent experiments. induction of AP-1 and Ets-2 transcription factors, and ul- timately transcriptional activation of specific target genes such as the urokinase plasminogen activator gene (uPA) [46]. It is a sequential cascade, since Ets-2 must be induced before it can be phosphorylated by activated MAP kinase [46]. Ets-2 phosphorylation is also induced by LPS [38] and probably relates to the ability of LPS to prevent apoptosis in macrophages [9]. In stimulated cells, the CSF-1R binds ligand, and is rapidly internalised and degraded. The CSF-1R mRNA (encoded by the c-fms proto-oncogene) is substantially down-modulated (an- The additive nature of signalling pathways in macrophages As discussed in the introduction, each of the three broad classes of agonist studied here apparently requires the http://www.biomedcentral.com/1471-2172/2/11 http://www.biomedcentral.com/1471-2172/2/11 BMC Immunology 2001, 2:11 adaptor protein MyD88 for maximal activity and acts to induce NF-κB. Despite the shared pathways, maximal activation of the TLR4 pathway, with either CD4-TLR4 or LPS, did not prevent an additive response to the TLR2/6 agonists lipopeptide and peptidoglycan. Con- versely, maximal activation of the TLR2/6 pathway with lipopeptide did not block an additive response to LPS. Bacterial DNA effects were additive with either of these classes of agonist. There are relatively few reports on the effects of the combined addition of microbial agonists. In recent studies, two groups studying early gene responses have reported "cross-tolerance" of TLR2 and TLR4-me- diated responses [25,26]. In other studies, responses were at least additive. Figure 6 For example, bacterial DNA and LPS were reported to exert more than additive effects on the iNOS gene in macrophages [50]. We have now shown that the effects on cytokines such as TNF-α, IL-6 and IL- 12 are also more than additive (Fig 6). Materials and Methods Promoter constructs There may be good reason for the innate immune system to retain the ability to respond maximally to different classes of infectious challenge. The global responses to TLR4 and TLR2/6 activation may not be identical (e.g. gram-positive organisms may not induce interferon-β [51]), and the innate immune system may quite com- monly respond to multiple classes of challenge in a mixed infection. For example, TLR4-deficient mice dis- play reduced pathology when challenged with mixed in- fections [52]. The human interleukin 1B promoter clones into the luci- ferase vector pGL3 was a gift from Dr. Matt Fenton [53]. We subcloned the promoter fragment into the corre- sponding sites of the Promega renilla luciferase vector. The IL-6 luciferase reporter (pIL6-luc651) was provided by Dr. Oliver Eickelberg [54]. The IL-12 reporter was constructed by us. The murine IL-12 p40 promoter re- gion from -349 to +56 [55] was generated by PCR and cloned into the pGL2-basic KpnI/XhoI site. The ELAM luciferase reporter is as previously described [33]. From a signaling perspective, the simplest explanation is that the response to any agonist is limited solely or pri- marily by the availability of surface receptor, and all the downstream effectors are available in excess. Based upon the ability of LPS to repress TLR4 mRNA expression [5], CD4-TLR4 and CD4-TLR2/6 probably act to repress sur- face expression of the corresponding full-length recep- tors, thereby preventing super-activation. An alternative is that each pathway associates with a separate function- al pool of signalling molecules that need not be identical. If this were the case, one might expect that overexpres- sion of CD4-TLR4 would result in the utilization of more downstream signaling molecules than full activation of endogenous TLR4 could assemble. This appears not to be so since maximal activation by LPS and CD4-TLR4 were remarkably similar. The fact that bacterial DNA ac- tion is additive with either CD4-chimaera, or with LPS or lipopeptide, implies that recognition does not absolutely require either TLR4 or TLR2/6. In keeping with this view, macrophages and B cells from knockout mice defi- cient in either TLR2 or TLR4 respond normally to bacte- rial DNA, whereas the recent knockout of TLR9 is selectively defective in DNA signalling [10]. Transfection 6 5 × l06 RAW264 cells were transfected by electropora- tion as described previously [56]. To avoid phenotypic drift in cell culture [23], all experiments were carried out using RAW264 cells that had been recently obtained from the American Type Culture collection, expanded immediately, and then frozen in aliquots. Cells were maintained in culture for no more than 4–6 weeks. All cell culture, including the electroporation procedure, was carried out in RPMI1640 medium with 10 mM HEPES (pH7.4) and 10% fetal bovine serum. The inclu- sion of HEPES is a departure from previous studies and appears to increase survival and transfection efficiency. In each transfection, we added 10 µg of the desired firefly luciferase reporter gene, 10 µg of the IL-1β promoter-re- nilla luciferase plasmid and 2 µg of the selective marker plasmid pNeoTak which also directs expression of the Tet represser [34]. In this system, only the Neo resist- ance is relevant, but the Tet represser could be used sub- sequently for studies using inducible modifier cassettes. After transfection, the cells were placed in a 100 mm square bacteriological petri dish (Sterilin) with approxi- mately 25 ml of medium. Following overnight incuba- BMC Immunology 2001, 2:11 http://www.biomedcentral.com/1471-2172/2/11 tion, most viable cells adhere weakly. The medium was changed, and G418 (200 µg/ml) was added. The cells were left in culture with one change of medium to re- move dying cells around day 4–5. By day 7–10, several hundred individual foci of stably transfected cells were evident in each dish. These were removed by washing of the surface using a syringe with an 18 g needle (the cells are weakly adherent to bacteriological plastic), expanded and frozen in aliquots for future experiments. tion, most viable cells adhere weakly. The medium was changed, and G418 (200 µg/ml) was added. The cells were left in culture with one change of medium to re- move dying cells around day 4–5. By day 7–10, several hundred individual foci of stably transfected cells were evident in each dish. These were removed by washing of the surface using a syringe with an 18 g needle (the cells are weakly adherent to bacteriological plastic), expanded and frozen in aliquots for future experiments. activating oligonucleotide-1 (AO-1; 5'-GCT CAT GAC GTT CCT GAT GCT G-3') and nonactivating oligonucle- otide (NAO-1; 5'-GCT CAT GAG CTT CCT GAT GCT G-3', [9]). Luciferase assays l f h For analyses of the response to various agonists, 2 × 105 of the transfected RAW264 cells were placed in 1 ml of medium in 24 well tissue culture plates and incubated overnight. In some experiments, the medium was re- placed with prewarmed medium. Agonist at the desired final concentration was added in a small volume, and the cells were incubated for the desired time at 37°C. Cells were harvested and analysed simultaneously for firefly and renilla luciferase activity using the Promega Dual Luciferase reporter assay reagents. In each experiment, duplicate wells were tested; results generally differed by less than 10% of the mean. 5. Nomura F, Akashi S, Sakao Y, Sato S, Kawai T, Matsumoto M, Nakan- ishi K, Kimoto M, Miyake K, Takeda K, Akira S: Cutting edge: en- dotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression. J Immunol 2000, 164:3476-3479 6. Aderem A, Ulevitch RJ: Toll-like receptors in the induction of the innate immune response. Nature 2000, 406:782-787 p 7. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Take- da K, Akira S: Differential roles of TLR2 and TLR4 in recogni- tion of gram-negative and gram-positive bacterial cell wall components. Immunity 1999, 11:443-451 p y 8. Stacey KJ, Sweet MJ, Hume DA: Macrophages ingest and are ac- tivated by bacterial DNA. J Immunol 1996, 157:2116-2122 9. Sester DP, Beasley SJ, Sweet MJ, Fowles LF, Cronau SL, Stacey KJ, Hume DA: Immunostimulatory DNA down-modulates CSF-1 receptor surface expression on murine macrophages with concomitant growth arrest and factor-independent survival. J. Immunol 1999, 163:6541-6550 References 1. Sweet MJ, Hume DA: Endotoxin signal transduction in macro- phages. J. Leukoc. Biol. 1996, 60:8-26 1. Sweet MJ, Hume DA: Endotoxin signal transduction in macro- phages. J. Leukoc. Biol. 1996, 60:8-26 p g J 2. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Take- da K, Akira S: Cutting edge: Toll-like receptor 4 [TLR4]-defi- cient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999, 162:3749-3752 3. Poltorak A, He X, Smimova I, Liu MY, Huffel CV, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B: Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998, 282:2085-2088 4. Qureshi ST, Lariviere L, Leveque G, Clermont S, Moore KJ, Gros P, Malo D: Endotoxin-tolerant mice have mutations in Toll-like receptor 4 [Tlr4] [see comments] [published erratum ap- pears in J Exp Med 1999 May 3; 189 [9]: following 1518]. J Exp Med 1999, 189:615-625 Transfection 6 RAW264 cells were plated out at 2 × 105 cells per well and BMM at 5 × 105 cells per well in 1 ml complete medium (in the case of BMM, medium contained 10% L929 medium) in 24 well plates on the evening before the experiment. The following day cells were stimulated for 24 h as outlined in the figure legends, supernatants were collected and IL-6, IL-12 and TNF-α levels were de- termined by ELISA using paired antibodies (Pharmin- gen, San Diego, CA). For studies of the dominant-positive TLR receptors, RAW264 cells were transiently transfected by electropo- ration using 2 µg of the desired expression plasmid en- coding the CD4-TLR chimaeric receptor described in Ozinsky et al. [35] together with 10 µg of the IL12 pro- moter luciferase vector. Cells from one transfection were plated into 1 ml of medium in 24 wells for 4–6 hours, the medium was changed to remove dead cells, and the cells were then incubated overnight. The desired agonist was added to the cells without further change of medium, and the cells were harvested 8 hrs later for luciferase deter- mination. http://www.biomedcentral.com/1471-2172/2/11 1 regulation by lipopolysaccharide pretreatment. Arch Surg 1994, 129:1263-1269 34. Underhill DM, Ozinsky A, Smith KD, Aderem A: Toll-like receptor- 2 mediates mycobacteria-induced proinflammatory signal- ing in macrophages. Proc Natl Acad Sci U S A 1999, 96:14459-14463 16. Makhlouf M, Ashton SH, Hildebrandt J, Mehta N, Gettys TW, Halush- ka PV, Cook JA: Alterations in macrophage G proteins are as- sociated with endotoxin tolerance. Biochim Biophys Acta 1996, 1312:163-168 35. g p g 35. Ozinsky A, Underhill DM, Fontenot JD, Hajjar AM, Smith KD, Wilson CB, Schroeder L, Aderem A: The repertoire for pattern recog- nition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors. Proc Natl Acad Sci U S A 2000, 97:13766-13771 17. Medvedev AE, Kopydlowski KM, Vogel SN: Inhibition of lipopoly- saccharide-induced signal transduction in endotoxin-toler- ized mouse macrophages: dysregulation of cytokine, chemokine, and toll-like receptor 2 and 4 gene expression. J Immunol 2000, 164:5564-5574 36. Qureshi ST, Lariviere L, Leveque G, Clermont S, Moore KJ, Gros P, Malo D: Endotoxin-tolerant mice have mutations in Toll-like receptor 4 [Tlr4] [see comments] [published erratum ap- pears in J Exp Med 1999 May 3; 189 [9]: following 1518]. J Exp Med 1999, 189:615-625 18. Kraatz J, Clair L, Rodriguez JL, West MA: In vitro macrophage en- dotoxin tolerance: defective in vitro macrophage map kinase signal transduction after LPS pretreatment is not present in macrophages from C3H/HeJ endotoxin resistant mice. Shock 1999, 11:58-63 37. Poltorak A, He X, Smimova I, Liu MY, Huffel CV, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B: Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998, 282:2085-2088 19. Fahmi H, Chaby R: Selective refractoriness of macrophages to endotoxin-induced production of tumor necrosis factor, elic- ited by an autocrine mechanism. J Leukoc Biol 1993, 53:45-52 38. Sweet MJ, Stacey KJ, Ross IL, Ostrowski MC, Hume DA: Involve- ment of Ets, rel and Spl-like proteins in lipopolysaccharide- mediated activation of the HIV-1 LTR in macrophages. J In- flamm 1998, 48:67-83 y J 20. Fahmi H, Charon D, Mondange M, Chaby R: Endotoxin-induced desensitization of mouse macrophages is mediated in part by nitric oxide production. Infect Immun 1995, 63:1863-1869 y p 21. Frankenberger M, Pechumer H, Ziegler-Heitbrock HW: Inter- leukin-10 is upregulated in LPS tolerance. 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https://openalex.org/W2551684864	https://opus.lib.uts.edu.au/bitstream/10453/105195/1/12904_2016_Article_167.pdf	English	null	Using telehealth to support end of life care in the community: a feasibility study	BMC palliative care	2,016	cc-by	6,095	© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tieman et al. BMC Palliative Care (2016) 15:94 DOI 10.1186/s12904-016-0167-7 Tieman et al. BMC Palliative Care (2016) 15:94 DOI 10.1186/s12904-016-0167-7 Open Access Abstract Background: Telehealth is being used increasingly in providing care to patients in the community setting. Telehealth enhanced service delivery could offer new ways of managing load and care prioritisation for palliative care patients living in the community. The study assesses the feasibility of a telehealth-based model of service provision for community based palliative care patients, carers and clinicians. Methods: This study was a prospective cohort study of a telehealth-based intervention for community based patients of a specialist palliative care service living in Southern Adelaide, South Australia. Participants were 43 community living patients enrolled in the Southern Adelaide Palliative Service. To be eligible patients needed to be over 18 years and have an Australian modified Karnofksy Performance Score > 40. Exclusion criteria included a demonstrated inability to manage the hardware or technology (unless living with a carer who could manage the technology) or non-English speaking without a suitable carer/proxy. Participants received video-based conferences between service staff and the patient/carer; virtual case conferences with the patient/carer, service staff and patient’s general practitioner (GP); self-report assessment tools for patient and carer; and remote activity monitoring (ACTRN12613000733774). Results: The average age of patients was 71.6 years (range: 49 to 91 years). All 43 patients managed to enter data using the telehealth system. Self-reported data entered by patients and carers did identify changes in performance status leading to changes in care. Over 4000 alerts were generated. Staff reported that videocalls were similar (22.3%) or better/much better (65.2%) than phone calls and similar (63.1%) or better/much better (27.1%) than face- to-face. Issues with the volume of alerts generated, technical support required and the impact of service change were identified. Conclusions: The trial showed that patients and carers could manage the technology and provide data that would otherwise not have been available to the palliative care service. Trial registration: Australian New Zealand Clinical Trials Registry ACTRN12613000733774 registered Trial registration: Australian New Zealand Clinical Trials Registry ACTRN12613000733774 registered on 02/07/2013. Trial registration: Australian New Zealand Clinical Trials Registry ACTRN12613000733774 registered on 02/07/2013. Keywords: Telemedicine, Palliative care, Home care services Keywords: Telemedicine, Palliative care, Home care services Keywords: Telemedicine, Palliative care, Home care services Using telehealth to support end of life care in the community: a feasibility study nnifer J. Tieman1, Kate Swetenham2, Deidre D. Morgan1, Timothy H. To2 and David C. Currow1 Correspondence: jennifer.tieman@flinders.edu.au 1Palliative and Supportive Services, Flinders University, Bedford Park, South Australia, Australia Full list of author information is available at the end of the article Methods Reviews of the feasibility and effectiveness of telehealth suggest that telehealth may offer benefits to patients with a range of conditions but that various factors may influence its usefulness and effectiveness such as the severity of the condition or the disease trajectory and how the intervention works within the service delivery model [8–11]. Reviews also indicate that there is satisfaction with the use of telehealth, mainly videoconferencing applications, by patients and by carers [12–14]. Even given the range of potential applications that could be useful within palliative care, there is limited re- search into the potential role, feasibility and effectiveness of telehealth applications for palliative care delivered into the community. Authors have acknowledged the potential contribution that telehealth could play in pal- liative care for patients in rural and remote areas [15], the role that mobile technologies could play [16], and opportunities for specific population groups such as paediatrics [17] or lung cancer [18]. However, reviews and trials have also highlighted the need to build the evi- dence around palliative care telehealth in the community [12–14, 19, 20]. Recent evidence also indicates that provider acceptance of telehealth within palliative care organisations plays a key role in ensuring its uptake and utilisation [21]. As such, there is a need to build the evidence base around the benefits and burdens of tele- health, together with its acceptability to patients and their families and to health providers. Understanding the ex- tent, likelihood, and manner in which such interventions can be implemented as planned and proposed is import- ant in determining the feasibility of telehealth as part of care delivery. This study investigates the feasibility of a tel- ehealth intervention for community based palliative care patients, carers and clinicians (ACTRN12613000733774). The components of the palliative care telehealth model to support patient and carer in the home environment and to enhance clinical feedback are outlined in Fig. 1. They in- cluded ongoing video-based conferences between service staff and the patient or carer, virtual case conferences with the patient and carer, service staff and the patient’s general practitioner (GP), self-report assessment tools for the patient and carer, and remote activity monitoring. The study design was a prospective cohort study of a telehealth-based intervention for SAPS palliative care patients based in the community. Methods home visits. As demand for palliative care increases as a result of an ageing population and progressive chronic ill- nesses, mechanisms that support home-based care need to be explored. Telehealth represents one avenue for in- vestigation, offering more regular engagement through videoconferencing, potentially continuous remote moni- toring to highlight changing performance status, and im- mediate access to resources and information for patients and families. For clinicians and services, a telehealth enhanced service for the community may enable them to allocate their staff resources more appropriately to pa- tients where self-reported symptom needs are high or where unanticipated changes are being identified. The study was conducted in the Southern Adelaide Palliative Care Service (SAPS), South Australia. Ethics approval for the study was granted by the Southern Adelaide Clinical Human Research Ethics Committee on 2 August 2013 (168.13). Five subsequent amendments were sought and all approved. g pp A Palliative Care Telehealth Research Team (PCTRT) was established to guide the development and imple- mentation of the telehealth model for use by the com- munity team of a specialist palliative care service. Membership of the PCTRT included the Director of the Clinical Service, clinical staff (medicine, nursing, allied health), and researchers with expertise in clinical trial design, health informatics, health services research, and evaluation. A Project Manager was appointed to support the project development. Input was sought and received during concept and module development from service providers, stakeholders, and patients and carers involved with the service. The PCTRT met regularly with the tel- ehealth technical team as well as with the external IT consultant and web provider across the course of the project. As telehealth resources must satisfy the utility and usability criteria of clinicians and consumers not just those of funders and system providers, meetings were held with the clinicians providing direct care to enable input and feedback on the proposals and ongoing management of the telehealth applications. Details on the development of the resources have been previously reported [22]. Telehealth uses information and communication tech- nologies to capture and transmit health data and to de- liver services and information [7]. Not all healthcare can be delivered through telehealth, however, many special- ties are using or have tested telehealth to augment or re- place some aspects of care and it is being delivered in various clinical service settings. Background palliative care service [1–3]. Home-based care often covers a period of time from weeks to months, or even years. Most palliative care patients indicate that they would prefer to be cared for, and to die, at home. How- ever, care in the community commonly requires support from family or friends who fulfil a vital caregiver role which enables the patient to remain at home [4, 5]. It also requires flexible and responsive health care provision that provides continuity of care and addresses changing health needs [6]. This can be difficult to deliver within resource-constrained services where contact may be limited to intermittent telephone calls and occasional Despite increasing investment in inpatient palliative care facilities, the vast majority of palliative care will continue to be provided in the home where patients and their families may be cared for by their primary care providers, in shared care arrangements with specialist palliative care services or as patients of the specialist Page 2 of 7 Tieman et al. BMC Palliative Care (2016) 15:94 Page 2 of 7 Tieman et al. BMC Palliative Care (2016) 15:94 Methods Patients were invited to participate in the study if they were not in bed more than 50% of the time as indicated by a performance score greater than 40 using Australia-modified Karnofsky Performance Status (AKPS) assessment at the point of entry to the study [23]. Participants needed to be 18 years of age or older. Potential participants who demonstrated inability to manage the hardware or tech- nology (unless living with a carer who could manage the technology) or who were non English speaking without a suitable carer/proxy were excluded. Participants were able to withdraw at any time. Potentially eligible participants were identified from the SAPS Client List during a weekly screening process. They were approached by the SAPS clinical team, Tieman et al. BMC Palliative Care (2016) 15:94 Page 3 of 7 Fig. 1 Components of Telehealth model health care utilisation monitoring via a self-report electronic diary planned responses if self-reported assessments exceeded pre-defined thresholds generally identified by alerts. Responses included face to face videoconference, home visit and/or virtual case conference with the family, GP and palliative care service access to appraised and structured online information resources for patients and carers were included in project specific pages held on the CareSearch website [28], and activity and weight monitoring using FitBit®™technology [29]. Applications enabled self-reported data to be entered and stored in the CareSearch website [28]. SAPS clinicians pro- viding direct care to study participants received an iPad for project use and training in the functionality of the iPad and the individual applications. Following each video interaction with a client, the participating clinicians completed a brief assessment on the quality of the technology and the signifi- cance of the interaction. Clinician perceptions on the tele- health project were collected in focus groups and interviews and have been reported elsewhere [30]. (usually the Clinical Nurse Consultant or Caregiver Network Facilitator) to consider participation in the study. Potential participants were then contacted by a member of the investigating team to discuss participa- tion in the study. An appointment was made to outline the study and obtain consent. Given a lack of familiarity with technology in many cases and potential burden given the stage of illness, a second appointment was routinely made to demonstrate the technology uses and to assist in entering the first set of data with the patient and/or carer. Results BMC Palliative Care (2016) 15:94 Page 4 of 7 Page 4 of 7 Table 1 Number of times tools accessed by patients and carers and number of alerts arising Application (Tool) Scale access Alerts arising Australian-modified Karnofsky Performance Scale (Function) 1058 611 Symptom Assessment Scale (Symptoms) 3009 4386 Caregiver Assessment Questionnaire-Caregiver Network Service (Carer self-assessment) 263 283 Table 1 Number of times tools accessed by patients and carers and number of alerts arising through CAQ-CNS entry (18.2%) or non-completion of any data for 2 days (14.6%). This indicates that clinical responses are being made to data being entered by the patient. With respect to the outcomes of clinical contacts, 111 notes were recorded in clinical records. A change in medication was the most common noted outcome. Data indicate that four admission recommendations were made following a video call (Table 2). The clinical staff made 121 ratings on conducting clin- ical assessments using videocalls compared to a phone call and 111 ratings comparing videocalls to face to face. The nurses reported that videocalls were similar to (22.3%) or better/much better than (65.2%) phone calls. Videocalls were also reported to be similar to (63.1%) or better/much better (27.1%) than face-to-face contacts (Table 3). Alerts were generated by the system when data en- tered by the patient or by the carer breached pre- determined thresholds. For example, the threshold for the SAS was ≥5 and ≤70 for the AKPS. There were 611 alerts arising from changes in the AKPS across the study and 4386 alerts generated through SAS. Of the 4386 alerts arising from the SAS, pain and fatigue were the most commonly reported symptoms generating 726 and 1205 alerts respectively. Data showed that patients were also using the ‘Other’ category in SAS to identify symp- toms not included in the scale that were causing them concern. Thirty-five of 38 carers completed the CAQ- CNS at least once (92.1%). A score above seven on any item in the CAQ-CNS was classified as an alert. Nurses entered responses about clinical outcomes in their clinical notes and identified instances in which tele- health made a difference to clinical practice. Results Forty-three community participants received active ser- vices between 6 June 2013 and 31 July 2014. Forty-one patients on the trial had a cancer diagnosis. On average, patients spent 128.9 days (range: 17 to 415) on the pro- gram. The average age of patients was 71.6 years (range: 49 to 91 years). Thirty-one patients were aged 65 years or older with 12 of these being over 80 years. Of the 43 participants, 38 were patient-carer dyads and five partici- pants had no carer. Seven participants lived alone with two having visiting rather than resident carers. Two of the carers were siblings, three were daughters and the remaining 33 were partners or spouses. Seventeen of the patients were women (39.5%) and 26 (60.5%) were men. Only the carer was consented in one dyad as the pa- tient’s level of cognition was insufficient for self- reporting of symptoms and function. Participants received a combined telecare and telemo- nitoring package using an iPad tablet for data entry [24]. The components of the package included: structured online video contacts between the patient and the Nurse Clinical Practice Consultant (CPC) and between the carer and the Nurse Clinical Practice Consultant and/or Caregiver Network Facilitator (CNF) patient self-assessment and online data entry using the following tools—AKPS [23], Assessment of Quality of Life (AQoL) [25] and Symptom Assessment Scale (SAS) [26]. Alerts were sent to the relevant health professional if scores breached pre-specified thresholds Data entry shows that patients and carers were able to use the technology and did self-report using the applica- tions. Table 1 reports the number of times the various tools were accessed for data entry. As the SAS comprises seven separate symptom reports, the number of alerts can exceed the number of times the scale was accessed as a patient may be reporting high scores for multiple symptoms. This is similar for the CAQ-CNS which also comprises multiple items that could lead to an alert. carer self-report using the service’s carer needs self-assessment tool, Caregiver Assessment Questionnaire-Caregiver Network Service (CAQ-CNS) which was developed for use in the community facilitator/caregiver pilot study and then adopted for use in SAPS [27], and assessment of the Tieman et al. Results Nurses also indicated that technology had been very effective in en- abling the patient or carer to be reassured (57.3%), quicker problem management (23.2%), identification of problems that may not have been recognised (20.5%), ability to resolve issues that would have previously re- quired a home visit (16.8%), and ability to share informa- tion with other health professionals (7.6%). Each of the tools had a specified frequency for patient and/or carer self-report. Patients and carers were instructed to complete the AKPS on a weekly basis. On average, patients entered data 1.25 times per week, or 25% more frequently than expected. The SAS was expected to be competed daily however, patients entered data less fre- quently, on average 0.73 times daily or 27% less than ex- pected. Patients generally entered data more closely to what was required at the beginning of their time on the program (i.e., the first 2 weeks). Of those who died while receiving the telehealth intervention (n = 15), the deceased had on average stopped entering data 16 days before death. On average, carers entered data 0.60 times per week. The data shows that the actual patterns of self- reporting by patients and carers varied from the expected rates of data entry. Patient and carer attitudes and responses to the tele- health initiative were sought through interviews and are being reported separately. Feasibility of telehealth This study sought to determine if a telehealth enhanced community service for palliative care patients was feasible. The trial showed that patients and carers, including pa- tients over 80 years, could manage the technology and provide data that would otherwise not have been available to the palliative care services. Self-reported data entered by patients and carers did identify changes in performance state and in symptom distress triggering alerts to the ser- vice provider. Scheduled videocall contacts and contacts made in response to triggers led to changes in care. Clini- cians reported that the quality of the telehealth contact was acceptable and in most case comparative to current In total, there were 180 recorded contacts made with patients, carers or both arising from scheduled calls or from contacts made in response to alerts. The primary mode of contact was video-conferencing (56.7%), followed by phone call (29.4%), then face-to-face (13.9%). Contact was most often made with both carers and patients (44.9%), followed by carer only 35.9%) and patient only (19.1%). Scheduled contact (67.3%) was more common than unscheduled contact (32.7%). Fifty seven unscheduled contacts were made. Reasons for un- scheduled contacts were: symptom trigger alerted through SAS entry (25.5%), followed by message from patient (20.0%), decline in functional status alerted through AKPS entry (21.8%), caregiver trigger alerted Table 2 Reported outcome of service contact by type of contact Phonecall Videocall Face to face Medication change 10 15 11 Admission recommendations 1 4 – Other 4 9 6 No change 12 33 6 Total 27 61 23 Tieman et al. BMC Palliative Care (2016) 15:94 Page 5 of 7 Table 3 Comparison of videocall to normal practice Video review Much worse Worse Similar Better Much better Compared to phone call (N = 121) 4 (3.3%) 11 (9.1%) 27 (22.3%) 59 (48.7%) 20 (16.5%) Compared to face to face (N = 111) 3 (2.7%) 7 (6.3%) 70 (63.1%) 30 (27.0%) 1 (0.1%) Abbreviations: N number remain high as a patient’s disease progresses. Such alerts will continue, even if the symptom cannot be fully cor- rected. Clinicians noted that their clinical knowledge of the patient also informed their interpretation of rou- tinely high symptom alerts. Their feedback suggests that review of the agreed clinical thresholds and a more so- phisticated dashboard that would place an alert in the context of the patient’s status over time could assist in prioritising and responding to alerts. Feasibility of telehealth Addressing the issue of burdensome alerts is important as in other sys- tems, particularly computerized provider order entry (CPOE) and clinical decision support (CDS), alert fatigue and ignoring alerts tends to increase with growing ex- posure to alerts and heavier use of the systems [32, 33]. modes of contact. These findings suggest that telehealth approaches to support community-based palliative care patients are feasible and valuable for clinical care. For palliative care patients, where changes in symptoms are not uncommon and relatively high scores can also be expected at some points or with some co-morbidities, telehealth provides a window into the community patient’s status. There are some suggestions that those receiving care based in the community may not always have the same outcomes as inpatient care. For example, summary data from the Australian Palliative Care Outcomes Collab- oration for the period 2010 to 2015 notes that inpatients are more likely to have a shorter time in the unstable phase than those in the community (86 to 77%). It also re- ports that those being cared for in inpatient settings are more likely to have no pain or mild pain at the end of a period of care provision than those receiving community based care (89 to 82%) [31]. Telehealth may provide a mechanism to reduce this variability between inpatient and community settings by enabling more consistent monitoring and more reactive management of symptoms. Variability in the patterns of self-reported data entry sug- gests that the impact of compliance needs to be considered in assessing feasibility. Patient compliance issues in home- based telehealth studies have been recognised and reported and it is not uncommon to find that compliance with data entry will diminish over time [34]. User training and user support have been identified as mechanisms to mitigate against this effect [34]. However, it is worth considering that in a palliative care context where changes and decline should be anticipated that variability in participation and compliance with the data entry regime should also be antici- pated [35]. Further work may help to identify the optimal point at which the introduction of telehealth in community- based care should occur and whether changes in data entry may also be a further indicator of a changing patient condi- tion. Telehealth support requirements Telehealth support requirements Implementing telehealth depends on a sophisticated in- frastructure given current interoperability consider- ations, and privacy and security concerns within the health system. These add to the complexity of planning for service delivery [36, 37]. Providing a telehealth en- hanced palliative care service required dedicated tech- nical resource to be made available to support clinical service delivery. However, some of these technical sup- port needs were related to the capture of research and evaluation data to assess feasibility of the approach ra- ther than the service delivery itself. Others related to meeting the specific processes and regulations needed to allow some level of integration with clinical record Feasibility of telehealth The possibility of normalising telehealth applications by introducing them in ambulatory clinic as a means of familiarising patients and caregivers with their functions and applications may also be worthwhile. Telehealth may also enable a more equitable management of palliative care resources by enabling clinical time to be di- rected to those in the community with the greater need. The current model results in limited contact between visits; the telehealth intervention captures escalating clinical need that otherwise remains hidden until a crisis arises. Given policy directions and consumer desires to remain at home, telehealth may also assist in managing the projected in- creased demand for palliative care services associated with ageing, progression of chronic diseases and more timely re- ferral to palliative care in the disease trajectory. Proactively monitoring symptoms, rather than responding to crises, could enable teams to deliver care in a more efficient man- ner by targeting community visits where symptom change or carer burden are being recorded. Authors’ contributions JJT has contributed to the study design, collected data, analysed the information and wrote the paper; KS, DDM, THT and DCC, have contributed to the study design, data collection and analysis, revised the draft papers and made intellectual contributions. All authors read and approved the final manuscript. Alerts and data reporting While this study has shown that telehealth enables self- reporting of symptoms and the generation of alerts, it also highlights complexity around the role of alerts. Alerts did not necessarily result in a clinical contact or response. In many instances they appeared to be provid- ing clinical information rather than acting as a clinical trigger. Moreover, even though the alerts were being triggered in accordance with agreed clinical thresholds, the number of ongoing alerts proved burdensome. For example, breathlessness scores may increase and may Tieman et al. BMC Palliative Care (2016) 15:94 Page 6 of 7 Page 6 of 7 Funding h l h g g There are also a variety of issues that need to be consid- ered in implementing telehealth changes from a service perspective. Telehealth represents a new form of practice and staff require support in developing skills and changing work practices. Reluctance to change practice to a tele- health option for monitoring of patients may be due, in part, to fear of missing something that would otherwise be picked up from a physical home assessment even though home visits continued to occur during the trial. Clinicians need to see that the change has patient and carer benefits not just system or service benefits [30]. For some services early adoption of technology may be rewarding while for other services new technologies will add significant stress. Therefore active change management is an inevitable part of telehealth planning and implementation. The Telehealth in the Home: Aged and Palliative Care in SA project is “An initiative funded by the Australian Government”. Availability of data and materials All data and material related to the manuscript have been archived by the first and third author (Jennifer Tieman and Deidre Morgan) at the Flinders University of South Australia. Raw data from the Telehealth in the Home: Aged and Palliative Care in SA project is still being analysed. The data pertinent to this publication is available on request from the first and third authors. Author details 1 1Palliative and Supportive Services, Flinders University, Bedford Park, South Australia, Australia. 2Southern Adelaide Palliative Services, Repatriation General Hospital, Daw Park, South Australia, Australia. Received: 22 May 2016 Accepted: 9 November 2016 Received: 22 May 2016 Accepted: 9 November 2016 Received: 22 May 2016 Accepted: 9 November 2016 Abbreviations l AKPS: Australia-modified Karnofsky Performance Status; AQoL: Assessment of Quality of Life; CAQ-CNS: Caregiver Assessment Questionnaire-Caregiver Network Service; CDS: Clinical decision support; CNF: Caregiver Network Facilitator; CPC: Nurse Clinical Practice Consultant; CPOE: Computerized provider order entry; GP: General practitioner; PCTRT: A Palliative Care Telehealth Research Team; SAPS: Southern Adelaide Palliative Care Service; SAS: Symptom Assessment Scale Ethics approval and consent to participate E hi l f h d d b h There are a number of limitations that need to be ac- knowledged. The number of participants was relatively small and as it was a feasibility study there was no con- trol group against which to compare outcomes. Recruit- ment related to a single area of service provision within a single metropolitan palliative care service. Participants were self-selecting. While the study model tried to min- imise additional study support to mimic “normal” ser- vice activity, there was additional technical and study support available to the service. However, the recruit- ment and participation rates suggest that telehealth is of sufficient maturity to support properly constructed and powered trials. Ethics approval for the study was granted by the Southern Adelaide Clinical Human Research Ethics Committee on 2 August 2013 (168.13). Written consent was obtained for all participants of this study. Conclusions systems in the health service. While privacy and security are important considerations for technology enabled so- lutions they may also limit flexibility in delivery of com- munity services if the patient’s personal IT resources are not able to be networked to the health service. This study has shown that palliative care patients and their carers living in the community were able to man- age the technology associated with a telehealth trial in- volving videoconferencing and remote monitoring of symptoms. Participants included old and very old pa- tients. While some level of technology problems are probably inevitable, it is possible to design and develop integrated systems that can be used in the community. The telehealth model offers new ways of supporting care at the end of life in the community and further applica- tions of these approaches should be investigated. Technology upgrades and cyber threats outside the con- trol of the project also influenced the project. For example, a transient security threat for technology platforms, The Heartbleed bug had a significant, albeit brief, impact on this project. Heartbleed is a weakness in the encryption security of programs such as email, internet sites and for specific programs such as FitBit®™. It has the potential to allow hackers to access data from a range of sites and services. As a security measure, FitBit®™forced a reset on all pass- words on their products. This required a manual reset of all FitBit®™passwords on the tablets. Each partici- pant was contacted and a time made for IT to visit to reset their passwords. Unfortunately this coincided with a scheduled roll out of upgrades to the apps. This created a further burden for participants and highlighted the need for ongoing IT support. Competing interests h h d l h The authors declare that they have no competing interests. Acknowledgements We would like to thank the Members of the Steering Committee, Palliative Care Telehealth Research Team and Operational Team, and patients and carers who participated in this trial. References G Stud health technol inform. 2014;204:1–6. 33. McCoy AB, Thomas EJ, Krousel-Wood M, Sittig DF. Clinical decision support alert appropriateness: a review and proposal for improvement. 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Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: 23. Abernethy AP, Shelby-James T, Fazekas BS, Woods D, Currow DC. The Australia-modified Karnofsky Performance Status (AKPS) scale: a revised scale for contemporary palliative care clinical practice [ISRCTN81117481]. BMC palliat care. 2005;4:7. 24. Apple Inc. iPad. http://www.apple.com/au/ipad/. Accessed 19 January 2016. 25. Richardson J, Iezzi A, Khan MA, Maxwell A. 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https://openalex.org/W4391399684	https://journal.uwhs.ac.id/index.php/jitk/article/download/606/pdf	English	null	THE RELATIONSHIP OF WORK STRESS LEVEL AND SLEEP QUALITY IN INPATIENT NURSES	Jurnal Ilmu dan Teknologi Kesehatan	2,024	cc-by	2,816	INTRODUCTION Nursing is a profession that is humanistic and adheres to standards of nursing service or care using the nursing code of ethics as the main guideline (Suara et al., 2018). Nurses have a heavy workload which results in a decline in their health, such as a decrease in daily activities, feeling tired, and a decrease in the body's immune system which has an impact on the quality of sleep and the amount of work can trigger stress for nurses because they feel anxiety and pressure due to this. (Agustina, 2022). Sleep is a process of recovery and restoring energy reserves in the body. Fulfilling the need for adequate sleep will help reduce stress in individuals. Lack of quality sleep can affect your mood and body and cause negative psychological effects (Kemenkes, 2019). In Indonesia, 73.3% of nurses have poor sleep quality in the care unit and 26.7% have good sleep quality, while in non-intensive pediatric wards 27.5% of nurses have poor sleep quality (Agririsky & Adiputra, 2018). Research conducted by Dimkatni et al in 2019 found that up to 30.3% of nurses had good sleep quality and 69.7% of nurses had poor sleep quality. Due to irregular work shifts, nurses' sleep patterns often change, resulting in fatigue at work (Dimkatni et al., 2020). Factors that can influence sleep quality are shown in an individual's ability to sleep and get the amount of rest that suits their needs. This can be influenced by several factors including disease, environment, motivation, exercise and fatigue, psychological stress, and lifestyle. Stress is one of the causes of poor sleep quality which is often not realized by individuals themselves (Saragih & Darmanik, 2022). Stress is a physical and mental disorder caused by changes and demands in life which are influenced by the environment and individual appearance. Work stress experienced by nurses is caused by excessive and continuous work demands which can affect the quality of nurses' sleep. This can affect work efficiency and the risk of work accidents, as well as lower production levels. When work stress is not managed well and effectively, it will affect the individual (Sanger & Lainsamputty, 2022). According to research by Bessie et al in 2021, 87.5% of male nurses experienced moderate stress and 12.5% experienced severe stress. Meanwhile, 25% of female nurses experienced mild stress, 66.66% experienced moderate stress and 8.33% of nurses experienced severe stress. (Bessie et al., 2021). ABSTRACT ABSTRACT Inpatient nurses have the ability and qualified knoledge in treating patients. Nurses often experience work stress due to caring for patient with various diseases, facing work situations and pressure at work. This research aims to determine the relationship betwenn work stress levels and sleep quality in inpatient nurses. This type of quantitative research with a correlational mwthod using a cross sectional approach. The sample in this study were nurses in the alamanda and mawar inpatient rooms. The sampling technoque used simple random sampling technique with a sample of 36 respondents with 18 respondents in each room. The bivariate test uses the rank speread test with research instruments in the form of standardized questionnaires, namely HSE and PSQI. In this study, it has gone through an ethical test with number 60/EC-LPPM/UWHS/VII-2023. The results showed that 22 respondents (61,1%) had moderate levels of work stress and 21 respondents (58,3%) has poor sleep quality. The result is a p value of 0,044<0,05 and a correlation coefficient = 0,338. This shows that the closeness of the correlation is in the low category with a positive direction where the higher the level of work stress, the worse the sleep quality of inpatient nurses. There is a relationship between the level of work stress and the quality of sleep in inpatient nurses. Keyword : Work stress, Sleep quality, Nurse Jurnal Ilmu dan Teknologi Kesehatan STIKES Widya Husada, Vol. 15 No. 1 Hal 12-15 ISSN 2086-8510 (Print), ISSN 2655-5875 (Online) Jurnal Ilmu dan Teknologi Kesehatan STIKES Widya Husada, Vol. 15 No. 1 Hal 12-15 ISSN 2086-8510 (Print), ISSN 2655-5875 (Online) https://doi.org/10.33666/jitk.v15i1.606 INTRODUCTION ) Based on a preliminary study conducted at RSUD dr. Gondo Suwarno Ungaran, by interviewing 4 nurses in the Alamanda and Mawar inpatient ward, the nurse said that the quality of his sleep was very poor and he couldn't sleep well every night, besides that the large number of patients resulted in several tasks piling up and having to be completed immediately, thus affecting the quality of his sleep. On average, nurses sleep <7 hours/day. With the increasing number of patients and increasing work, nurses often have a lot on their minds which ultimately causes work stress for nurses. The Relationship Of Work Stress Level And Sleep Quality In Inpatient Nurses Erna Filiasari, Menik Kustriyani, Wijanarko Heru Pramono The Relationship Of Work Stress Level And Sleep Quality In Inpatient Nurses Erna Filiasari, Menik Kustriyani, Wijanarko Heru Pramono 12 Jurnal Ilmu dan Teknologi Kesehatan STIKES Widya Husada, Vol. 15 No. 1 Hal 12-15 ISSN 2086-8510 (Print), ISSN 2655-5875 (Online) https://doi.org/10.33666/jitk.v15i1.606 https://journal.uwhs.ac.id/index.php/jitk/article/view/606 Not only because of work problems, but the relationships between co-workers, superiors and the work environment also have an influence on their psychological condition. METHODS This research was carried out at RSUD dr. Gondo Suwarno Ungaran in July-August 2023. This type of research is quantitative research with correlational methods using a cross sectional approach. The sampling technique in this research used a simple random sampling technique with 36 samples. Inclusion criteria are executive nurses and nurses who have worked >1 year. The instruments in this research used standard questionnaires, namely the HSE (Health and Safety Executive) questionnaire which consisted of 35 questions and the PSQI questionnaire which contained 7 components. The results of this study were analyzed using SPSS 25, univariate analysis in this study used the frequency distribution and percentage of each variable, namely stress level and sleep quality. This research has gone through ethical testing number 60/EC-LPPM/UWHS/VII-2023. RESULTS RESULTS LTS Table 1. Frequency Distribution of Respondents based on Level of Work Stress Job Stress Level Frequency Percentage (%) Low Currently Tall 10 22 4 27,8 61,1 11,1 Total 36 100 Table 1. Frequency Distribution of Respondents based on Level of Work Stress Job Stress Level Frequency Percentage (%) Based on frequency distribution table 4.1 above, it can be seen that of the total number of respondents, the majority had moderate levels of stress, namely 22 respondents (61.1%). Table 2. Frequency Distribution of Respondents based on Sleep Quality Sleep Quality Frequency Percentage Table 2. Frequency Distribution of Respondents based on Sleep Quality Sleep Quality Frequency Percentage (%) able 2. Frequency Distribution of Respondents based on Sleep Quality Sleep Quality Frequency Percentage (%) Good Sleep Quality Poor Sleep Quality 15 21 41,7 58,3 Total 36 100 The data in table 4.2 shows the amount of sleep quality among nurses in the inpatient room at RSUD dr. The majority of Gondo Suwarno Ungaran had a higher quality of poor sleep, namely 21 respondents (58.3%). Table 3. Relationship between Work Stress Level and Sleep Quality Job Stress Level Sleep Quality P value rho Good Sleep Quality Poor Sleep Quality Total F % F % F % Low Currently Tall 7 7 1 19,4 19,4 2,8 3 15 3 8,3 41,7 8,3 10 22 4 27, 8 61, 1 11, 1 0,044 0,338 Total 15 21 36 100 Table 3. Relationship between Work Stress Level and Sleep Quality J b St Sleep Quality Based on table 4.3, it is known that 15 respondents (41.7%) had moderate levels of work stress and poor sleep quality. The results of the analysis using the Spearman rank test, for the variables of work stress level and sleep quality, showed a p value of 0.044 <0.05, so Ho was rejected and Ha was accepted. So it can be concluded that there is a relationship between work stress levels and sleep quality among inpatient nurses at RSUD dr. Gondo Suwarno Ungaran. Meanwhile, the results of the correlation coefficient = 0.338 show that the correlation is in the low category, namely 0.20-0.399 in a positive direction, where the lower the level of work stress, the better the sleep quality of inpatient nurses. PEMBAHASAN The results of the analysis showed that 27.8% of respondents experienced low levels of work stress. Judging from the results of the questionnaire, respondents clearly know what duties and responsibilities must be carried out and are their obligations. Respondents work based on a work system and can easily find out schedule changes that occur in the room. This is in line with previous research, namely that 62.2% of respondents experienced low stress and 33.8% experienced severe stress, where work stress had an effect on nurse performance, nurses with mild work stress were more likely to have good performance. (Hakman et al., 2021). The results of research on data on respondents' work stress levels were that most of the respondents were at moderate stress levels, with 22 respondents with a percentage of 61.1%. Based on the questionnaire, differences of opinion between colleagues can trigger an uncomfortable environment. Tasks that are too many and beyond their capabilities and short time limits make respondents feel that working under long time pressure and lack of rest can result in work fatigue, risk of injury at work, and disturbed sleep quality. This research is in line with research by Hikmawati et al (2020), namely 84.8% of respondents experienced moderate work stress and 15.2% of respondents experienced high work stress. 22 nurses experienced work stress that occurred in the inpatient room at Nur Hidayah Hospital Bantul Yogyakarta due to the heavy workload (Hikmawati et al., 2020). In line with research by Runtu et al (2018) that work stress that occurs in nurses is in the moderate category, namely 70.7%. Stress occurs due to the many demands of work and being faced with several tasks that are beyond one's abilities as a nurse. Not only that, nurses are also required to have creative thinking and versatile energy (Runtu et al., 2018). ( ) As many as 11.1% of respondents experienced high levels of stress. The results of the questionnaire showed that respondents received some unfavorable treatment from colleagues and superiors. In carrying out a lot of work, respondents experienced difficulty in completing the work in the allotted time. So respondents often ignore some of the tasks that are their responsibilities. PEMBAHASAN This is in line with research conducted by Badri 2020, the results showed that 25 or 53.2% of respondents experienced high work stress where this stress was caused by a lot of work that was not in line with what had been planned by some nurses. Apart from that, there is pressure which causes nurses to lose concentration in completing their tasks so that quite a few nurses feel bored with their work in the ICU room which is the same every day. (Badri, 2020). Based on research results, it was 41.7%. Most respondents had never used sleeping pills to help them fall asleep. Respondents have quite good sleep efficiency where respondents need time to rest or sleep for 6-7 hours. The results of research conducted by Karlinda et al (2021) showed that 39 nurses or 62.9% of nurses on duty in the Covid-19 isolation ward had good sleep quality, while 23 nurses or 37.1% had poor sleep quality. In this study, the average respondent needed around 15-30 minutes to start sleeping (Karlinda et al., 2021). Based on the analysis that has been carried out, the results of the hypothesis test show that the p-value is 0.044 (p=<0.05), so Ho is rejected and Ha is accepted, which means there is a significant relationship between the level of work stress and the quality of sleep among inpatient nurses at RSUD Dr. Gondo Suwarno Ungaran. In this study, the results of the correlation coefficient show a value of 0.338, which means that the relationship between the level of work stress and the quality of sleep among nurses has a positive relationship, where the higher the level of stress, the worse the quality of sleep experienced by nurses. Based on the results of the questionnaire filled out by respondents, it was found that 41.7% of respondents experienced stress due to job demands which required respondents to work quickly and seriously and continuously in carrying out their responsibilities as a nurse. RESULTS The Relationship Of Work Stress Level And Sleep Quality In Inpatient Nurses Erna Filiasari, Menik Kustriyani, Wijanarko Heru Pramono The Relationship Of Work Stress Level And Sleep Quality In Inpatient Nurses Erna Filiasari, Menik Kustriyani, Wijanarko Heru Pramono 13 Jurnal Ilmu dan Teknologi Kesehatan STIKES Widya Husada, Vol. 15 No. 1 Hal 12-15 ISSN 2086-8510 (Print), ISSN 2655-5875 (Online) https://doi.org/10.33666/jitk.v15i1.606 https://doi.org/10.33666/jitk.v15i1.606 https://journal.uwhs.ac.id/index.php/jitk/article/view/606 PEMBAHASAN y y y g p The results of this study were supported by Agustina (2022) in her research entitled Nurses' Sleep Quality can be influenced by Nurses' Job Stress Levels, after carrying out statistical tests, they obtained a p value = 0.009 and showed that 83.3% of nurses experienced moderate stress with poor sleep quality, Likewise, nurses who experience low work stress are followed by good sleep quality among nurses, namely only 66.7% of the 30 respondents (Agustina, 2022). In line with research by Oktaviani et al (2021), the results show that the p value = 0.001 with r = 0.445, which means there is a significant relationship between stress levels and sleep quality and with a strong level of closeness in a positive direction (Oktaviani et al., 2021). https://doi.org/10.33666/jitk.v15i1.606 https://doi.org/10.33666/jitk.v15i1.606 ( ) ( ) https://journal.uwhs.ac.id/index.php/jitk/article/view/606 had good sleep quality, There is a relationship between the level of work stress and the quality of sleep among inpatient nurses at RSUD dr. Gondo Suwarno Ungaran with the Spearman rank statistical test results p=0.044≤ 0.05, then Ho is rejected and Ha is accepted so there is a significant relationship with r=0.338. CONCLUSION Based on the research results, it can be concluded as follows The level of work stress experienced by inpatient nurses at RSUD dr. Gondo Suwarno Ungaran was mostly in the medium category, namely 22 respondents (61.1%) while the other 10 respondents (27.8%) experienced low stress, Sleep quality among inpatient nurses at RSUD dr. Gondo Suwarno Ungaran mostly had poor sleep quality, namely 21 respondents (58.3%) and 15 respondents (41.7%) The Relationship Of Work Stress Level And Sleep Quality In Inpatient Nurses Erna Filiasari, Menik Kustriyani, Wijanarko Heru Pramono The Relationship Of Work Stress Level And Sleep Quality In Inpatient Nurses Erna Filiasari, Menik Kustriyani, Wijanarko Heru Pramono 14 Jurnal Ilmu dan Teknologi Kesehatan STIKES Widya Husada, Vol. 15 No. 1 Hal 12-15 ISSN 2086-8510 (Print), ISSN 2655-5875 (Online) Jurnal Ilmu dan Teknologi Kesehatan STIKES Widya Husada, Vol. 15 No. 1 Hal 12-15 ISSN 2086-8510 (Print), ISSN 2655-5875 (Online) REFERENCES Agririsky, I. A. C., & Adiputra, I. N. (2018). Gambaran Kualitas Tidur Perawat Dengan Shift Kerja di Ruang Rawat Inap Anak RSUP Sanglah Denpasar Tahun 2016. E-Jurnal Medika, 7(11), 1–8. Agustina, M. (2022). Kualitas Tidur Perawat dapat di Pengaruhi oleh Tingkat Stress Kerja Perawat. Journal of Management Nursing, 1(02), 52–59. https://doi.org/10.53801/jmn.v1i02.20 Badri, I. A. (2020). Hubungan Beban Kerja dan Lingkungan Kerja dengan Stres Kerja Perawat Ruangan ICU Dan IGD. Human Care Journal, 5(1), 379. https://doi.org/10.32883/hcj.v5i1.730 Dimkatni, N. W., Sumampouw, O. J., & Manampiring, A. E. (2020). Apakah Beban Kerja, Stres Kerja dan Kualitas Tidur Mempengaruhi Kelelahan Kerja pada Perawat di Rumah Sakit? Sam Ratulangi Journal of Public Health, 1(1), 009. https://doi.org/10.35801/srjoph.v1i1.27273 Hakman, Suhadi, & Nani, Y. (2021). Pengaruh Beban Kerja , Stres Kerja , Motivasi Kerja Terhadap Kinerja. Nursing Care and Health Technology Journal, 1(2), 47–54. Hikmawati, A. N., Maulana, N., & Amalia, D. (2020). Beban Kerja Berhubungan dengan Stres Kerja Perawat. Jurnal Ilmiah …, 2(3), 95–102. http://jurnal.rs-amino.jatengprov.go.id/index.php/JIKJ/article/view/23 Karlinda, L., Aini, F., & Wakhid, A. (2021). Gambaran Kualitas Tidur Perawat Pasien COVID-19 Setelah Satu Tahun Pandemi COVID-19. Jurnal Ilmu Keperawatan Jiwa, 4(3), 645–652. Kemenkes. (2019). Apa saja gejala Stres? Germas. https://p2ptm.kemkes.go.id/infographic-p2ptm/stress/apa-saja- gejala-stres Oktaviani, F. T., Apriliyani, I., Yudono, D. T., & Dewi, F. K. (2021). Hubungan Tingkat Stres dengan Kualitas Tidur pada Mahasiswa Tingkat Akhir Dimasa Pandemi Penyakit COVID-19 di Universitas Harapan Bangsa, Purwokerto. Health Information: Jurnal Penelitian, 13(2), 54–60. Runtu, V. R., Pondaag, L., & Hamel, R. (2018). Hubungan Beban Kerja Fisik Dengan Stres Kerja Perawat di ruang Instalasi Rawat Inap Rumah Sakit Umum GMIM Pancaran Kasih Manado. Jurnal Keperawatan, 6(1), 1–7. Sanger, A. Y., & Lainsamputty, F. (2022). Stres dan kualitas tidur pada perawat rumah sakit di Sulawesi Tengah. Holistik Jurnal Kesehatan, 16(1), 61–73. https://doi.org/10.33024/hjk.v16i1.5905 Saragih, J., & Darmanik, D. W. (2022). Keperawatan Dasar (M. Deswita (ed.); Cetakan Pe). Penerbit Mitra Cendekia Media. https://www.google.co.id/books/edition/Keperawatan_Dasar/F_2GEAAAQBAJ?hl=id&gbpv=1&dq=faktor- faktor+yang+mempengaruhi+kualitas+tidur+perawat&pg=PA82&printsec=frontcover Media. https://www.google.co.id/books/edition/Keperawatan_Dasar/F_2GEAAAQBAJ?hl=id&gbpv=1&dq=faktor- faktor+yang+mempengaruhi+kualitas+tidur+perawat&pg=PA82&printsec=frontcover Suara, M., Dalami, E., Rochimah, Raenah, E., & Rusmiyati. (2018). Konsep Dasar Keperawatan (Jusirm@n/Sapto (ed.); Cetakan 10). CV. Trans Info Media. www.traninfotim.blogspot.com The Relationship Of Work Stress Level And Sleep Quality In Inpatient Nurses Erna Filiasari, Menik Kustriyani, Wijanarko Heru Pramono The Relationship Of Work Stress Level And Sleep Quality In Inpatient Nurses Erna Filiasari, Menik Kustriyani, Wijanarko Heru Pramono 15
https://openalex.org/W4368371144	https://figshare.com/articles/journal_contribution/Figure_S1_from_Abatacept_Ruxolitinib_and_Screening_for_Concomitant_Respiratory_Muscle_Failure_to_Mitigate_Fatality_of_Immune-Checkpoint_Inhibitor_Myocarditis/22758896/1/files/40438283.pdf	English	null	Figure S2 from Abatacept/Ruxolitinib and Screening for Concomitant Respiratory Muscle Failure to Mitigate Fatality of Immune-Checkpoint Inhibitor Myocarditis	null	2,023	cc-by	160	Supplementary Figure 1. Examples of cardiac and skeletal muscle pathology findings on autopsies of 2 patients. Panels A-E are from a patient dead acutely from a fulminant ICI-myocarditis. Inflammatory infiltrates surrounding cardiomyocyte necrosis (A, black arrow, hematoxylin and eosin). These inflammatory infiltrates are composed by CD3+ T-cells (B) and CD68+ macrophages (C). Skeletal muscles were also affected by endomysial lympho-histiocytic inflammatory infiltrates and myocyte necrosis (white arrows) affecting the diaphragm (D, hematoxylin and eosin) and the psoas muscles (E, hematoxylin and eosin). Panel F is from a patient dead from COVID-19 approximatively one-year after an ICI-myocarditis episode complicated by appearance of an irreversible high-grade sinus node dysfunction requiring pacemaker implementation. Heart pathology (F, hematoxylin and eosin) revealed a complete sinus node destruction replaced by a yellowish fibrous tissue surrounding the sinoatrial nodal artery (visible within the small black square) instead of cardiomyocytes normally present. The sinus node spot is located between the superior vena cava (arrow) and the pericardium (arrowhead).
https://openalex.org/W4393987264	https://ejournal.itn.ac.id/index.php/jati/article/download/8929/5164	Indonesian	null	IMPLEMENTASI TRANSFER LEARNING MENGGUNAKAN CONVOLUTIONAL NEURAL NETWORK DALAM KLASIFIKASI PENYU	JATI	2,024	cc-by-sa	4,039	IMPLEMENTASI TRANSFER LEARNING MENGGUNAKAN CONVOLUTIONAL NEURAL NETWORK DALAM KLASIFIKASI PENYU Gede Eka Okta Putra, Ketut Queena Fredlina, I Nyoman Yudi Anggara Wijaya Teknik Informatika, Universitas Primakara Ekap231001@gmail.com JATI (Jurnal Mahasiswa Teknik Informatika) JATI (Jurnal Mahasiswa Teknik Informatika) Vol. 8 No. 1, Februari 2024 ABSTRAK Pemanfaatan teknologi kecerdasan buatan, khususnya Machine Learning, telah menjadi solusi inovatif dalam konteks pelestarian fauna, terutama di negara maritim seperti Indonesia. Keanekaragaman hayati di perairan Indonesia, yang melimpah dan menjadi daya tarik wisata, menyoroti perlunya konservasi. Penyu, sebagai salah satu spesies laut yang mencolok, memiliki peran krusial dalam menjaga ekosistem perairan dunia selama puluhan juta tahun. Meskipun memiliki enam dari tujuh spesies penyu di dunia, data dari IUCN Red List of Threatened Species menunjukkan bahwa keenam spesies ini berstatus rentan terhadap kepunahan. Ancaman terhadap populasi penyu, seperti penangkapan telur untuk diperdagangkan, penangkapan induk penyu, dan aktivitas perburuan yang merusak, menjadi tantangan serius dalam upaya pelestarian. Dalam konteks ini, kami mengusulkan pemanfaatan teknologi Machine Learning, khususnya Convolutional Neural Network dan Transfer Learning, untuk klasifikasi jenis atau spesies penyu. Dua model yang dikembangkan diukur tingkat akurasinya, dengan tujuan menciptakan model akurat yang dapat mendeteksi spesies penyu melalui citra input. Pendekatan ini diharapkan dapat memberikan kontribusi signifikan dalam mencegah ancaman kepunahan terhadap penyu di Indonesia. Penelitian ini tidak hanya menghadirkan solusi teknologi yang inovatif, tetapi juga bertujuan meningkatkan kesadaran masyarakat terhadap pentingnya pelestarian spesies penyu. Dengan menyatukan pemanfaatan teknologi Machine Learning dan edukasi masyarakat, diharapkan dapat tercipta kesadaran yang lebih luas dan berkelanjutan terhadap pelestarian spesies ini di perairan Indonesia. 1. PENDAHULUAN data dari IUCN Red List of Threatened Species, Enam species ini berstatus rentan kepunahan Di era kemajuan teknologi kecerdasan buatan yang semakin kompleks, Machine Learning menjadi salah satu teknologi yang dapat dijadikan solusi dalam permasalahan konservasi atau pelestarian fauna. Salah satu implementasinya yaitu penerapan Machine Learning untuk klasifikasi spesies hewan, khususnya di perairan Indonesia. Indonesia merupakan negara dengan julukan negara maritim. Luasnya lautan Indonesia memberikan kekayaan sumber daya laut yang melimpah. Keanekaragaman hayati di lautan Indonesia menjadi daya tarik wisata. Salah satu hewan laut yang menjadi pusat perhatian wisatawan adalah penyu[1]. Berbagai kasus melatarbelakangi masalah ini, seperti telur penyu yang diambil untuk diperdagangkan, penangkapan induk penyu dan ancaman lain seperti kegiatan penangkapan ikan yang menyebabkan banyak penyu yang mati[1]. Tidak hanya itu, perburuan penyu juga dilakukan untuk diambil karapasnya dan dijadikan aksesoris seperti gelang, liontin, hingga diawetkan untuk dijadikan pajangan, sehingga sangat berpengaruh terhadap populasi penyu di alam. Hal ini mengakibatkan penyu di Indonesia menjadi salah satu satwa endemik konservasi yang menghadapi ancaman kepunahan serius. Untuk mencegah ancaman kepunahan yang semakin membesar, dapat dimulai dengan meningkatkan kesadaran masyarakat akan spesies penyu. Salah satu solusinya yaitu menggunakan pemanfaatan teknologi[2]. Penyu merupakan hewan yang berperan penting dalam menjaga kesehatan lautan dunia selama lebih dari 100 juta tahun. Tugas-tugas ini termasuk menjaga fungsi terumbu karang untuk mempertahankan produktivitas dan mentransfer nutrisi penting dari perairan ke darat. Karena terdapat keanekaragaman habitat perairan di berbagai wilayah, hal ini menciptakan evolusi dan adaptasi sehingga terdapat beragam spesies penyu. Enam dari tujuh spesies penyu di dunia berada di Indonesia karena Indonesia merupakan habitat untuk mencari makan, berkembang biak dan bermigrasi dari Samudra pasifik ke Samudra hindia atau sebaliknya. Enam spesies penyu tersebut adalah Penyu Hijau (Chelonia mydas), Penyu Sisik (Eretmochelys imbricata), Penyu Pipih (Natator depressus), Penyu Lekang (Lepidochelys olivacea), Penyu Tempayan (Caretta caretta), dan Penyu Belimbing (Dermochelys coriacea). Namun, menurut Pemanfaatan teknologi Machine Learning dapat digunakan untuk melakukan klasifikasi jenis atau spesies penyu. Pembuatan model menggunakan lebih dari satu sistem agar mendapatkan model yang memiliki akurasi tertinggi, yaitu menggunakan algoritma Convolutional Neural Network dan Transfer Learning, dimana hasilnya berupa 2 model yang dapat melakukan klasifikasi jenis penyu. Kedua model ini akan diukur tingkat akurasinya sehingga didapat satu model yang dapat mendeteksi spesies penyu melalui input citra dengan sangat akurat. Dengan dikembangkannya teknologi ini, diharapkan mampu menciptakan kesadaran dan edukasi dalam dua sisi, 1077 Vol. 8 No. 2.1. Penyu Penyu-penyu merupakan jenis reptil yang sangat penting bagi keseimbangan ekosistem laut, tetapi mereka juga merupakan salah satu jenis hewan yang paling terancam punah di dunia. Penyebab utama kepunahan penyu adalah aktivitas manusia yang merusak habitat mereka, seperti penebangan hutan mangrove dan pembangunan pantai, serta perburuan ilegal dan perikanan yang tidak bertanggung jawab. Penyu juga sering terperangkap dalam alat tangkap ikan yang tidak sengaja, yang dapat menyebabkan kematian mereka. Penyu juga sering terkena dampak dari perubahan iklim, seperti perubahan suhu laut dan perubahan pasokan makanan. Untuk membantu menyelamatkan penyu dari kepunahan, diperlukan tindakan yang terpadu dari pemerintah, perusahaan, dan masyarakat, termasuk pengelolaan yang lebih baik dari habitat penyu, perlindungan terhadap penyu dari kegiatan manusia yang merusak, dan pengawasan yang lebih ketat terhadap perburuan dan perikanan yang tidak bertanggung jawab. Menurut Sukresno (1997), semua penyu terdaftar dalam daftar kota Appendix I (Convention on International Trade in Endangered Species). konvensi tersebut melarang semua perdagangan internasional produk yang berasal dari penyu, baik berupa telur, daging maupun cangkang[1] optimization, dan kemajuan arsitektur[7] Gambar 1. Convolutional Neural Network Gambar 1. Convolutional Neural Network Convolutional layer adalah sebuah layer yang terbentuk dari beberapa kernel atau filter convolutional yang digunakan untuk menghitung feature maps yang berbeda. Tujuan utama dari penggunaan convolutional layers adalah untuk mencari atau mencari feature representations dari suatu input. Setelah diterapkan filter tersebut maka ukuran citra input juga akan berkurang atau megecil, hal ini akan mempermudah jaringan-jaringan neuron untuk mempelajari features dari input tersebut. Untuk menghitung ukuran hasil input setelah diterapkan kernel atau filter tersebut dapat menggunakan rumus berikut. 2.2. Machine Learning Dunia digital memiliki banyak data, seperti data Internet of Things (IoT), data keamanan siber, data seluler, data bisnis, data media sosial, data kesehatan, dll. Untuk menganalisis data ini secara cerdas dan mengembangkan aplikasi cerdas dan otomatis yang sesuai, pengetahuan tentang kecerdasan buatan, khususnya, machine learning adalah kuncinya[3]. Saat ini hampir semua yang berkaitan dengan intelligent systems yang menawarkan fitur dengan kemampuan kecerdasan buatan kebanyakan mengandalkan machine learning. Machine learning secara singkat dapat dijelaskan sebagai sebuah kapasitas dari sebuah sistem yang dapat belajar dari problem-specific training data untuk mekalukan otomatisasi sebuah proses analytical model building dan menyelsaikan tugas terkait. Algoritma machine learning biasanya memproses data untuk mempelajari pola baik itu tentanng individu, proses bisnis, transaksi, peristiwa, dan sebagainya. Ada beberapa jenis atau tipe dari machine learning yaitu supervised, unsupervised, semi-supervised, dan reinforcement learning[4] Pooling layer adalah layer yang berfungsi untuk mengurangi beban komputasi dengan cara mengurangi jumlah koneksi antara convolutional layers. Metode pooling yang paling sering dipakai adalah max- pooling dan average-pooling. Max-pooling hanya mengambil nilai terbesar dari hasil features extraction, sedangkan average-pooling mengambil nilai rata-rata dari hasil features extraction. Fully connected layers adalah layer neural network yang sangat mirip seperti yang ada pada arsitektur neural network tradisional, karena setiap node yang ada pada Fully connected layers saling terhubung satu sama lain. Setiap node yang berada di barisan paling akhir pasti terhubung sebagai vektor ke layer pertama. Fully connected layers adalah parameter yang paling banyak digunakan pada arsitektur CNN, dan yang paling membutuhkan banyak waktu untuk proses training[8]. 1. PENDAHULUAN 1, Februari 2024 JATI (Jurnal Mahasiswa Teknik Informatika) ada permintaan yang signifikan untuk mengenali objek secara otomatis[5]. Convolutional Neural Network telah banyak membantu kemampuan computer vision dengan menghasilkan tingkat akurasi yang sangat baik dalam pemrosesan video, pengenalan objek, klasifikasi dan segmentasi gambar, natural language processing, pengenalan suara, dan banyak bidang lainnya. Selanjutnya, pengenalan sejumlah besar data dan perangkat keras yang tersedia telah membuka jalan baru untuk studi Convolutional Neural Network[6]. Beberapa konsep inspiratif untuk kemajuan Convolutional Neural Network telah dikembangkan sampai sekarang, termasuk fungsi aktivasi alternatif, regularisasi, parameter optimization, dan kemajuan arsitektur[7] yaitu dari sisi pemanfaatan teknologi Machine Learning untuk klasifikasi dan pengenalan spesies penyu di Indonesia kepada masyarakat yang lebih luas. 2.5. Klasifikasi Klasifikasi adalah proses mengelompokkan sesuatu ke dalam kelompok-kelompok yang berbeda berdasarkan karakteristik yang sama atau mirip. Klasifikasi dapat digunakan dalam berbagai bidang, seperti ilmu pengetahuan, seni, dan bisnis. Misalnya, dalam ilmu pengetahuan, klasifikasi dapat digunakan untuk mengelompokkan hewan dan tumbuhan ke dalam kelompok yang sesuai dengan ciri-ciri fisik atau genetik mereka. Dalam seni, klasifikasi dapat digunakan untuk mengelompokkan lagu ke dalam genre musik yang berbeda. Dalam bisnis, klasifikasi dapat digunakan untuk mengelompokkan produk ke dalam kategori yang sesuai dengan jenis atau kegunaannya. Klasifikasi sering digunakan untuk mempermudah pengorganisasian dan pemahaman terhadap suatu hal yang ingin diklasifikasikan[9]. 2. 2. Langkah selanjutnya adalah melakukan studi pustaka atau studi literatur, tahapan ini merupakan langkah penting bagi setiap peneliti karena memungkinkan untuk memperkaya referensi dalam bidang penelitian yang akan dilakukan. Dengan menelaah penelitian terdahulu yang telah dilakukan dan memiliki topik yang serupa, peneliti dapat lebih mudah mengembangkan dan menemukan temuan baru yang dapat digali. 2. Langkah selanjutnya adalah melakukan studi pustaka atau studi literatur, tahapan ini merupakan langkah penting bagi setiap peneliti karena memungkinkan untuk memperkaya referensi dalam bidang penelitian yang akan dilakukan. Dengan menelaah penelitian terdahulu yang telah dilakukan dan memiliki topik yang serupa, peneliti dapat lebih mudah mengembangkan dan menemukan temuan baru yang dapat digali. 2. Langkah selanjutnya adalah melakukan studi pustaka atau studi literatur, tahapan ini merupakan langkah penting bagi setiap peneliti karena memungkinkan untuk memperkaya referensi dalam bidang penelitian yang akan dilakukan. Dengan menelaah penelitian terdahulu yang telah dilakukan dan memiliki topik yang serupa, peneliti dapat lebih mudah mengembangkan dan menemukan temuan baru yang dapat digali. 3. 3. Pada tahap ketiga dilakukan pengumpulan dan pengolahan data. Pengumpulan data diawali dengan mengumpulkan data yang relevan sesuai dengan topik penelitian yang telah ditentukan. Selanjutnya, data tersebut akan menjalani proses preprocessing yang meliputi penyesuaikan ukuran citra agar dapat dimasukan ke dalam algoritma, jika tidak ada data yang di rubah maka tidak perlu melakukan proses preprocessing pada data, selanjutnya dilakukan perancangan arsitektur Convolutional Neural Network, arsitektur Convolutional Neural Network terbaik. 2.3. Convolutional Neural Network Transfer learning adalah teknik yang digunakan dalam pembelajaran mesin untuk mengambil pengetahuan yang telah diperoleh dari satu tugas dan menerapkannya ke tugas lain yang terkait. Hal ini Computer vision menjadi kata yang semakin sering dibahas pada bidang pemrosesan gambar. Dengan banyaknya pengaplikasian computer vision, 1078 Vol. 8 No. 1, Februari 2024 JATI (Jurnal Mahasiswa Teknik Informatika) 1. Tahap pendahuluan dalam penelitian ini diawali dengan penentuan topik penelitian yaitu klasifikasi jenis penyu berdasarkan class. Setelah itu dilakukan identifikasi masalah untuk menjawab permasalahan utama yang dapat dipecahkan. Tujuan penelitian ditetapkan untuk mengarahkan penelitian agar mampu menjawab permasalahan yang ada. Selanjutnya ditentukan batasan masalah untuk menentukan ruang lingkup penelitian, sedangkan perancangan metodologi dirancang untuk mempersiapkan langkah-langkah yang diperlukan dalam penelitian ini, mulai dari pengumpulan data hingga analisi yang efektif. Dengan tahap pendahuluan yang baik, maka penelitian memiliki landasan yang kokoh untuk dilanjutkan ke tahap selanjutnya, berguna karena seringkali membutuhkan banyak data dan waktu untuk mengumpulkan dan melatih model pembelajaran mesin dari awal untuk menyelesaikan tugas tertentu[9]. Dengan menggunakan transfer learning, model yang sudah dilatih pada tugas yang terkait dapat digunakan sebagai titik awal untuk menyelesaikan tugas baru, yang biasanya membutuhkan lebih sedikit data dan waktu untuk dilatih ulang[10]. Metode penelitian ini dilakukan sesua dengan gambar berikut. Metode penelitian ini dilakukan sesua dengan gambar berikut. gambar berikut. Gambar 2. Metode Penelitian 4. Petelah di proses dan diprediksi oleh Convolutional Neural Network, langkah selanjutnya adalah menginterpretasikan hasilnya[11]. Pada model Convolutional Neural Network, hasil yang diinterpretasikan untuk mengetahui kemampuan model dalam mengklasifikasikan jenis penyu dengan benar. g j p y g 5. Pada akhir penelitian dibuat rangkuman dari keseluruhan penelitian dan ditarik kesimpulan berdasarkan hasil yang diperoleh. Kesimpulan ini meliputi pencapaian tujuan penelitian dan jawaban atas rumusan masalah yang telah ditetapkan sebelumnya. Dalam kesimpulan ini penting untuk memaparkan hasil dan menjelaskan sejauh mana model atau metode yang digunakan telah berhasil menyelesaikan masalah yang diteliti, selain itu, saran juga diberikan sebagai rekomendasi bagi peneliti selanjutnya untuk mengatasi kesenjangan atau keterbatasan yang ditemukan selama penelitian. Saran tersebut dapat berupa pengembangan metode, perluasan data atau sampel, perbaikan teknik pengolahan data, atau aspek lain yang dapat meningkatkan kualitas Gambar 2. Metode Penelitian 1079 JATI (Jurnal Mahasiswa Teknik Informatika) Vol. 8 No. 1, Februari 2024 f1-score adalah metrik evaluasi yang digunakan untuk mengukur performa model klasifikasi. penelitian di masa mendatang. Langkah terakhir ini penting karena melibatkan sintesis dan evaluasi dari semua penelitian yang dilakukan. Dengan ringkasan dan kesimpulan yang jelas, peneliti dapat menyajikan gambaran menyeluruh tentang hasil penelitian dan relevansinya dengan tujuan awal. Selain itu, peneliti dapat memberikan landasan bagi pengembangan dan peningkatan ilmu pengetahuan di bidang yang sama, melalui saran-saran bagi peneliti selanjutnya. Berikut adalah hasil evaluasi pada model VGG16, dapat dilihat bahwa model VGG16 mendapatkan accuracy sebesar 80% pada data testing. Tabel 1. Clasification Report Vgg16 Precision Recall F1-Score Support 0 0.78 0.70 0.74 10 1 0.90 0.90 0.90 10 2 0.73 0.80 0.76 10 accuracy 0.80 30 Tabel 1. Clasification Report Vgg16 4. HASIL DAN PEMBAHASAN Hasil dari training bukan menjadi satu-satunya tolak ukur untuk menilai kemampuan suatu model. Untuk melakukan evaluasi performa model lebih detail maka digunakan data testing yaitu data yang sebelumnya belum pernah dilihat model sebelumnya dengan begini kemampuan model dapat dilihat dengan skenario yang lebih nyata, kedua model akan diuji menggunakan data testing yang sama. Berikut adalah rumus dan perhitungan manual untuk mencari precision, recall, dan F1-Score dari confusion matrix dari model VGG16. Precision mengukur sejauh mana prediksi positif yang dilakukan oleh model benar. Dalam kata lain, precision menghitung beberapa persen prediksi positif yang sebenarnya benar dari semua prediksi positif yang dilakukan oleh model. Untuk melakukan evaluasi pada model digunakan confusion matrix. Confusion matrix adalah sebuah tabel yang digunakan untuk mengevaluasi kinerja suatu model klasifikasi. Tabel ini berisikan empat kompenen utama yaitu True Positive (TP), True Negative (TN), False positive (FP), False Negative (FN). TP menunjukkan jumlah kasus dimana model memprediksi kelas positif dengan benar, TN menunjukkan jumlah kasus dimana model memprediksi kelas negatif dengan benar, FP menunjukkan jumlah kasus dimana model salah memprediksi kelas positif, dan FN menunjukkan jumlah kasus dimana model salah memprediksi kelas negatif. Recall mengukur sejauh mana model mampu menemukan semua contoh positif yang ada dalam dataset. Recall menghitung berapa persen contoh positif yang sebenarnya berhasil ditemukan oleh model dari semua contoh positif yang ada dalam dataset. F1 score adalah penggabungan antara precision dan recall menjadi satu ukuran yang mencerminkan keseluruhan performa model. F1 Score adalah rata-rata harmonic dari precision dan recall. F1 score berguna untuk memperhitungkan keseimbangan antara precision dan recall dalam suatu model. F1 score memiliki hak terbaik ketika precision dan recall memiliki nilai yang seimbang. Berikut adalah hasil confusion matrix dari model VGG16 ternyata memiliki hasil tidak begitu baik melihat hasil akurasi dan loss pada tahap training yang dimana model berhasil mendapatkan hasil true positif (TP) sebanyak 24. model VGG16 mengalami kekeliruan dalam mengklasifikasi jenis penyu hijau dan lekang. Bahkan dari 10 data uji pada kelas jenis penyu hijau model VGG16 hanya benar memprediksi 7 data testing. 𝑃𝑟𝑒𝑠𝑖𝑠𝑖= 𝑇𝑃 𝐹𝑃+ 𝑇𝑃∗100% 𝑅𝑒𝑐𝑎𝑙𝑙= 𝑇𝑃 𝐹𝑁+ 𝑇𝑃∗100% 𝑓1 −𝑠𝑐𝑜𝑟𝑒= 2 ∗ 𝑝𝑟𝑒𝑠𝑖𝑠𝑖∗𝑟𝑒𝑐𝑎𝑙𝑙 𝑝𝑟𝑒𝑠𝑖𝑠𝑖+ 𝑟𝑒𝑐𝑎𝑙𝑙∗100% 𝑓1 −𝑠𝑐𝑜𝑟𝑒= 2 ∗ 𝑝𝑟𝑒𝑠𝑖𝑠𝑖∗𝑟𝑒𝑐𝑎𝑙𝑙 𝑝𝑟𝑒𝑠𝑖𝑠𝑖+ 𝑟𝑒𝑐𝑎𝑙𝑙∗100% 1. Precision a. Class 0 = 7 / (2+7) = 7/9 = 0.78 b. Class 1 = 9 / (1+9) = 9/10 = 0.90 c. Class 2 = 8 / (3+8) = 8/11 = 0.73 2. Recall a. 4. HASIL DAN PEMBAHASAN Class 0 = 7 / (3+7) = 7/10 = 0.70 b. Class 1 = 9 / (1+9) = 9/10 = 0.90 c. Class 2 = 8 / (2+8) = 8/10 = 0.80 3. F1-Score a. Class 0 = 2 / (0.78 x 0.70/ 0.78 + 0.70) = 0.74 b. Class 1 = 2 / (0.90 x 0.90/ 0.90 + 0.90) = 0.90 c. Class 2 = 2 / (0.73 x 0.80/ 0.73 + 0.80) = 0.76 4. Accuracy 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦= 𝐽𝑢𝑚𝑙𝑎ℎ 𝑝𝑟𝑒𝑑𝑖𝑘𝑠𝑖 𝑏𝑒𝑛𝑎𝑟 𝑡𝑜𝑡𝑎𝑙 𝑗𝑢𝑚𝑙𝑎ℎ 𝑑𝑎𝑡𝑎 𝑓1 −𝑠𝑐𝑜𝑟𝑒= 2 ∗ 𝑝𝑟𝑒𝑠𝑖𝑠𝑖∗𝑟𝑒𝑐𝑎𝑙𝑙 𝑝𝑟𝑒𝑠𝑖𝑠𝑖+ 𝑟𝑒𝑐𝑎𝑙𝑙∗100% 1. Precision a. Class 0 = 7 / (2+7) = 7/9 = 0.78 b. Class 1 = 9 / (1+9) = 9/10 = 0.90 c. Class 2 = 8 / (3+8) = 8/11 = 0.73 2. Recall a. Class 0 = 7 / (3+7) = 7/10 = 0.70 b. Class 1 = 9 / (1+9) = 9/10 = 0.90 c. Class 2 = 8 / (2+8) = 8/10 = 0.80 3. F1-Score a. Class 0 = 2 / (0.78 x 0.70/ 0.78 + 0.70) = 0.74 b. Class 1 = 2 / (0.90 x 0.90/ 0.90 + 0.90) = 0.90 c. Class 2 = 2 / (0.73 x 0.80/ 0.73 + 0.80) = 0.76 4. Accuracy 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦= 𝐽𝑢𝑚𝑙𝑎ℎ 𝑝𝑟𝑒𝑑𝑖𝑘𝑠𝑖 𝑏𝑒𝑛𝑎𝑟 𝑡𝑜𝑡𝑎𝑙 𝑗𝑢𝑚𝑙𝑎ℎ 𝑑𝑎𝑡𝑎 Gambar 3. Confusion Matrix VGG16 1. Precision a. Class 0 = 7 / (2+7) = 7/9 = 0.78 b. Class 1 = 9 / (1+9) = 9/10 = 0.90 c. Class 2 = 8 / (3+8) = 8/11 = 0.73 Gambar 3. Confusion Matrix VGG16 Dari confusion matrix dapat diperoleh hasil precision, recall, dan f1-score pada masing-masing kelas dan juga hasil keseluruhan akurasi model yang diuji menggunakan data testing. Precision, recall, dan 1080 JATI (Jurnal Mahasiswa Teknik Informatika) Vol. 8 No. 1, Februari 2024 Accuracy = 24 / 30 = 0.80 Tabel 3. Perbandingan Akurasi Model Prediksi Akurasi Benar Salah VGG16 24 6 80% VGG19 25 5 83% Diagram dibawah adalah confusion matrix dari model VGG19. Dari gambar tersebut dapat dilihat bahwa model VGG19 tidak mengalami kesuliatan dalam memprediksi jenis penyu. Tetapi hasil tersebut masih lebih baik jika di bandingkan hasil testing model VGG16, walaupun ada kesalahan dalam memprediksi jenis penyu nanum dari masing masing 10 gambar yang diuji pada tiap kelas model VGG19 jumlah kesalahan prediksi lebih sedikit jika dibandingkan model VGG16. 4. HASIL DAN PEMBAHASAN Dari hasil confusion matrix dari kedua model yang telah diuji dapat di lakukan perhitungan secara manual untuk mendapatkan akurasi yang diperoleh masing-masing model saat mencoba mengprediksi data testing, nilai akurasi keseluruhan model dapat dihitung dengan cara membagi jumlah hasil prediksi yang benar lalu dibagi dengan jumlah data yang diuji. Dari tabel dibawah dapat dilihat bahwa model VGG19 menghasilkan performa yang paling bagus. Gambar 4. Confusion Matrix VGG19 5. KESIMPULAN DAN SARAN Hasil dari pemodelan dilakukan dengan metode transfer learning, dimana Transfer learning adalah teknik yang digunakan dalam pembelajaran mesin untuk mengambil pengetahuan yang telah diperoleh dari satu tugas dan menerapkannya ke tugas lain yang terkait. Hal ini berguna karena seringkali membutuhkan banyak data dan waktu untuk mengumpulkan dan melatih model pembelajaran mesin dari awal untuk menyelesaikan tugas tertentu. dan menggunakan dua model yaitu model vgg16 dan juga vgg19, Dimana VGG16 adalah arsitektur ini pertama kali diperkenalkan dalam Kontes Klasifikasi Gambar ImageNet 2014 dan menduduki peringkat tinggi dalam kompetisi tersebut. VGG16 terkenal karena keunikannya yang sederhana namun efektif. Arsitektur VGG16 ini terdiri dari 16 lapisan yaitu 13 lapisan konvolusi dan 3 lapisan fully connected. Sedangkan VGG19 juga diperkenalkan dalam Kontes Klasifikasi Gambar ImageNet 2014 dan memiliki performa yang baik. Dimana VGG19 itu adalah turunan dari VGG16 yang memiliki 19 lapisan dengan penambahan lapisan tersebut bertujuan untuk meningkatkan kapasitas representasi dan memungkinkan model untuk memahami fitur-fitur yang lebih kompleks dalam gambar. Karena terdapat dua model yang digunakan hasil dari pengujiannya tersebut. Dimana kedua model tersebut mendapatkan hasil training dan validation yang sangat baik. VGG16 mendapatkan hasil training accuracy sebesar 85% dan validation accuracy sebesar 83%. VGG19 memiliki hasil accuracy dengan training accuracy sebesar 87% dengan validation accuracy sebesar 70%. Namun walaupun mendapatkan accuracy yang tinggi pada tahap training nyatanya performa model pada tahap testing menunjukkan hasil yang berbeda-beda. Setelah melakukan tahap testing model VGG16 hanya mendapatkan accuracy pada data testing sebesar 81%, ini merupakan hasil yang paling rendah dari model VGG19. Model VGG19 mendapatkkan accuracy yang baik dengan angka mencapai 85% dibandingkan dengan VGG16, jadi dapat di simpulkan bahwa diabandingkan kedua model VGG16 dan VGG19 yang memiliki performa yang paling baik untuk Gambar 4. Confusion Matrix VGG19 Berdasarkan confusion matrix diatas berikut diperoleh hasil evaluasi seperti berikut. Model VGG19 berhasil mendapatkan hasil yang cukup bagus dengan akurasi mencapai angka 83%. Tabel 2. Clasification Report VGG19 Precision Recall F1-Score Support 0 0.86 0.60 0.71 10 1 0.91 1.00 0.95 10 2 0.75 0.90 0.82 10 accuracy 0.83 30 Tabel 2. Clasification Report VGG19 Berikut adalah rumus dan perhitungan manual untuk mencari precision, recall, dan F1-Score dari confusion matrix dari model VGG19. 1. Precision a. Class 0 = 6 / (1+6) = 6/7 = 0.86 b. Class 1 = 10 / (1+10) = 10/11 = 0.91 c. Class 2 = 9 / (3+9) = 9/12 = 0.75 2. Recall a. 5. KESIMPULAN DAN SARAN Class 0 = 6 / (4+6) = 6/10 = 0.60 b. Class 1 = 10 / (0+10) = 10/10 = 1.00 c. Class 2 = 9 / (1+9) = 9/10 = 0.90 3. F1-Score a. Class 0 = 2 / (0.86 x 0.60 / 0.86 + 0.60) = 0.71 b. Class 1 = 2 / (0.91 x 1.00 / 0.91 + 1.00) = 0.95 c. Class 2 = 2 / (0.75 x 0.90 / 0.75 + 0.90) = 0.82 4. Accuracy 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦= 𝐽𝑢𝑚𝑙𝑎ℎ 𝑝𝑟𝑒𝑑𝑖𝑘𝑠𝑖 𝑏𝑒𝑛𝑎𝑟 𝑡𝑜𝑡𝑎𝑙 𝑗𝑢𝑚𝑙𝑎ℎ 𝑑𝑎𝑡𝑎 Accuracy = 25 / 30 = 0.83 1. Precision a. Class 0 = 6 / (1+6) = 6/7 = 0.86 b. Class 1 = 10 / (1+10) = 10/11 = 0.91 c. Class 2 = 9 / (3+9) = 9/12 = 0.75 2. Recall a. Class 0 = 6 / (4+6) = 6/10 = 0.60 b. Class 1 = 10 / (0+10) = 10/10 = 1.00 c. Class 2 = 9 / (1+9) = 9/10 = 0.90 3. F1-Score a. Class 0 = 2 / (0.86 x 0.60 / 0.86 + 0.60) = 0.71 b. Class 1 = 2 / (0.91 x 1.00 / 0.91 + 1.00) = 0.95 c. Class 2 = 2 / (0.75 x 0.90 / 0.75 + 0.90) = 0.82 4. Accuracy 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦= 𝐽𝑢𝑚𝑙𝑎ℎ 𝑝𝑟𝑒𝑑𝑖𝑘𝑠𝑖 𝑏𝑒𝑛𝑎𝑟 𝑡𝑜𝑡𝑎𝑙 𝑗𝑢𝑚𝑙𝑎ℎ 𝑑𝑎𝑡𝑎 Accuracy = 25 / 30 = 0.83 1. Precision a. Class 0 = 6 / (1+6) = 6/7 = 0.86 b. Class 1 = 10 / (1+10) = 10/11 = 0.91 c. Class 2 = 9 / (3+9) = 9/12 = 0.75 2. Recall a. Class 0 = 6 / (4+6) = 6/10 = 0.60 b. Class 1 = 10 / (0+10) = 10/10 = 1.00 c. Class 2 = 9 / (1+9) = 9/10 = 0.90 1081 JATI (Jurnal Mahasiswa Teknik Informatika) Vol. 8 No. 1, Februari 2024 Dalam Pendeteksian Jenis Daging Hewan Ternak,” 2022. [Online]. Available: https://doi.org/10/25047/jtit.v9i1.278 memprediksi jenis penyu adalah VGG19. Berdasarkan hasil klasifikasi jenis penyu, terdapat perbedaan yang signifikan pada performa model VGG16 dan VGG19. pada model VGG16 mendapatkan akurasi 80%, sedangkan model VGG19 mendapatkan akurasi 83%. Dapat disimpulkan bahwa model VGG19 mengklasifikasikan jenis penyu lebih baik daripada model VGG16 dalam konteks dataset yang digunakan. 5. KESIMPULAN DAN SARAN Berdasarkan hasil penelitian yang telah dilakukan, maka peneliti memberikan saran untuk memperbanyak jumlah dataset dan juga jenis penyu agar fungsionalitas model menjadi lebih bagus dan juga performa model menjadi lebih baik. [6] [6] O. Saputra, D. Iskandar Mulyana, and M. B. 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https://openalex.org/W2981622118	https://dipot.ulb.ac.be/dspace/bitstream/2013/294931/3/PaG7.4.pdf	English	null	Linking Food Democracy and Sustainability on the Ground: Learnings from the Study of Three Alternative Food Networks in Brussels	Politics and governance	2,019	cc-by	8,308	Issue This article is part of the issue “New Perspectives on Food Democracy” edited by Basil Bornemann (University of Basel, Switzerland) and Sabine Weiland (Université Catholique de Lille, France). This article is part of the issue “New Perspectives on Food Democracy” edited by Basil Bornemann (University of Basel, Switzerland) and Sabine Weiland (Université Catholique de Lille, France). © 2019 by the authors; licensee Cogitatio (Lisbon, Portugal). This article is licensed under a Creative Commons Attribu- tion 4.0 International License (CC BY). tors that embody alternatives to the more standard- ized industrial mode of food supply” (Renting, Marsden, & Banks, 2003, p. 394). Examples of AFNs include short food supply chains, solidarity purchasing groups, farmer’s market, and community-supported agriculture or consumers food co-operatives and foods with a geo- graphical indication of origin. Linking Food Democracy and Sustainability on the Ground: Learnings from the Study of Three Alternative Food Networks in Brussels 1 Institute for Environmental Management and Land-Use Planning, Center for Studies on Sustainable Development, Université Libre de Bruxelles, 1050 Brussels, Belgium; E-Mails: frlohest@ulb.ac.be (F.L.), tbauler@ulb.ac.be (T.B.) 2 Institute for Environmental Management and Land-Use Planning, Environmental Management, Society and Territory, Université Libre de Bruxelles, 1050 Brussels, Belgium; E-Mails: ssureau@ulb.ac.be (S.S.), joris.van.mol@ulb.ac.be (J.V.M.), wouter.achten@ulb.ac.be (W.A.) * Corresponding author Submitted: 4 April 2019 \| Accepted: 7 July 2019 \| Published: 28 October 2019 Politics and Governance (ISSN: 2183–2463) 2019, Volume 7, Issue 4, Pages 21–31 DOI: 10.17645/pag.v7i4.2023 Keywords Keywords alternative food networks; food democracy; sustainability assessment; sustainability transition Keywords alternative food networks; food democracy; sustainability assessment; sustainability transition Abstract The article explores and discusses, both conceptually and empirically, the exercise of food democracy in the context of three alternative food networks (AFNs) in Brussels, Belgium. It demonstrates that food democracy can be described as a “vector of sustainability transition”. The argumentation is built on the results of a 3.5-year participatory-action research project that configured and applied a sustainability assessment framework with the three local AFNs under study. Firstly, the article presents a localized understanding of food democracy. Food democracy is defined as a process aiming to trans- form the current food system to a more sustainable one. This transformation process starts from a specific point: the people. Indeed, the three AFNs define and implement concrete processes of power-configuration to alter the political, economic, and social relationships between consumers and producers as well as between retailers and producers. Sec- ondly, the article assesses and discusses how the three AFNs perform these practices of food democracy and what effects these have on the actors concerned. The assessment shows that the three AFNs distinguish themselves along a gradient of their transformative potential in terms of practices. However, this variation in their interpretation of food democracy does not translate into a gradient of performance. 1. Introduction: Alternative Food Networks, Sustainability, and Democracy Third, AFNs experiment with new gov- ernance schemes and reconfigure power relationships along the food chain. While the first two sets of inno- vative practices target ecological and socio-economic im- pacts, the third dimension directly addresses the issue of food democracy. Although AFNs could only choose one of these three archetypal sets of innovative activi- ties, they generally combine two or all of them, with dif- ferent intensities. This means that AFNs hold a core set of sustainability promises with which food democracy is intrinsically associated. However, the literature on AFNs remains unclear in identifying clearly and systematically how AFNs concep- tualize, operationalize, and implement food democracy, and what effects are exerted on the actors/citizens in- volved. This article attempts to fill this gap and suggests giving specific content to food democracy in the context of AFNs daily sustainability practices. Based on the re- sults of 3.5 years of participatory research (CosyFood, 2019) which configured and applied a sustainability as- sessment framework for local AFNs, this article concep- tually and empirically explores the relationship between sustainability and democracy in the context of three AFNs in Brussels, Belgium. This article demonstrates how food democracy forms a cornerstone of the sustainabil- ity project of these AFNs but also that the idea of food democracy shapes a diversity of practices. Moreover, this article explores whether and how local AFNs in Brussels allow for the emergence and navigation of food democ- racy within their very own alternative practices. Introduced in the 1990s by Lang (1999), food democ- racy was precisely developed to describe such grassroots experiments, alternative to the global food system con- trolled by big companies and framed by the agricultural (production) agendas. The core idea of food democracy is then to give more power to all the actors involved in the food chain; it is a call for more consumer/citizen partic- ipation in the management and the control of the food system (Booth & Coveney, 2015; Dubuisson-Quellier & Lamine, 2008; Hassanein, 2003; Levkoe, 2006; Lockie, 2009; Renting, Schermer, & Rossi, 2012; Wilkins, 2005). Similarly, Hassanein (2003) defined food democracy as a means for collective action, and more precisely as a prag- matic and gradual method for the transformation of the food system to sustainability. 1. Introduction: Alternative Food Networks, Sustainability, and Democracy Alternative food networks (AFNs) are under deep scrutiny since they emerged as concrete attempts to counter the negative externalities of the dominant global and industrial food system (Deverre & Lamine, 2010; Le Velly, 2016; Maye & Kirwan, 2010; Tregear, 2011). Commonly, the concept of AFNs “cover[s] newly emerg- ing networks of producers, consumers and other ac- On the ground, AFNs are mainly alternative food dis- tribution systems, their primary focus being to experi- ment with and drive the transition towards sustainabil- Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 21 ure power relationships along the food chains they pro- mote in order to build a more sustainable food system. If using a sustainability transition perspective (Geels & Schot, 2007), this definition of food democracy allows AFNs to be linked to the developing concept of “trans- formative social innovation” defined as “a process of change in social relations, involving new ways of doing, organizing, framing and/or knowing, which challenge, al- ter and/or replace established (dominant) institutions in a specific socio-material context” (Haxeltine et al., 2016, p. 8). It means that AFNs have a high transformative po- tential linked to the new ways of organizing and govern- ing the entire food chains they promote; this potential to transform sustainability relates to their practices of food democracy/democratic processes, and that is concerned with the power-relationships they configure. ity of the current food system (Kirwan, Ilbery, Maye, & Carey, 2013; Maye & Duncan, 2017; Rossi, 2017). A re- cent literature review states that AFNs intend to build sustainable food systems by way of three types of innova- tive practice (Forssell & Lankoski, 2015). First, AFNs pro- mote ecological ways of growing food by marketing and distributing high-quality foodstuffs. Positive impacts on the environment, especially on soils and biodiversity, as well as on human health, are expected. Second, AFNs experiment with new types of food chain configuration reducing the spatial and social distance between pro- ducers and consumers involving minimal geographical transport distances, minimal value chain length (num- ber of intermediaries) and minimal informational dis- tance. Socioeconomic impacts are projected, such as im- proved income for producers and stronger social ties be- tween food chain actors, improving territorial/rural de- velopment (Praly, Chazoule, Delfosse, & Mundler, 2014; Renting et al., 2003). Mutual trust is targeted, too, as well as ecological benefits from reduced food miles (Mundler & Rumpus, 2012). 1. Introduction: Alternative Food Networks, Sustainability, and Democracy The concept of “civic food networks” equally emerged to designate AFNs as the “ex- pression of the revitalized role of civil society-based gov- ernance mechanisms” and “a source of dynamism and innovation” (Renting et al., 2012, p. 297). From this spe- cific point of view, the project of AFNs concerns the trans- formation of the food regime from a particular starting point: the people. The article is structured as follows. In the next section (Section 2), the research context is described, empirical materials, and method. We will focus on the participa- tory aspects of our research. In Section 3, the indicators linked to food democracy and the results are presented. The particular practices that promote the construction of food democracy will be described and discussed (Section 3.1), as well as the impacts of those processes on each AFN stakeholder (Section 3.2). In Section 4, based on the results, the link between food democracy and sustainability transformation is discussed. Section 5 presents the conclusions. Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 2.1. The CosyFood Project: Partners and Goals 2.2. Materials and Method The first set of materials used in this article is the con- tent of the sustainability assessment framework. This content has been co-constructed by the four project partners for nearly two years. To do this, the “principle- criteria-indicators” framework was used (Rey-Valette et al., 2008). By starting from a blank page, this method takes into account the actors’ representations, values, beliefs, and knowledge. Concretely, a series of partici- patory workshops and co-creational activities were con- ducted with each type of AFN stakeholder: leaders and employees, producers, and consumers. By the end of the process, the sustainability assessment tool contained 14 sustainability principles, 55 sustainability criteria, and 105 indicators (the full framework with the detailed description of all principles is available in French at CosyFood, 2019). The three AFNs involved in the CosyFood project are quite different. The first AFN is a brand (and partially a franchise) of neighbourhood organic shops with a co- operative status (“the organic shops” in the following sections). This AFN exclusively retails organic foodstuffs favouring local products and shorter supply chains. It started in 2013 with only one small shop. It is now (early 2019) a bigger cooperative network involving nine or- ganic shops in Brussels. Total sales were around €15.5 million in 2017 and it employs around 100 staff. The second AFN is a non-profit organization which networks solidarity purchasing groups for peasant agri- culture (“the Gasap” in the following sections). Since 2008, organic local farmers deliver their products every (two) week(s) directly to ninety (in 2019) small groups of consumers. Every group organizes themselves to take collective care of the foodstuffs’ distribution as well as fi- nancial operations. Each local farmer and each purchas- ing group is a legal member of the organization. The orga- nization supports the system in multiple ways: the selec- tion of farmers, organization of meetings between farm- ers and consumer groups, dissemination of the model. The Gasap receives a public subvention for sustaining these support activities. The total sales by the local farm- ers involved were around €1.3 million in 2017. y , ) The principles designate the most critical sustainabil- ity goals for the (alternative) food system. They reveal a shared and collective vision in terms of values, beliefs, and ethics. 2.1. The CosyFood Project: Partners and Goals This vision embraces fundamental values of sustainability such as solidarity, economic viability, fair- ness and justice, sensitization and transparency, and of course elements regarding strong respect for the eco- logical limits of the planet. The criteria define the pre- cisely elaborated conditions for respecting those princi- ples in the context of a food network. The whole set of criteria represents a shared roadmap towards a sustain- able (alternative) food system. It is important to men- tion that the three AFNs wanted to distinguish “perfor- mance criteria” from “practice criteria”. Performance cri- teria designate “levels of performance” (e.g., the level of participation) that can be more or less defined as impacts. Practice criteria identify the concrete practices that AFNs have to implement to be able to perform at the desired level (e.g., promoting participation [or not] in the decision-making process). Finally, the indicators translate each criterion into precise measurements and allow them to assess whether AFNs conform to their roadmap. In Section 3.1, the criteria and indicators of food democracy identified for this article are presented and discussed. The third AFN is an online shop which exclusively sells local foodstuffs from organic or conventional small/medium-scale farmers and transformers (“the on- line shop” in the following sections). Farmers and food processors must participate in the weekly 2-hour distri- bution event. The digital tool provider, which is a start- up located in France, manages the online shop centrally. However, at the local level, it is always a person who is in charge of configuring the network of producers and consumers, and of organizing and hosting the weekly dis- tribution. For the Brussels case under study, it is a co- operative that hosts this configuring task. In 2017, sales were around €0.5 million. To involve the three AFNs in the whole research pro- cess, the research methodology was built on the ex- isting good practices for participatory-action research (Chevalier, Buckels, & Bourassa, 2013) and participatory evaluation (Sébastien, Lehtonen, & Bauler, 2017). The five people funded by the project together managed all of the research activities and considered themselves as equally skilled citizen-researchers, with a mutually recog- nized field expertise related to food chains and food sus- tainability. Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 2. Research Context and Empirical Materials This article is based on (a part of) the results of the CosyFood project. In this participatory-action research project, we worked for 3.5 years with three AFNs in Brussels. Each project partner (i.e., us, a university part- ner, and the three AFNs) was funded by the research pro- gram (1.5 full-time employees for the university partner and 1 full-time employee for each AFN partner). In this article, we argue that in the context of AFNs, food democracy mainly acts as a “vector of [sustain- ability] transition” (Chiffoleau, Millet-Amrani, & Canard, 2016; Kropp, 2018; Rossi, 2017). It is a core set of in- novative practices implemented by AFNs that reconfig- 22 Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 2.1. The CosyFood Project: Partners and Goals The goals of the project were as follows: 1) to configure a shared sustainability assessment framework for 2) assessing and comparing the impacts of the three AFNs on local sustainability and 3) to apply the content of the shared sustainability framework into specific sus- tainability improvement-tools for each AFN. This article is built on the results linked to the two first objectives, for which we gathered distinctive materials correspond- ing to particular methods. The second set of materials used in this article is the data collected for the measurement of the identified food democracy indicators. This data was produced fol- lowing a qualitative survey conducted between February and June 2018. Data was collected through anonymous online questionnaires with the relevant actors of each AFN (farmers, wholesalers if any, food processors, con- sumers) and consolidated with semi-structured inter- views with three leading actors (“managers”) in each AFN. The number of questionnaires sent varies in each AFN because they vary in terms of size (see Table 2 and its footnote). Using these two sets of materials, the following sec- tions of the article consist of, on the one hand, in an in- ductive re-construction and ex-post re-interpretation of Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 23 a localized understanding of food democracy in the con- text of the three AFNs; an interpretation of the materi- als in the light of a food democracy perspective. On the other hand, the data collected by the survey allows as- sessment of whether the three AFNs conform to their un- derstanding of food democracy, understood as a vector of transformation towards sustainability. tem, which is currently wholly framed and structured by “state-market” interests and agendas (De Schutter, Mattei, Vivero-Pol, & Ferrando, 2019). It also refers to a demand for more citizen participation in the manage- ment and control of the food system. Based on this, we consider that food democracy leads to transforma- tion towards sustainability through the reconfiguration of power-relationships. 3. Reconstructing Perspectives on Food Democracy in the Context of Three AFNs in Brussels For this article and with these very generic and gen- eral considerations in mind, we scrutinized the whole set of sustainability criteria and indicators. We identified and selected 12 (of 55) criteria linked to the field of food democracy as broadly defined above: six performance criteria and six practice criteria. Subsequently, we linked 15 (of 105) indicators to the notion of food democracy as described in the introduction (see Table 1). 3.1. Food Democracy: Vector of Transition by Giving Back Power to the Food Chains Actors 3.2. Food Democracy on the Ground: Food Democracy Performances in 3 Contextualized AFNs The results compiled in Table 2 show if and to what ex- tent the three AFNs implement (some of) the identified practices for building food democracy and the effect of these on their performance. The first category of indicators relates to the config- uration of political power within the food chains. The re- sults reveal that when the stakeholders involved were asked if they were satisfied with their political power, the average satisfaction score (Indicator A4) is very similar for each of the three AFNs. This even though the daily im- plemented practices (Indicators A1 to A3) vary between the three AFNs. Furthermore, the above selection of criteria and in- dicators illustrates that the three AFNs want to assert explicitly that the three different dimensions of power are interrelated. As we observed during the research pro- cess, the three AFNs are aware that, for example, giv- ing a formal right to vote to a producer to participate in the decision-making process does not necessarily mean that he/she will use it. For this producer, gaining the le- gal power to configure the AFNs functioning (political power) does not mean either that they will be free from all constraints when they set the prices of their foodstuffs (economic power) or that they will feel part of the AFN community (social power). Though, for the three AFNs which developed the sustainability framework, as far as food democracy is also part of the general sustainability endpoint, the ideal target is that all the conditions linked to democracy and listed in Table 1 must occur. In their perfect world, sustainability would only be fully achieved if those three sets of power-configuration practices arose and if they led to the expected performance. The online shop is hosted by a co-operative recog- nized by the Belgian Centre National de la Coopération, which means, among other things, that each member gets one vote at the Annual General Meeting (AGM). Formally, the consumers and farmers who invested and bought some shares get an equal power in the decision- making process. Informally, in this AFN, the general as- sembly tends to validate decisions and choices made by the project manager. The project manager thus has the power to decide about the day-to-day practices as far as informal consultations of AFNs members nurture the most important choices. 3.1. Food Democracy: Vector of Transition by Giving Back Power to the Food Chains Actors As mentioned in the introduction, food democracy as a concept was developed to promote and apprehend grassroots experiments such as AFNs that give back con- trol and power to the actors involved in the food sup- ply chains. Food democracy is about re-engaging citizens and food actors into the governance of the food sys- By analyzing the content of the 12 criteria and the 15 corresponding indicators, it is possible to reconstruct the vision of food democracy shared by the three AFNs involved in the project. This was done by connecting food Table 1. Criteria and indicators linked to democracy. Assessment criteria Indicators Practices Funding resources A1. Ownership properties of the retailer/facilitator Participation in the decision-making process A2. Existence of participative and/or cooperative decision-making processes A3. The formal/legal distribution of power in the decision-making process Sensitization practices B1. Existence of formal sensitization programs for consumers B2. Which information is offered to the consumers and by which means Knowledge transmission and learning B3. Existence of frequent and formal meetings between processes producers and consumers Terms of trade C1. Level of pricing power for producers/suppliers C2. The basis for setting prices Competition management by the C3. Commitment modalities between sellers retailer/facilitator (producers/suppliers) and buyers (retailer or consumers) Performances Level of participation of the stakeholders A4. Whether the stakeholders are satisfied with their power and inclusion in the decision-making processes Quality of social relations between B4. Whether the producers/suppliers trust in the stakeholders reliability of the relationships with the retailer/facilitator Level of recognition of the work of the B5. Whether the supplier feels recognized and valued producers/suppliers for his/her work by the client Level of consumers’ sensitization B6. Whether the consumers feel more about sustainable food and producers’ and more aware and conscious about the sustainability daily realities of food systems and producers’ labor conditions Level of the economic viability C4. Level of monthly income in regards to the minimal of producers local income to live Affordability C5. Level of feeling about economic affordability (consumers) Table 1. Criteria and indicators linked to democracy. 24 democracy to their overall common sustainability frame- work. Regarding this, food democracy for them does not solely represent an isolated “fourth pillar” of sustain- ability. It instead appears as a transversal starting point for diverse practices aiming to reach a certain global level of sustainability. 3.1. Food Democracy: Vector of Transition by Giving Back Power to the Food Chains Actors Indeed, when scrutinizing the cri- teria and indicators linked to food democracy listed in Table 1, the interdependence between practices and per- formances can quite easily be observed. Also, it can be seen that the identified criteria and indicators address the idea of power-(re)configuration. In this respect, the practices that give back power to the actors involved are envisioned along three dimensional comprehensions of power: political power, understood as the “power to decide and to participate in the decision-making pro- cess” (indicators labelled with an A in Table 1); power, in terms of social resources, understood as gaining capacity through learning and building a confident social network (indicators labelled with a B), and economic power un- derstood as gaining commercial and economic capacity (indicators labelled with a C). issues around food. The food democracy elements they have put on the table is empirical proof of their aware- ness of the need to implement participatory processes and more balanced power-configurations within their daily practices of food distribution and consumption. Such shared attention to food democracy illustrates that the grassroots actors are already on their way to building a more democratic, and as a result, a more sustainable food system. However, the following section explores in more detail what effects each AFN has concerning food democracy. Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 3.2. Food Democracy on the Ground: Food Democracy Performances in 3 Contextualized AFNs Even though they are called “co-operative”, the or- ganic shops are not recognized as such because they do not respect all basic principles of co-operatives (e.g., mechanisms to limit the power of controlling partners and distribution of dividends). Indeed, at the AGM, the members’ voting power depends on the number of eco- nomic shares owned. Because a few investors own the majority of shares, the final decisions belong to them, and they are more potent than the other “co-op mem- bers” (consumers, farmers, suppliers, managers, work- ers). Nevertheless, they have a more balanced power when it comes to the daily management of the supermar- kets. At the level of the board, investors cannot force a decision even if they were to join together and oppose all the other represented categories of cooperators. These elements illustrate the result of a Brussels- contextualized inquiry about the grassroots significance of food democracy, understood in relationship to food sustainability. Moreover, we assert that the participa- tory process has produced and defined a formal set of food democratization practices. These practices appear as necessary conditions and vectors of sustainability. So, it becomes now possible to extend and give concrete con- tent to the notion of “food democracy”. The sustainabil- ity assessment framework includes the contextualized conditions (the criteria) for building food democracy; at the same time, it offers a way to assess the effectiveness of its implementation and related effects (the indicators). Finally, based on the criteria and indicators they co- constructed during the CosyFood project, it is evident that the three AFNs involved are, at least in terms of intentions, democracy-led and aware of the democracy The Gasap is a non-profit organization. This official status implies that each member at the general meeting must have equal voting power (“one man, one vote”). However, in the way that the organization performs Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 25 Table 2. Measurement of the indicators linked to food democracy in three Brussels-based AFNs. Power type Indicators Organic shops Gasap Online shop Political power- configuration practices A1. Ownership properties of the retailer/facilitator Non-recognized co-operative; ownership by investors Nonprofit organization, no capital ownership Host within a recognized co-operative; ownership by cooperators A2. Existence of participative and/or cooperative decision-making processes Structural, formal participative decision-making processes Structural, formal participative decision-making processes Informal participation and consultation A3. 3.2. Food Democracy on the Ground: Food Democracy Performances in 3 Contextualized AFNs The formal/legal distribution of power in the decision-making process One share, one vote One member (group or farmer), one vote One member (farmer or consumer), one vote Political power- configuration performances A4. Whether the stakeholders are satisfied with their power and inclusion in the decision-making processes Average 3.2/5 Average 3.6/5 Average 3/5 Social power- configuration practices B1. Existence of formal sensitization programs for consumers No Yes. One employee is dedicated to this. Specific “discussions” 4 times a year for the members No B2. Which information is offered to the consumers and by which means Label, newsletter, digital social networks Visits on farms, Label, dedicated communication, participative quality control, free conversations with farmers during the distribution Dedicated communication, open discussions with farmers during the distribution B3. Existence of frequent and formal meetings between producers and consumers No Yes, every (two) week(s) Yes, weekly Social power- configuration performances B4. Whether the producers/suppliers trust in the reliability of the relationships with the retailer/facilitator Average 4.1/5 Average 4/5 Average 4.4/5 B5. Whether the supplier feels recognized and valued for his/her work by his clients Average 3.4/5 Average 4.1/5 Average 4.5/5 B6. Whether the consumers feel more and more aware and conscious about the sustainability of food systems and producers’ labor conditions Average 3.7/5 Average 4.2/5 Average 4.3/5 Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 26 Table 2. (Cont.) Measurement of the indicators linked to food democracy in three Brussels-based AFNs. Power type Indicators Organic shops Gasap Online shop Economic power- configuration practices C1. Level of pricing power for producers/suppliers Negotiation with suppliers, no negotiation with farmers High High C2. The basis for setting prices 94% of farmers who sell directly to the AFN set prices based on production costs 47% of farmers set prices based on production costs 95% of farmer set prices based on production costs C3. Commitment modalities between sellers (producers/suppliers) and buyers (retailer or consumers) No commitment Mutual commitment No commitment Economic power- configuration performances C4. Level of monthly income in regards to the minimal local income to live 65% of farmers have monthly income higher than the minimal income 52% of farmers have monthly income higher than the minimal income 71% of farmers have monthly income higher than the minimal income C5. 3.2. Food Democracy on the Ground: Food Democracy Performances in 3 Contextualized AFNs In the case of perfor- mance criteria and corresponding indicators, the respondents were asked to evaluate the theme/subject at stake on a satisfaction scale, from 1 (not satisfied at all) to 5 (very satisfied). The final scores, as compiled in Table 2, refer to the average score of the all concerned actors’ level of satisfaction. In the case of practice criteria and corresponding indicators, the results summarize the qualitative data that was gathered. In this way, the organic shops and the Gasap have im- plemented structural participative decision-making pro- cesses, contrary to the online shop, which consults value chain actors only informally. Simultaneously, the mea- surements of the performance show that even though each AFN implements different practices, this makes no difference in terms of the effects observed. Within the three political power-configurations, the actors (farmers and suppliers in our data) do feel involved but are not completely satisfied with their involvement. day-to-day, the board takes all the important decisions, together with the employees, while the AGM approves the annual budget and the annual action plan. As a significant innovation on the path towards greater levels of food democracy, we see that every type of actor in each of the AFNs gets an opportunity to partic- ipate in the decision-making process. Providers (farmers, transformers, wholesalers) and consumers hold an influ- ence over the configuration of the system and the way it operates. In the case of the organic shops, the formal power is less balanced between actors. For now, a few investors own most of the capital and have the last word. However, in the cases of the Gasap and the online shop, if the distribution of political power seems to be fairer and better balanced, the main decisions are taken by board members, employees, volunteers, or manager(s). The second category of indicators relates to social power. The central insight resulting from the assessment concerns the direct contact between farmers and con- sumers, a characteristic which differentiates the organic shops from the two other AFNs. In terms of performance, the results show that when there are frequent direct meetings between farmers and consumers, as in the Gasap and the online shop, the farmers feel more rec- ognized for their work (Indicator B5). Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 3.2. Food Democracy on the Ground: Food Democracy Performances in 3 Contextualized AFNs Level of feeling about economic affordability (consumers) Average 3/5 4/5 3.7/5 Notes: For the organic shops, the questionnaires were sent to seven wholesalers and six farmers and eight transformers who deliver the stores directly. Together, these actors represent 50% of the total supply of the organic shops. The response rate was 95%: All the wholesalers and farmers responded and seven of the eight transformers. For the indicators regarding consumers, 122 consumers volun- tary responded to the survey, online or on paper. For the Gasap, the questionnaires were sent to 20 farmers and three transformers, in other words, all the network providers. The global response rate is 73%: 16 farmers and one transformer. In this AFN, 186 consumers answered the online survey. For the online shop, we went the questionnaires to all the providers (17 farmers and four transformers), and only one farmer did not respond. So, the response rate was 90%. 76 consumers answered the online survey. In the case of perfor- mance criteria and corresponding indicators, the respondents were asked to evaluate the theme/subject at stake on a satisfaction scale, from 1 (not satisfied at all) to 5 (very satisfied). The final scores, as compiled in Table 2, refer to the average score of the all concerned actors’ level of satisfaction. In the case of practice criteria and corresponding indicators, the results summarize the qualitative data that was gathered. Notes: For the organic shops, the questionnaires were sent to seven wholesalers and six farmers and eight transformers who deliver the stores directly. Together, these actors represent 50% of the total supply of the organic shops. The response rate was 95%: All the wholesalers and farmers responded and seven of the eight transformers. For the indicators regarding consumers, 122 consumers volun- tary responded to the survey, online or on paper. For the Gasap, the questionnaires were sent to 20 farmers and three transformers, in other words, all the network providers. The global response rate is 73%: 16 farmers and one transformer. In this AFN, 186 consumers answered the online survey. For the online shop, we went the questionnaires to all the providers (17 farmers and four transformers), and only one farmer did not respond. So, the response rate was 90%. 76 consumers answered the online survey. 3.2. Food Democracy on the Ground: Food Democracy Performances in 3 Contextualized AFNs However, this AFN is not transformative in terms of the configuration of its economic exchanges because of the mainstream market rules. The Gasap makes one more significant step beyond this. This AFN tries to alter the dominant regime through the governance of its chain. It builds solidarity through a community of people and im- plements disruptive commercial relationships between actors. Then, the Gasap holds the most potential to en- act a radical version of transformation, because it is the most challenging to the dominant regime in each cate- gory of power-configuration. Social ties and relationships between actors could also be associated with power dynamics in economic terms. If looking at commercial relationships (Indicator C1), none of the AFNs negotiate the prices of the food- stuffs with the farmers and the organic shops negotiate solely when the supplier is a wholesaler. The Gasap in the only one that operates with a mutual commitment between the buyers (the consumers directly) and the farmers (Indicator C3). However, if relating this to the scores concerning the farmers’ level of economic viabil- ity (Indicator C4), and the ratings about the freedom to set prices based on production costs, we identify a para- dox for the Gasap. Indeed, mutual commitment to a fair price is formally much stronger than in the two other AFNs, but it does not lead to economic viability for farm- ers. Moreover, prices seem to be implicitly constrained by this mutual commitment because farmers do not dare to increase them. This situation contrasts with the case in the online shop: More farmers are profitable and are able to set prices based on their real production costs. Moreover, last but not least, the scores for the three AFNs regarding affordability are positive. A big dilemma remains: how to generate increased profitability for farm- ers while maintaining affordability for consumers. Considering food democracy as a “vector of transi- tion towards sustainability”, the distinctive deepness of the transformative practices implemented by the three AFNs could have led to some differences in terms of per- formance. However, as we can see, none of the three AFNs performs in a perfectly democratic way. The ob- served and measured effects of the three distinctive power-configurations do not distinguish the three AFNs in terms of their performance, except for profitability where there is a more distinctive score. 3.2. Food Democracy on the Ground: Food Democracy Performances in 3 Contextualized AFNs However, in terms of practices, the legal position and the ownership configuration of the AFNs are still only symbolic signals about the formal opportunity to partici- pate given to the actors in the value chain. Indeed, each AFN can operate formal and informal participative pro- cesses, whatever its legal status and ownership property. However, regarding the two other scores, the differ- ences between the three AFNs are not significant. Firstly, concerning sensitization (Indicator B6), the frequent di- 27 Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 rect meetings between farmers and consumers should help to make consumers more involved and aware of sustainability and agricultural conditions. However, this does not produce significantly different effects. This prac- tice is the leading information channel about produc- tion processes and the quality of foodstuffs used by the Gasap and the online shop. The organic shops use more delegation practices such as labels and passive informa- tion, which seem to perform equally. Secondly, regarding the level of trust in the reliability of the relationship be- tween providers and retailers/facilitators, there is little evidence of a difference. The Gasap has the lowest score in terms of trust and reliability. This result is puzzling; indeed, the Gasap implements the most coherent and complete program of sensitization in comparison to the other two. It also gives priority to the building of ties be- tween categories of actors. In summary, as in the case of the decision-making processes, different practices gen- erate no significant difference in terms of performances, except maybe for the farmers’ quest for recognition of their work. deep democratic processes, lots of initiatives to recon- nect individuals, especially farmers and consumers, as well as mutual economic commitment. Between these two AFNs, the online shop operates a more or less hy- brid configuration. Indeed, in terms of food democracy, the organic shops are only able to alter and challenge the dominant food system through their governance processes and practices, tending to reproduce the mainstream meth- ods of organizing economic relationships and social ties. The online shop goes one step further: its transformative potential appears in the food chain’s governance. Most of the democratic practices are informal, but it seems to perform well, especially if we look at the second category of indicators, regarding the social ties that are developed. 3.2. Food Democracy on the Ground: Food Democracy Performances in 3 Contextualized AFNs We may ex- plain this constatation by the fact that the actors who responded in the survey are very diverse. It means that they could participate in each AFN for various reasons due to their diverse backgrounds. For example, the farm- ers’ profiles are very distinctive, in terms of activity, size, means of production (e.g., manual vs. motorized), or longevity. Of course, these explanations need more investiga- tion. However, in the end, the results further contribute to illustrating that each AFN achieves at least a part of its objectives and generates (positive) effects on the ac- tors who are involved. In this sense, the three categories of power-configuration processes can clearly label the three AFNs as an alternative to the mainstream regarding food democracy. They are all participative, formally as well as informally. So, if we look at the broader situation for the sustainability of the Brussels food system/regime, the results tend to show that the three AFNs participate in the transformation of the current food system, despite different transformative potentials (more or less strin- gent) but also despite their having different scales of ac- tivity. In the context of the broader field of research on Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 References Booth, S., & Coveney, J. (2015). Food democracy: From consumer to food citizen. Singapore: Springer. Chevalier, J., Buckels, D. J., & Bourassa, M. (2013). Guide de la recherche-action, la planification et l’évaluation participatives [Guide to participatory action research, planning and evaluation]. Ottawa: SAS2 Dialogue. Furthermore, the article brings to the fore a new pro- posal that helps to assess aspects of food democracy practices in the context of AFNs. The set of criteria and indicators opens an interesting analytical tool around the three kinds of power-configuration processes at stake. Regarding this, the article fills a gap in the literature on food democracy and AFNs. It gives and proposes a concrete but potentially generic content to the concept of food democracy that as a result becomes more use- able. Moreover, in terms of methodology and discussion, the developed analytical/assessment tool allows discus- sion of the potential interdependence between the three categories. For example, could it be possible for the organic shops to change their economic (commitment) practices, regarding their commercial scale and model, to achieve more direct contacts and learning between farm- ers and consumers? Chiffoleau, Y., Millet-Amrani, S., & Canard, A. (2016). From short food supply chains to sustainable agricul- ture in urban food systems: Food democracy as a vec- tor of transition. Agriculture, 6(4), 57. CosyFood. (2019). CosyFood. Action Co-Create. Re- trieved from http://www.cocreate.brussels/- CosyFood- De Schutter, O., Mattei, U., Vivero-Pol, J. L., & Ferrando, T. (2019). Food as commons: Towards a new relation- ship between the public, the civic and the private. In J. S. Vivero-Pol, T. Ferrando, O. De Schutter, & U. Mat- tei, Routledge handbook of food as a commons (pp. 372–395). London and New York, NY: Routledge. 372–395). London and New York, NY: Routledge. Deverre, C., & Lamine, C. (2010). Les systèmes agroali- mentaires alternatifs: Une revue des travaux anglo- phones en sciences sociales [Alternative agri-food systems: A review of english-language social sci- ence works]. Économie Rurale: Agricultures, Alimen- tations, Territoires, 2010(317), 57–73. Finally, the results illustrate that, in the context of a sustainability project, none of the three grassroots ex- periments are perfect, although they all achieved some great results, whatever the practices involved. On this basis, we classify the thee AFNs as transformative so- cial innovations although they have different potentials for transformation. Dubuisson-Quellier, S., & Lamine, C. (2008). Consumer involvement in fair trade and local food systems: Delegation and empowerment regimes. Acknowledgments The authors wish to express gratitude to the three AFNs they worked with during this 3.5-year research project, especially the employees and volunteers dedicated to the CosyFood project: Aurélie Labarge, Julie Minne, Isadora Meersseman, Christophe Nothomb, Florian Delespesse, and Camille Latin. 4. Food Democracy as a Vector Sustainability Transformation: Three Gradients of Transformative Potential If we return to the concept and definition of “transfor- mative social innovation”, we could say that the three AFNs distinguish themselves by their position along a gra- dient of their transformative potential. At one end of the axis, the organic shops implement the least stringent practices for each category of power-configuration. At the other end of the axis, the Gasap has the most am- bitious targets and appears the most coherent system: Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 28 democracy and food sustainability in the context of un- connected AFNs in terms of localization. Secondly, more detailed and empirical research should be conducted regarding the relationship between AFNs as social in- novations which merely constitute a “niche”, and the broader food “regime” (Seyfang & Longhurst, 2013) at a regional or national level. Indeed, even if our article shows promising results about the effects of food democ- racy processes on the actors and individuals, it still lack- ing in the measures and explanations offered about how these transformations could lead to the transition of the regional food system. sustainability transition, this assessment leads to further discussion and reflection on how coordinating and up- scaling a diversity of food democracy practices can con- tribute to food sustainability. 5. Conclusions The article showed that the three AFNs conceptualize and define food democracy as the way to reach some sustainability goals, mainly in governance and socio- economic terms. In this respect, AFNs connect their daily practices and normative beliefs to the sustainabil- ity transition approach. Our participatory sustainability- focused assessment approach allowed us to reconstruct a grounded and socially constructed “vision” of food democracy that the three grassroots AFNs share in common. The identified criteria and indicators illus- trate this vision and show a rather pragmatic relation- ship between food democracy and sustainability. Power- configurations need to transform, and new ways of or- ganizing must be implemented to achieve sustainability goals. Democracy dimensions as such are part of the sus- tainability landscape but are also part of the pathway to more sustainable practice. Food democracy is a means for collective and transformative action. It consists of a set of processes that give more power to the actors in- volved in the food chains, at the three political, social, and economic levels. The authors proposed in this arti- cle to provide concrete and operational content to the concept of food democracy that can surely open new em- pirical research and investigations. The authors declare no conflict of interests. The authors declare no conflict of interests. Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 References GeoJournal, 73(1), 55–65. However, our results appeal for more investigation. Firstly, it would be interesting to repeat the same pro- cess in other places to compare the visions of food ( ) Forssell, S., & Lankoski, L. (2015). The sustainability Forssell, S., & Lankoski, L. (2015). The sustainability Forssell, S., & Lankoski, L. (2015). The sustainability Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 29 Sociology of Agriculture and Food, 20, 383–389. promise of alternative food networks: An examina- tion through alternative characteristics. Agriculture and Human Values, 32(1), 63–75. Mundler, P., & Rumpus, L. (2012). The energy effi- ciency of local food systems: A comparison between different modes of distribution. Food Policy, 37(6), 609–615. Geels, F. W., & Schot, J. (2007). Typology of sociotech- nical transition pathways. Research Policy, 36(3), 399–417. 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Lang, T. (1999). Food policy for the 21st century: Can it be both radical and reasonable? In M. Koc̦, R. MacRae, L. J. Mougeot, & J. Welsh (Eds.), For hunger-proof cities: Sustainable urban food systems. (pp. 216–224). Ot- tawa: International Development Research Centre. Sébastien, L., Lehtonen, M., & Bauler, T. (2017). Introduc- tion: Les indicateurs participatifs tiennent-ils leurs promesses? [Introduction: Are participatory indica- tors keeping their promise?]. Participations, 2(18), 9–38. Le Velly, R. (2016). Sociologie des systèmes alimentaires alternatifs: Une promesse de différence [Sociology of alternative food systems: A promise of difference]. Paris: Presses des Mines. Seyfang, G., & Longhurst, N. (2013). Desperately seek- ing niches: Grassroots innovations and niche devel- opment in the community currency field. Global En- vironmental Change, 23(5), 881–891. Levkoe, C. Z. (2006). Learning democracy through food justice movements. Agriculture and Human Values, 23(1), 89–98. 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Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 About the Authors François Lohest, a Political Scientist by training (MSc 2009), has been a researcher at the Université Libre de Bruxelles since 2016. A former organic vegetable producer, he is now involved in research in the field of sustainability transitions, mainly regarding alternative food chains, food governance, and the upscaling of grassroots innovations. 30 Politics and Governance, 2019, Volume 7, Issue 4, Pages 21–31 Tom Bauler (Prof.), an Ecological Economist leaning towards Science, Technology & Society studies (STS), holds the Chair “Environment & Economy” at the Université Libre de Bruxelles. Tom explores the roles of policy instruments and tools for environmental policy. Emphasis is given to questions re- lating to the place, insertion, use and impact of policy dynamics, discourses, instruments, and tools (e.g., indicators, scenario exercises, policy concepts). Solène Sureau is a PhD student at the Institute for Environmental Management and Land-Use Planning, Université Libre de Bruxelles. Her research focuses on the assessment of social and socio-economic impacts of Alternative Food Networks and the methodological development of social life cycle assess- ment (S-LCA). She holds a master’s degree in Macroeconomics (Université de Strasbourg, 2007) and Environmental Management (Université Libre de Bruxelles, 2014). Joris Van Mol has been working on monitoring and evaluation in both public and private sector settings for over 10 years. He’s been concentrating mainly on issues related to food and agriculture, with partic- ular attention to social dimensions. Initially, his work focused on the position of smallholder farmers in the South and their integration in globalized value chains. Gradually, he became interested in the sustainability promise of more locally embedded alternative food systems. He is currently focusing on the role and contribution of reporting systems in the transition to a more sustainable economy. Wouter M. J. Achten, a Bio-Science Engineer by training (MSc 2005, PhD 2010), has been Professor of Environmental Impact Management at the Université Libre de Bruxelles since 2013. He is active on methodological and applied research in the field of sustainability science, mainly focusing on life cycle thinking tools. He hosts methodological research on land-use LCA, life cycle sustainability assessment, social LCA, input-output LCA, territorial LCA, consequential LCA, and currently applies this in the fields of agricultural production, bio-products, circular economy, waste management, and forestry. 31
https://openalex.org/W3036333679	https://journal.trunojoyo.ac.id/rechtidee/article/download/6890/pdf	Indonesian	null	PIDANA PELATIHAN KERJA PENGGANTI DENDA YANG BERKEMANFAATAN DALAM PERSPEKTIF PERLINDUNGAN ANAK	Deleted Journal	2,020	cc-by-sa	4,313	Abstract This research is aimed to find out, understand, and analyse ratio legis of regulation concerning job training as mentioned in Law concerning Juvenile Court System. Statute approaches were employed in this research, supported by legal materials that were analysed and interpreted based on systematic, grammatical, and teleological interpretation technique. g g p q The research result shows the following: (1) the ratio legis of job training mentioned in UU SPPA is that the sanction should be given based on the levels of punishment severity, where the sanction should be delivered based on the structure of punishment in UU SPPA as follows: warning; conditional punishment; job training; training delivered in the Department of Corrections; and imprisonment. Jail sentence is positioned in the last structure to make it the least option taken by the judge in terms of delivering sanction to ABH due to consideration that it may negatively affect the child’s future. (2) Sanction where job training is implemented for the child concerned regulated as primary criminal sanction in UU SPPA is deemed irrelevant, recalling that there is no element of disapprobation. In terms of the regulation for job training as in UU SPPA and Law concerning Labour Affairs, the job training is more considered as a sanction aimed to discipline the child. Pidana Pelatihan Kerja Pengganti Denda yang Berkemanfaatan dalam Perspektif Perlindungan Anak NuriniAprilianda FakultasHukumUniversitasBrawijaya aprilianda@yahoo.co.id / nurini.aprilianda@ub.ac.id Submit: 16-03-2020; Review: 06-05-2020; Terbit: 17-06-2020 Submit: 16-03-2020; Review: 06-05-2020; Terbit: 17-06-2020 Submit: 16-03-2020; Review: 06-05-2020; Terbit: 17-06-2020 Kata Kunci: Anak, Pidana Pelatihan Kerja, Pidana. tahun 2016 mencapai 4.622 kasus, selanjutnya tahun 2017 berjumlah 4.579 kasus dan tahun 2018 mencapai 4.885 kasus. Di tahun 2018 kasus Anak Berhadapan Hukum masih menduduki urutan pertama yaitu mencapai 1.434 kasus. Kasus ABH didominasi kasus kekerasan seksual yang mana laki-laki mendominasi sebagai pelaku dibandingkan dengan anak pe rempuan. Sepanjang tahun 2018, pelaku berjumlah 103, sedangkan pelaku berjenis kelamin perempuan berjumlah 58 anak ( http://www.kpai.go.id/berita/kpai- sebut-pelanggaran-hak-anak-terus- meningkat ). tahun 2016 mencapai 4.622 kasus, selanjutnya tahun 2017 berjumlah 4.579 kasus dan tahun 2018 mencapai 4.885 kasus. Di tahun 2018 kasus Anak Berhadapan Hukum masih menduduki urutan pertama yaitu mencapai 1.434 kasus. Kasus ABH didominasi kasus kekerasan seksual yang mana laki-laki mendominasi sebagai pelaku dibandingkan dengan anak pe rempuan. Sepanjang tahun 2018, pelaku berjumlah 103, sedangkan pelaku berjenis kelamin perempuan berjumlah 58 anak ( http://www.kpai.go.id/berita/kpai- sebut-pelanggaran-hak-anak-terus- meningkat ). Abstrak Tulisan merupakan hasil penelitian yang bertujuan untuk menggali dan menganalisis ratio legis pembentuk undang-undang menetapkan pidana pelatihan kerja sebagai pengganti denda bagi anak. Untuk menjawab tujuan tersebut, digunakan metode penelitian normatif, dengan pendekatan peraturan perundang- undangan dan dianalisis berdasarkan metode penafsiran sistematis, gramatikal dan intrepetasi teleologis. Hasil penelitian menunjukkan bahwa ratio legis penetapan pidana pelatihan kerja sebagai pengganti denda dalamUndang-undang No. 11 Tahun 2012 Tentang Sistem Peradilan Pidana Anak adalah sebagai berikut: (1) pidana denda tidak dikenakan pada anak-anak di bawah umur, karena asumsinya mereka belum bekerja. Bila denda dijatuhkan pasti yang akan membayar adalah orang tuanya. Oleh sebab itu, tidak ada pidana denda di dalam Undang-Undang ini. (2) diharapkan agar lebih baik anak sendiri bertanggung jawab terhadap perbuatan yang dilakukannya. Oleh karena itu, mungkin karena anak tidak bias bekerja atau 57 RechtIdee, Vol. 15, No. 1, Juni 2020 58 tidak punya uang, harus dicari alternatif lain supaya anak merasa dia bertanggung jawab terhadap perbuatannya yaitu pelatihan kerja. Pendahuluan Negara Indonesia masih memiliki angka yang cukup signifikan dari segi permasalahan hukum yang melibatkan anak. Persoalan pada anak dewasa ini dianggap cukup beragam. Anak Berhadapan Hukum atau biasa disebut dengan ABH pada tahun semester pertama 2018 terdapat 1.885 kasusanak yang mana ada 504 kasus ABH. Sepanjang tahun 2011- 2017 terdapat 9.266 kasus. Pertahun, jumlah paling banyak terjadi pada tahun 2014 dimana kasus anak mencapai jumlah 2.208. Urutan yang kedua ada di tahun 2013 dengan jumlah kasu ssebanyak 1.428 dan yang terakhir pada tahun 2012 dengan jumlah 1.413 kasus (http://www.kpai.go.id/berita/kpai- kurun-waktu-6-tahun-abh-sudah- mencapai-9-266-kasus). Data Permasyarakatan untuk di daerah Jawa Timur pada akhir tahun 2015 jumlah nara pidana anak sebanyak 2.017 anak. Kemudian akhir tahun 2016 jumlahnya meningkat sebanyak 2.123 anak. Sedangkan di bulan Juni tahun 2017 jumlah narapidana anak sebanyak 3.983. ( http://smslap.ditjenpas.go.id) Komisi Perlindungan Anak Indonesia (KPAI) menyatakan Kasus Pengaduan yang masuk di KPPAI, tahun 2015 berjumlah 4.309 kasus, RechtIdee, Vol. 15, No. 1, Juni 2020 59 perubahan atas UU RI No. 23 tahun 2002 tentang Perlindungan Anak. Di antara narapidana anak yang menjalani pidana penjara tersebut, terdapat narapidana anak yang selain dijatuhi pidana penjara juga dijatuhi pidana pelatihan kerja sebagai penggan tidenda. Hal ini dijatuhkan hakim sesuai dengan ketentuan pasal 71 (3) Undang-UndangNomor 11 tahun 2012 tentang Sistem Peradilan Pidana Anak (UU SPPA) bahwa : Apabila dalam hukum materiil diancam pidana kumulatif berupa penjara dan denda, pidana denda diganti dengan pelatihan kerja. 2) Putusan PN Jombang Nomor 02/Pid.Sus-Anak/2016/PN.JBG, menyatakan mengadili Anak EP yang telah terbukti secara sah dan meyakinkan bersalah melakukan tindak pidana “Membujuk anak untuk melakukan persetubuhan dengannya terus menerus sebagai perbuatan yang dilanjutkan”. Karenanya EP dijatuhi pidana penjara selama 1 (satu) tahun dan pelatihan kerja selama 3 (tiga) bulan di Lembaga Perlindungan dan Pendamping Anak (LP2A) Jombang. EP terbukti melakukan tindak pidana sebagaimana diatur dalam pasal 81 ayat (2) UU RI No 35 tahun 2014 tentang perubahan atas UU RI No. 23 tahun 2002 tentang PerlindunganAnak jo pasal 64 ayat (1) KUHP, UU RI No. 11 tahun 2012 tentang Sistem Peradilan Pidana Anak. Beberapa narapidana anak yang dijatuhi pidana penjara dan pidana pelatihan kerja pengganti denda dapat dilihat dalam beberapa putusan sebagaiberikut. Pendahuluan 1) Putusan PN Kabupaten Kediri Nomor : 23/Pid.Sus/An/2015/PN.Gpr.Hak im Anak menjatuhkan pidana penjara selama 2 (dua) tahun 8 (delapan) bulan dan pelatihan kerja selama 6 (enam) bulan terhadap DP (14 tahun), karena terbukti melakukan tindak pidana pencabulan sebagaimana diatur dalam Pasal 82 (1) jo Pasal 76 E UU RI No 35 tahun 2014 tentang 1) 3) Putusan PN Kendal Nomor: 2/Pid.SUS.Anak/2016/PN.K. Hakim anakdi PN Kendal menjatuhkan sanksi pidana 3) Putusan PN Kendal Nomor: 2/Pid.SUS.Anak/2016/PN.K. Hakim anakdi PN Kendal menjatuhkan sanksi pidana RechtIdee, Vol. 15, No. 1, Juni 2020 60 penjara selama 2 tahun 6 bulan di LPKA KUTOARJO serta pelatihan kerja selama 3 bulan di Lembaga Pelatihan Kerja pada KU (12 tahun) karena melakukan tindak pidana pencabulan sebagaimana diatur dalam Pasal 82 ayat (1) UU No. 35 tahun 2014 Tentang Perubahan terhadap UU No. 23 Tahun 2002 Tentang Perlindungan anak UU SPPA tidak memberikan pengertian dan penjelasan lebih lanjut mengenai pidana pelatihan kerja sebagai pengganti denda. Pelatihan kerja sendiri menurut Pasal 1 angka 9 UU 13 tahun 2003 tentang Ketenagakerjaan adalah keseluruhan kegiatan untuk memberi, memperoleh, meningkatkan, serta mengembangkan kompetensi kerja, produktivitas, disiplin, sikap, dan etos kerja pada tingkat keterampilan dan keahlian tertentu sesuai dengan jenjang dan kualifikasi jabatan atau pekerjaan. Lebih lanjut, dalam UU Ketenagakerjaan ditegaskan bahwa Pengusaha dilarang mempekerjakan anak (Pasal 68), kecuali bagi anak yang berumur antara 13 (tigabelas) penjara selama 2 tahun 6 bulan di LPKA KUTOARJO serta pelatihan kerja selama 3 bulan di Lembaga Pelatihan Kerja pada KU (12 tahun) karena melakukan tindak pidana pencabulan sebagaimana diatur dalam Pasal 82 ayat (1) UU No. 35 tahun 2014 Tentang Perubahan terhadap UU No. 23 Tahun 2002 Tentang Perlindungan anak tahun untuk melakukan pekerjaan ringan sepanjang tidak mengganggu perkembangan dan kesehatan fisik, mental, dan sosial (Pasal 69). Hal ini dapat dipahami karena sesuai dengan prinsip perlindungan anak, anak tidak boleh dieksploitasi. Namun demikian, tidak terdapat penjelasan apakah ketentuan dalamUU ketenagakerjaan dapat diberlakukan bagi Anak yang dijatuhi pidana pelatihan kerja pengganti denda. UU SPPA tidak memberikan pengertian dan penjelasan lebih lanjut mengenai pidana pelatihan kerja sebagai pengganti denda. Pelatihan kerja sendiri menurut Pasal 1 angka 9 UU 13 tahun 2003 tentang Ketenagakerjaan adalah keseluruhan kegiatan untuk memberi, memperoleh, meningkatkan, serta mengembangkan kompetensi kerja, produktivitas, disiplin, sikap, dan etos kerja pada tingkat keterampilan dan keahlian tertentu sesuai dengan jenjang dan kualifikasi jabatan atau pekerjaan. Pendahuluan Lebih lanjut, dalam UU Ketenagakerjaan ditegaskan bahwa Pengusaha dilarang mempekerjakan anak (Pasal 68), kecuali bagi anak yang berumur antara 13 (tigabelas) tahun sampai dengan 15 (lima belas) Berdasarkan uraian di atas, tulisan ini membahas tentang: ratio legis pembentuk undang-undang menetapkan pidana pelatihan kerja sebagai pengganti denda bagi anak dan konsep pidana pelatihan kerja sebagai pengganti denda yang berkemanfaatan dalam perspektif perlindungan anak. Untuk menjawab 2 hal tersebut, digunakan penelitian hukum normatif dengan pendekatan perundang-undangan. Metode Penelitian Penelitian ini merupakan penelitian normatif yaitu penelitian yang beranjak dari isu yang ada dalam peraturan perundangan- undang. Sebagai penelitian normatif RechtIdee, Vol. 15, No. 1, Juni 2020 61 pemuda yang berguna bagi bangsanya (Djamil, 2013: 4) Mengacu pada hal tersebut penting untuk menyepakati model penanganan ABH. maka pendekatan yang digunakan adalah perundang-undangan. Sumber bahan hukum berasal dari data sekunder berupa peraturan perundang-undangan yang relevan, teori dan pendapat para pakar. Analisis yang digunakan adalah analisis berdasarkan metode penafsiran sistematis, gramatikal dan intrepetasi teleologis. Sejak tanggal 3 Januari 1998 melalui Undang-Undang Nomor 3 Tahun 1997 terdapat unifikasi hokum melalui peradilan anak. Dalam undang-undang tersebut mengakomodasi mengenai teori dan praktek tentang peradilan anak yang sebelumnya sudah ada. Tetapi karena perkembangan zaman dan teknologi, undang-undang ini dirasakan sudah tidak relevan lag iuntuk diterapkan. Dengan pertimbangan: (Konsiderans UU 11/2012 tentang Sistem Peradilan Pidana Anak). Hasil dan Pembahasan Ratio Legis Pembentuk Undang- Undang Menetapkan Pidana Pelatihan Kerja Sebagai Pengganti Denda Bagi Anak Perlindungan terhadap anak merupakan salah satu tanggung jawab Negara. Hal ini dijelaskan dalam Pasal 28B UUD Tahun 1945 bahwa setiap anak memperoleh hak untuk kelangsungan hidupnya, serta memperoleh perlindungan daritindakan diskriminasi. Oleh karena itu, penanganan bagi Anak yang berkonflik dengan hukum (selanjutnya ABH) janganlah sampai menimbulkan stigmatisasi atau labeling dan kurangnya atau bahkan ketiadaan pembinaan terhadap mereka sehingga membuyarkan harapan-harapan mereka menjadi a) Bahwa anak adalah amanah dan juga karunia dari Tuhan Yang Maha Esa yang mempunyai harkat dan mertabat sebagai manusia seutuhnya. b) Bahwa untuk melindungi harkat dan juga martabatnya, anak memiliki hak terhadap perlindungan khusus, terutama perlindungan hukum dan system peradilan. RechtIdee, Vol. 15, No. 1, Juni 2020 62 c) Bahwa Indonesia sebagai salah satu Negara yang ikut dalam Konvensi Hak-Hak Anak (Convention on the Rights of the Child), mengatur tentang prinsip perlindungan hokum bagi anak memiliki kewajiban untuk memberikan perlindungan khusus bagi anak yang berhadapan dengan hukum. SPPA). UU SPPA ini membawa beberapa perubahan di antaranya ialah konsep diversi. Serta menggunakan model restorative justice, yakni model penyelesaian perkara yang mengutamakan pengembalian keadaan seperti semula, keadaan sebelum terjadinya sutau tindak pidana. Anak harus ditangani secara berbeda dengan orang dewasa. Oleh sebab itu, secara paradigma model penanganan yang berlaku melalui Undang-undang Pengadilan Anak, adalah sama dengan orang dewasa, dengan model Retributive Justice, yakni pidana sebagai pilihan utama atau pembalasan atas tindak pidana yang telah dilakukan. Model ini sudah tidak sesuai, ketidaksesuainnya tersebut berdasarkan 3 (tiga) pertimbangan: pertama, alasan karakteristi kanak. Undang-Undang Perlindungan Anak (Undang-undang Nomor 23 Tahun 2002) menyebutkan untuk tumbuh dan berkembang secara optimal, baikfisik mental maupun sosial, dan berahlak mulia. Anak belum dapat menentukan pilihan tindakan secara benar, hal ini dikarena kananak d) Bahwa dalam Undang-undang No. 3 Tahun 1997 tentang Pengadilan Anak (selanjutnya disebut dengan Undang-undang Pengadilan Anak) sudah tidak cocok lagi dengan kebutuhan dan perkembangan masyarakat dikarenakan belum dapat memberikan perlindungan kepada anak yang berhadapan dengan hokum secara menyeluruh sehingga perlu diganti dengan Undang-undang baru. Berdasarkan pertimbangan tersebut, maka perlu dibentuk Undang-undang baru yang mengatur mengenai Anak, dan karena itu diberlakukan Undang-undang No. 11 Tahun 2012 tentang Sistem Peradilan Pidana Anak (selanjutya disebut UU RechtIdee, Vol. 15, No. 1, Juni 2020 63 merupakan individu yang masih harus tumbuh dan berkembang dalam segala aspek. Kedua, alasan masa depan anak. Hasil dan Pembahasan Anak yang dipidana terlabel dan terstigmatisasi selepas pemidanaan sehingga Menyulitkan pertumbuhan psikis dan sosial anak kedepan. Ketiga, memulihkan hubungan antara ABH, korban dan masyarakat. (Undang- undang Nomor 23 Tahun 2002 tentang Perlindungan Anak) e) penjara. (2) Pidana tambahan terdiri atas: a) perampasan keuntungan yang diperoleh dari tindak pidana; atau b) pemenuhan kewajiban adat. (3) Apabila dalam hukum materiil diancam pidana kumulatif berupa penjara dan denda, pidana denda diganti dengan pelatihan kerja. Hukuman yang diberikan kepada anak tidak berorientasi pada pembalasan, hukuman yang diberikan kepada anak wajib mempertimbangkan prinsip- prinsip perlindungan anak. Pidana pokok dalam UU SPPA diatur dalam Pasal 71: (4) Pidana yang dijatuhkan kepada Pidana yang dijatuhkan kepada Anak dilarang melanggar harkat dan martabat Anak. Salah satu bentuk pidana yang terdapat dalam pasal tersebut adalah pidana denda. Namun memberikan pidana denda kepada ABH tidaklah tepat mengingat ABH biasanya belum memiliki pekerjaan. Oleh karena itu dalam Pasal 71 ayat (3) UU SPPA menyebutkan apabila dalam hokum materiil diancam pidana kumulatif berupa penjara dan denda, pidana denda diganti dengan pelatihan kerja. Pidana pelatihan (1) Pidana pokok bagi Anak terdiri atas: a) pidana peringatan; b) pidana dengan syarat: 1) pembinaan di luar lembaga; 2) pelayanan masyarakat; atau 3) pengawasan. c) pelatihan kerja; d) pembinaan dalam lembaga; dan RechtIdee, Vol. 15, No. 1, Juni 2020 64 kerja dijadikan pidana pengganti terhadap penjatuhan pidana denda bagi ABH. Namun apakah dengan menggantikan pidana denda dengan pidana pelatihan kerja dapat memberikan suatu perlindungan dan kemanfaatan bagi ABH. Apa yang menjadi dasar penggantian pidana denda kepidana pelatihan kerja. kehidupan lebih baik setelah kembalinya menjadi bagian dari masyarakat (Anggraini, 2013:11). Pasal 71 ayat (3) UU SPPA menyebutkan bahwa “apabila dalam hokum materiil diancam pidana kumulatif berupa penjara dan denda, pidana denda diganti dengan pelatihan kerja”. Digantinya pidana denda kepidana pelatihan kerja tentu memiliki alasan tersendiri. Untuk dapat mengetahui sebab dan alas an pidana denda bagi ABH diganti dengan pelatihan kerja, maka akan dipaparkan ratio legis dari Pasal 71 ayat (3) UU SPPA sebagai berikut: Pidana pelatihan kerja sebagai pengganti pidana denda telah dikenal sejak diundangkannya UU No. 3 Tahun 1997 yang kemudian diganti dengan Undang-Undang Nomor 11 Tahun 2012 tentang Sistem Peradilan Pidana Anak. Dalam UU Pengadilan Anak menyatakan bahwa anak yang diberikan putusan subside wajib latihan kerja adalah mereka yang dijatuhi pidana kumulatif penjara dan denda. Demikian kemudian anak yang tidak membayar denda dan melaksanakan wajib latihan kerja. Hasil dan Pembahasan Sebagaimana tujuan system peradilan pidana anak untuk mewujudkan kesejahteraan anak sebagai landasan perlindungan hokum bagi anak yang bermasalah dengan hokum ini adalah memberikan keterampilan hinggaia dapat mandiri dan mempunyai 1) Kalau usulan dari Gerinda ini memang pidana denda tidak dikenakan pada anak-anak di bawah umum, karena asumsinya merekakan belum bekerja. Jadi, kalau ada denda pasti yang bayarorangtuanya. Oleh sebab itu, tidak ada pidana denda di dalamUndang-Undang ini (Harkristuti dalam Risalah UU No. 11 Tahun 2012 tentang SPPA). 1) Kalau usulan dari Gerinda ini memang pidana denda tidak dikenakan pada anak-anak di bawah umum, karena asumsinya merekakan belum bekerja. Jadi, kalau ada denda pasti yang bayarorangtuanya. Oleh sebab itu, tidak ada pidana denda di dalamUndang-Undang ini (Harkristuti dalam Risalah UU No. 11 Tahun 2012 tentang SPPA). 2) Pertanggungjawaban Orang Tua Di negara-negara lain orang tua bertanggung jawab, biasanya RechtIdee, Vol. 15, No. 1, Juni 2020 65 UU No. 11 Tahun 2012 tentang SPPA). UU No. 11 Tahun 2012 tentang SPPA). secara ekonomis, berarti untuk ganti rugi dan denda. Orang tua yang biasanya harus pertanggungjawaban, tetapi di banyak negara sebenarnya lebih baik anak sendiri bertanggungjawab. Oleh karena itu, mungkin karena anak tidak bias bekerja atau tidak punya uang, kita harus cari alternatif lain. Misalnya, apakah anak bias membantu selama beberapa bulan korban. Misalnya, jika korban adalah orang yang bekerja di took apakah anak bias tidak membantu korban. Berarti lebih baik daripada denda yang atau uang, sanksinya lain supaya anak merasa dia bertanggungjawab dan dia yang harus merespon (Arosio Asnar dalam Risalah UU No. 11 Tahun 2012 tentang SPPA). secara ekonomis, berarti untuk ganti rugi dan denda. Orang tua yang biasanya harus pertanggungjawaban, tetapi di banyak negara sebenarnya lebih baik anak sendiri bertanggungjawab. Oleh karena itu, mungkin karena anak tidak bias bekerja atau tidak punya uang, kita harus cari alternatif lain. Misalnya, apakah anak bias membantu selama beberapa bulan korban. Misalnya, jika korban adalah orang yang bekerja di took apakah anak bias tidak membantu korban. Berarti lebih baik daripada denda yang atau uang, sanksinya lain supaya anak merasa dia bertanggungjawab dan dia yang harus merespon (Arosio Asnar dalam Risalah UU No. 11 Tahun 2012 tentang SPPA). Dari risalah pembentukan Pasal 71 ayat (3) UU SPPA di atas dapat ditarik kesimpulan bahwa terdapat dua pendapat, pertama.bahwa pidana denda merupakan sanksi yang tidak tepat bagi ABH, karena ABH belum memiliki penghasilan untuk membayar pidana denda tersebut. Hasil dan Pembahasan Kedua orang tua dapat bertanggung jawab atas perbuatan yang ABH lakukan, jadi orang tua yang membayar pidana denda yang dijatuhkan terhadap ABH. Seperti yang dikemukakan oleh Sudarto bahwa tujuan utama perlindungan hokum bagi anak adalah untuk mewujudkan kesejahteraan Anak disamping kepentingan masyarakat. Kepentingan Anak tidak boleh dikorbankan demi kepentingan masyarakat. Untuk hal tersebut, terdapat dua asas penting yang harus diperhatikan adalah; 3) Pasal 79, pidana pelatihan kerja sebagaimana dimaksud dalam Pasal 72 Ayat (1) huruf c dilaksanakan di lembaga yang melaksanakan pelatihan kerja yang sesuai dengan usia anak. Tidak ada masalah saya pikir (Aziz Syamsudin, dalam Risalah 1) Memajukan Kesejahteraan Anak (the promotion of the well being of the juvenile) Hal ini merupakan focus utama system hokum dalam menangani RechtIdee, Vol. 15, No. 1, Juni 2020 66 Anak yang melakukan pelanggaran, khususnya dalam sistem hukum yang mengikuti model peradilan pidana, system hokum harus lebih menekankan atau mengutamakan kesejahteraan Anak. Ditegaskan pula bahwa prinsip ini berarti menolak prinsip penggunaan sanksi yang hanya bersifat pidana atau yang bersifat menghukum semata (the avoidance of merely punitive sanctions). should it function merely as a social agency) (Sudarto, 1981:140). should it function merely as a social agency) (Sudarto, 1981:140). Sistem hukum yang menangani anak haruslah menekankan perlindungan kesejahteraan terhadap anak tersebut, bukan sebalikya. Pidana denda bagi ABH merupakan hal yang sangat tidak tepat. Bagaimana mungkin ABH bias membayar denda sedangkan ABH itu sendiri belum memiliki penghasilan sendiri. Tentu hal tersebut sangat merugikan ABH. Maka solusi yang terbaik adalah dengan mengganti pidana denda dengan pidana pelatihan kerja. Namun dalam pidana pelatihan kerja, harus tetap memperhatikan prinsip-prinsip perlindungan terhadap anak. 2) Prinsip Proposionalitas (the Prinsip proposionalitas, yaitu prinsip yang merupakan alat untuk dapat mengekang penggunaan sanksi yang hanya bersifat menghukum dalam arti pembalasan (just desort). Walaupun penekanan ini ditujukan pada permasalahan kesejahteraan Anak, namun perlu dicatat pendapat Paul W. Tappan yang menyatakan bahwa peradilan Anak janganlah hanya berfungsi sebagai suatu pengadilan pidana dan tidak pula harus berfungsi semata-mata sebagai lembaga sosial (the juvenile court should not function merely as a criminal court for children, not Berdasarkan konvensi Hak Anak yang kemudian diadopsi dalam UU No. 23 Tahun 2002 Tentang Perlindungan Anak yang sekarang dirubah oleh UU No. 35 Tahun 2014, ada empat prinsip umum perlindungan Anak yang menjadi dasar bagi setiap Negara dalam menyelenggarakan perlindungan anak, yaitu: 1) Prinsip Non diskriminasi RechtIdee, Vol. 15, No. 1, Juni 2020 67 Perspektif perlindungan anak adalah cara pandangan terhadap semua persoalan dengan menempatkan posisi anak sebagai yang pertama dan utama. Implementasi cara pandang demikian adalah ketika selalu menempatkan anak sebagai hal yang paling utama. Berikut gambar mengenai perlindungan anak. 2) Prinsip Kepentingan Terbaik Bagi Anak (Best Interests of The Child. 2) Prinsip Kepentingan Terbaik Bagi Anak (Best Interests of The Child. 3) Prinsip Hak Hidup, Kelangsungan Hidup, dan Perkembangan (The Right to life, Survival, and Development) 4) Prinsip Penghargaan Terhadap Pendapat Anak (Respect for the views of the Child) Gambar : Perlindungan bagi anak. KepentinganTerbaik bagi anak Nondiskriminasi Partisipasi Anak (Djamil, 2013 :31). Kelangsungan Hidup dan Perkembangan Anak Gambar : Perlindungan bagi anak. KepentinganTerbaik bagi anak Kelangsungan Hidup dan Perkembangan Anak Nondiskriminasi Partisipasi Anak (Djamil, 2013 :31). Partisipasi Anak (Djamil, 2013 :31). Nondiskriminasi (sumber: bahan hukum sekunder) perlindungan hukum yang sepatutnya diberikan kepada anak. (Muladi dan Barda, 2010:119). perlindungan hukum yang sepatutnya diberikan kepada anak. (Muladi dan Barda, 2010:119). Membicarakan perlindungan hokum bagi anak dalam proses peradilan tidak dapat dilepaskan dari apa sebenarnya tujuan atau dasar pemikiran dari peradilan anak itu sendiri. Bertolak dari tujuan dan dasar pemikiran inilah baru dapat ditentukan apa dan bagaimana hakikat serta wujud dari Pidana pelatihan kerja sebagai pengganti pidana denda sebagaiman diatur dalamPasal 71 ayat (3) UU SPPA, harus memperhatikan empat prinsip perlindungan anak. Pidana pelatihan kerja juga harus RechtIdee, Vol. 15, No. 1, Juni 2020 68 mempertimbangkan pendapat anak terhadap pekerjaan yang dilakukan oleh ABH. Pidana pelatihan kerja yang diberikan harus mendengar pendapat ABH dan mempertimbangkat bakat minat dari ABH itu sendiri. 2) Prinsip Proposionalitas (the Karena semua berjenis kelamin laki-laki maka pelaksanaan pidana subsider latihan kerja di Bengkel Las Intan sebagai badan usaha pengelasan teralis. 2) Tidak ada kualifikasi untuk menentukan tempat pelaksanaan latihan kerja, hanya didasarkan pada hubungan baik. Seperti yang terjadi di Lembaga Pembinaan Khusus Anak Kelas II B Pontianak. Dalam proses serah terima dari Lembaga Pembinaan Khusus Anak Kelas II B Pontianak kepada Dinas Sosial Kalimantan Barat, tidak ada pembahasan mengenai bakat, perkembangan jiwa dan bagaimana anak harus melaksanakan pidana subside latihan kerja (Anggraini, 2016 :20). Hasil wawancara dengan Kepala Bidang Pelayanan dan Rehabilitasi Sosial (PRS) Dinas Sosial Kalimantan Barat sebagai berikut : (Anggraini, 2016:20). 3) Tidak ada perjanjian tertulis yang menyatakan keberalihan tanggungjawab atas anak yang melaksanakan pidana subside latihan kerja. 4) Dalam menitipkan anak melaksanakan pidana subside latihan kerja, Dinas Sosial Kalimantan Barat memberitahukan lama latihan kerja oleh anak dan batasan sebagaimana dimaksud dalam Pasal 28 ayat (3) UU No. 3 Tahun 1997 tentang Pengadilan Anak lama latihan kerjahanya 4 (empat) jam sehari serta tidak dilakukan pada malam hari. 4) Dalam menitipkan anak melaksanakan pidana subside latihan kerja, Dinas Sosial Kalimantan Barat memberitahukan lama latihan kerja oleh anak dan batasan sebagaimana dimaksud dalam Pasal 28 ayat (3) UU No. 3 Tahun 1997 tentang Pengadilan Anak lama latihan kerjahanya 4 (empat) jam sehari serta tidak dilakukan pada malam hari. 1) Tidak adanya dana untuk melaksanakan pidana subside latihan kerja secara mandiri, anak dititipkan di “Bengkel Las Intan” bagi yang berjenis kelamin laki- laki dan “Erna Swiss Salon” bagi perempuan, keduanya terletak di Jl. Suwignyokota Pontianak. 5) Tidak ada pengawasan intensif dari DinasS osial Kalimantan Barat dalam pelaksanaan latihan kerja atapun bentuk absensi lainnya. RechtIdee, Vol. 15, No. 1, Juni 2020 69 6) Tidak ada evaluasi atas pelaksanaaan pidana subside latihan kerja. Buku Buku M. Nasir Djamil, Anak Bukan Untuk Dihukum, Catatan Pembahasan UU Sistem Peradilan Pidana Anak (UU-SPPA), Sinar Grafika, Jakarta, 2013. Undang-undangRepublik Indonesia Nomor 35 Tahun 2014 tentang Perubahan Atas Undang-undangNomor 23 tahun 2002 tentang Perlindungan Anak Peraturan Pemerintah Pengganti Undang- undang Republik Indonesia Nomor 1 Tahun 2016 tentang Perubahan Kedua atas Undang- undangNomor 23 Tahun 2002 tentang Perlindungan Anak Muladi dan Barda Nawawi Arif, Bunga Rampai Hukum Pidana, Alumni, Bandung, 2010. Sudarto, Kapita Selekta Hukum Pidana, Alumni, Bandung, 1981. Simpulan Simpulan 1) Ratio Legis Penetapan Pidana Pelatihan Kerja Sebagai Pengganti Denda DalamUndang- Undang No. 11 Tahun 2012 Tentang Sistem Peradilan Pidana Anak adalah sebagai berikut: a) Pidana denda tidak dikenakan pada anak-anak di bawah umur, karena asumsinya mereka belum bekerja. Bila denda dijatuhkan pasti yang akan membayar adalah orang tuanya. Oleh sebab itu, tidak ada pidana denda di dalamUndang-Undang ini. 1) Ratio Legis Penetapan Pidana Pelatihan Kerja Sebagai Pengganti Denda DalamUndang- Undang No. 11 Tahun 2012 Tentang Sistem Peradilan Pidana Anak adalah sebagai berikut: 7) Dinas Sosial melaporkan kepada Lembaga Pembinaan Khusus Anak Kelas II B Pontianak bahwa anak telah melaksanakan pidana subside latihan kerja dalam bentuk berita acara serah terima. 7) Dinas Sosial melaporkan kepada Lembaga Pembinaan Khusus Anak Kelas II B Pontianak bahwa anak telah melaksanakan pidana subside latihan kerja dalam bentuk berita acara serah terima. a) Pidana denda tidak dikenakan pada anak-anak di bawah umur, karena asumsinya mereka belum bekerja. Bila denda dijatuhkan pasti yang akan membayar adalah orang tuanya. Oleh sebab itu, tidak ada pidana denda di dalamUndang-Undang ini. Pidana pelatihan kerja Lembaga Pembinaan Khusus Anak Kelas II B yang bekerja sama dengan Dinas Sosial Kalimantan Barat adalah salah satu contoh pidana pelatihan kerja yang kurang memperhatikan pendapat anak. Tentu hal ini bertentang dengan prinsip perlindungan anak yakni penghargaan terhadap pendapat anak. Namun terdapat kendala di lapangan yang membuat Lembaga Pembinaan Khusus Anak Kelas II B yang bekerjasama dengan Dinas Sosial Kalimantan Barat melakukan pidana pelatihan kerja terhadap ABH, yakni kendala lokasi atau tempat anak menjalani pidana pelatihan kerja. b) Di beberapa Negara, pertanggungjawaban Orang Tua terhadap anaknya yang melakukan tindak pidana bersifat ekonomi dalam bentuk pemberian ganti kerugian kepada korban. Namun dibanyak negara disarankan agar lebih baik anak sendiri bertanggungjawab. Oleh karena itu, mungkin karena anak tidak bias bekerja atau tidak punya uang, kita harus cari alternatif lain supaya anak merasa dia bertanggungjawab terhadap perbuatannya. b) RechtIdee, Vol. 15, No. 1, Juni 2020 70 Putusan PN Jombang Nomor 02 / Pid . Sus-Anak / 2016 / PN. Jurnal Pemerintah/Dirjen Ham (Harkristuti Harkrisnowo): Risalah UUNo. 11 Tahun 2012 tentang Sistem Peradilan Pidana Anak. Lina Anggraini, Wajib Latihan Kerja Sebagai Hukuman Alternatif dalam Sistem Peradilan Pidana Anak (Studi Kasus Pada Lembaga Pembinaan Khusus Anak Pontianak),Jurnal Nestor, Vol. 3, No. 3, 2016. Juru Bicara Unicef (AROSIO ASNAR), Risalah UU No. 11 Tahun 2012 tentang Sistem Peradilan Pidana Anak. Peraturan Perundang-undangan Undang- undang No. 4 Tahun 1979 tentang Kesejahteraan Anak Undang-undang Nomor 13tahun 2003 tentang Ketenagakerjaan Undang-undang Nomor 23 tahun 2002 tentang Perlindungan Anak Undang-undang Nomor 11 Tahun 2012 tentang Sistem Peradilan Pidana Anak Peraturan Perundang-undangan Undang- undang No. 4 Tahun 1979 tentang Kesejahteraan Anak Ketua Rapat (H. Aziz Syamsuddin, , Risalah UU No. 11 Tahun 2012 tentang Sistem Peradilan Pidana Anak. Putusan PN Kabupaten Kediri Nomor : 23/Pid.Sus/An/2015/PN.Gpr Undang-undang Nomor 23 tahun 2002 tentang Perlindungan Anak Putusan PN Jombang Nomor 02 / Pid . Sus-Anak / 2016 / PN. Undang-undang Nomor 11 Tahun 2012 tentang Sistem Peradilan Pidana Anak RechtIdee, Vol. 15, No. 1, Juni 2020 71 Putusan PN Kendal Nomor: 2 / Pid.SUS.Anak/2016/PN.K Internet http://www.kpai.go.id/berita/kpai- kurun-waktu-6-tahun-abh- sudah-mencapai-9-266- kasus diakses pada tanggal 15 Februari 2020, pukul 20.30. http://www.kpai.go.id/berita/kpai- sebut-pelanggaran-hak- anak-terus-meningkat http://smslap.ditjenpas.go.id diakses pada tanggal 15 Februari 2020, pukul 20.39. Putusan PN Kendal Nomor: 2 / Pid.SUS.Anak/2016/PN.K Internet http://www.kpai.go.id/berita/kpai- kurun-waktu-6-tahun-abh- sudah-mencapai-9-266- kasus diakses pada tanggal 15 Februari 2020, pukul 20.30. http://www.kpai.go.id/berita/kpai- sebut-pelanggaran-hak- anak-terus-meningkat http://smslap.ditjenpas.go.id diakses pada tanggal 15 Februari 2020, pukul 20.39. Putusan PN Kendal Nomor: 2 / Pid.SUS.Anak/2016/PN.K Putusan PN Kendal Nomor: 2 / Pid.SUS.Anak/2016/PN.K Internet http://www.kpai.go.id/berita/kpai- kurun-waktu-6-tahun-abh- sudah-mencapai-9-266- kasus diakses pada tanggal 15 Februari 2020, pukul 20.30. http://www.kpai.go.id/berita/kpai- sebut-pelanggaran-hak- anak-terus-meningkat http://smslap.ditjenpas.go.id diakses pada tanggal 15 Februari 2020, pukul 20.39.
https://openalex.org/W4311867133	https://biblio.ugent.be/publication/01GKSHSKT9MJ5WM0NDS4M3FN3G/file/01GKSK4JRJBME7052P1TQN9P0J	English	null	Editorial: Environmental contaminants and animal health: Analysis, toxicity, and mitigation	Frontiers in veterinary science	2,022	cc-by	1,953	TYPE Editorial PUBLISHED 08 December 2022 DOI 10.3389/fvets.2022.1102836 TYPE Editorial PUBLISHED 08 December 2022 DOI 10.3389/fvets.2022.1102836 TYPE Editorial PUBLISHED 08 December 2022 DOI 10.3389/fvets.2022.1102836 KEYWORDS feed contaminants, renal toxicity, hepatic toxicity, mitigation, animal health, oxidative stress, omics Abdallah MF, Xu W and Abdeen A (2022) Editorial: Environmental contaminants and animal health: Analysis, toxicity, and mitigation. Front. Vet. Sci. 9:1102836. doi: 10.3389/fvets.2022.1102836 OPEN ACCESS OPEN ACCESS EDITED AND REVIEWED BY Arturo Anadón, Complutense University of Madrid, Spain CORRESPONDENCE Mohamed F. Abdallah mohamed.fathi@ugent.be SPECIALTY SECTION This article was submitted to Veterinary Pharmacology and Toxicology, a section of the journal Frontiers in Veterinary Science RECEIVED 19 November 2022 ACCEPTED 29 November 2022 PUBLISHED 08 December 2022 CITATION Abdallah MF, Xu W and Abdeen A (2022) Editorial: Environmental contaminants and animal health: Analysis, toxicity, and mitigation. Front. Vet. Sci. 9:1102836. doi: 10.3389/fvets.2022.1102836 OPEN ACCESS EDITED AND REVIEWED BY Arturo Anadón, Complutense University of Madrid, Spain CORRESPONDENCE Mohamed F. Abdallah mohamed.fathi@ugent.be SPECIALTY SECTION This article was submitted to Veterinary Pharmacology and Toxicology, a section of the journal Frontiers in Veterinary Science RECEIVED 19 November 2022 ACCEPTED 29 November 2022 PUBLISHED 08 December 2022 CITATION Abdallah MF, Xu W and Abdeen A (2022) Editorial: Environmental contaminants and animal health: Analysis, toxicity, and mitigation. Front. Vet. Sci. 9:1102836. doi: 10.3389/fvets.2022.1102836 Mohamed F. Abdallah1*, Wang Xu2 and Ahmed Abdeen3,4 1Department of Food Technology, Safety and Health, Ghent University, Ghent, Belgium, 2National Reference Laboratory of Veterinary Drug Residues, Ministry of Agriculture Key Laboratory for the Detection of Veterinary Drug Residues in Foods, Huazhong Agricultural University, Wuhan, China, 3Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Benha University, Toukh, Egypt, 4Center of Excellence in Screening of Environmental Contaminants (CESEC), Benha University, Toukh, Egypt Editorial on the Research Topic Environmental contaminants and animal health: Analysis, toxicity, and mitigation COPYRIGHT © 2022 Abdallah, Xu and Abdeen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. A wide range of environmental contaminants are potentially toxic to animals. This includes natural toxins (1, 2), pesticides (3), heavy metals (4) and other environmental contaminants such as microplastics (5). Animals are exposed to these pollutants mainly via consumption of contaminated feed and water, inducing variable deleterious eﬀects including hepatotoxicity, nephrotoxicity, neurotoxicity, genotoxicity, immunosuppression, etc. Despite the great success in developing several tools for their detection, as well as investigating their toxic mechanisms, still many aspects remain unclear along with those related to the “emerging contaminants”. Moreover, in the last decade, the Toxicology ﬁeld has become more interested in unraveling the combined toxicity of chemical mixtures under chronic exposure scenario. Finally, development of eﬀective and realistic strategies regarding biomonitoring, remediation, and protective medication are still required to minimize the health hazards of such contaminants. This Research Topic “Environmental Contaminants and Animal Health: Analysis, Toxicity, and Mitigation” aimed at collecting papers, which implement cutting-edge techniques, to improve our knowledge and understanding on the toxic mechanisms of the commonly exposed (natural) toxins in (farm) animals and novel approaches to alleviate their harmful eﬀects. In this special e-collection, there are seven papers covering many aspects of the above mentioned topic. In Egypt, El-Sappah et al. tracked the presence of ﬁve trace elements (zinc, manganese, copper, cadmium, and lead) in Nile Tilapia (Oreochromis niloticus) from three diﬀerent areas (Alexandria, Cairo and Aswan) as a bioindicator of environmental pollution. With the implementation of; (1) atomic absorption spectrophotometer analysis; (2) micronucleus frequency test; and (3) expression level of the Hsp70 gene assay, they concluded that the contents of cadmium, and lead Frontiers in Veterinary Science Frontiers in Veterinary Science 01 frontiersin.org 10.3389/fvets.2022.1102836 Abdallah et al. were higher than the safe levels recommended by many international organizations such as FAO, WHO, and the European Commission. Editorial on the Research Topic Indeed, more surveys are required to have a better insight into the accumulation of these metals in other aquatic organisms. On the other hand, Wei et al. performed a whole-transcriptome sequencing (RNA-seq) of liver samples from heifers to unravel the toxic eﬀects due to a chronic exposure of cadmium present in feed. The obtained results showed several diﬀerentially expressed genes involved in autophagy regulation, apoptosis, lipid metabolism, anti- inﬂammation, and antioxidant enzyme activity. Doubtless to say, such ﬁndings are useful to predict the cadmium related toxicities and to propose appropriate treatments. Although the paper published by Sun et al. in this special issue was mainly focusing on the antibiotic resistance genes and its relation with the integrative conjugative elements, the used methodology could be followed in other toxin producing bacteria, such as Clostridium botulinum, Listeria monocytogenes, Staphylococcus aureus, and Vibrio cholerae, that represents an issue in terms of food and feed safety. industries. Acrylamide represents a major threat to human health as it classiﬁed by IARC as human carcinogens. El-Shehawi et al. and Soliman et al. studied the protective eﬀects of Taify Pomegranate juice and Salsola imbricata leaf extract against acrylamide, respectively. Results from experiments using male rats indicate that both substances are promising as antitoxic agents. This was supported by biochemical, real-time PCR, histopathological, and immunohistochemical analysis. An increase in levels of the endogenous antioxidative enzymes, including SOD, catalase, and GSH were signiﬁcant after the exposure to either Taify Pomegranate juice or Salsola imbricata leaf extract. Additionally, anti-inﬂammatory eﬀects through reduction of the inﬂammatory cytokines (TNF-α and IL-6) secretion and the enhancement of the inﬂammatory cytokine IL-10 level were recorded. industries. Acrylamide represents a major threat to human health as it classiﬁed by IARC as human carcinogens. El-Shehawi et al. and Soliman et al. studied the protective eﬀects of Taify Pomegranate juice and Salsola imbricata leaf extract against acrylamide, respectively. Results from experiments using male rats indicate that both substances are promising as antitoxic agents. This was supported by biochemical, real-time PCR, histopathological, and immunohistochemical analysis. An increase in levels of the endogenous antioxidative enzymes, including SOD, catalase, and GSH were signiﬁcant after the exposure to either Taify Pomegranate juice or Salsola imbricata leaf extract. Additionally, anti-inﬂammatory eﬀects through reduction of the inﬂammatory cytokines (TNF-α and IL-6) secretion and the enhancement of the inﬂammatory cytokine IL-10 level were recorded. Funding Aﬂatoxin B1 (AFB1) is the most natural carcinogenic substance in the history. This fungal toxin is a frequent contaminant of many agricultural commodities and it targets liver, kindly and other body organs. In chicken, the consumption of AFB1 contaminated feed may lead to a reduced growth performance and tremendous economic losses (1). Through their investigation to ﬁnd a natural eco-friendly solution to reduce the toxic eﬀect of AFB1 in broilers, Damiano et al. presented their ﬁndings on the protective eﬀect on curcumin against the renal oxidative stress induced by the dietary exposure to low levels of AFB1. After 10 days of AFB1 exposure and curcumin, the known pathological eﬀects due to AFB1 toxicity in broiler kidneys were not observed. It would be interesting to investigate the curcumin eﬀect on a larger scale of broiler and formulate the curcumin as a feed supplement to commercialize it. Other natural substances were also studied for their protective eﬀects in this special issue, showing the increasing interest of reporting safe products that are able to decrease the toxic eﬀects of feed and food contaminants. Among these contaminants, acrylamide which is a widely used chemical material in several MFA was supported by the Ghent University Special Research Fund (BOF) postdoc mandate, Grant No. BOF20/PDO/032. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Author contributions MFA wrote the editorial. All authors reviewed, edited, and approved the submitted version. Editorial on the Research Topic In summary, the results of the above mentioned studies represent an enormous amount of new relevant data on toxic mechanism of common environmental and feed contaminants as well as the potential of many natural substances to play a crucial role as protective agents against these toxic substances. However, there is still a long way to go in order to better understand the mechanisms of action and that makes our hopes on the young researchers and new technologies to enrich our knowledge in this discipline and other related ﬁelds. It is well known that selenium shows a unique capability as an antioxidant via scavenging free radicals and nano- selenium (NSe) poses antioxidant roles through the alleviation of the oxidative stress caused by heavy metal such as mercury, cadmium, lead, and other substances. Based on that, Du et al. explored the role of NSe against the cadmium- induced acute hepatic toxicity in male Kunming mice. Their published results show that NSe has a clear eﬀect in reducing the toxic eﬀect of cadmium by diminishing the generation of reactive oxygen species and activating the Nrf2 pathway. Several questions remain unanswered regarding the underlying mechanism of nano-selenium which acts against Cd-induced hepatotoxicity. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Frontiers in Veterinary Science Frontiers in Veterinary Science frontiersin.org 02 10.3389/fvets.2022.1102836 10.3389/fvets.2022.1102836 Abdallah et al. 4. Jaishankar M, Tseten T, Anbalagan N, Mathew BB, Beeregowda KN. Toxicity, mechanism and health eﬀects of some heavy metals. Interdisc Toxicol. (2014) 7:60. doi: 10.2478/INTOX-2014-0009 Microb. (2016) 1:253–269. doi: 10.1007/978-3-319-27455-3_13/ FIGURES/2 Microb. (2016) 1:253–269. doi: 10.1007/978-3-319-27455-3_13/ FIGURES/2 4. Jaishankar M, Tseten T, Anbalagan N, Mathew BB, Beeregowda KN. Toxicity, mechanism and health eﬀects of some heavy metals. Interdisc Toxicol. (2014) 7:60. doi: 10.2478/INTOX-2014-0009 5. Zolotova N, Kosyreva A, Dzhalilova D, Fokichev N, Makarova O. Harmful eﬀects of the microplastic pollution on animal health: a literature review. PeerJ. (2022) 10:e13503. doi: 10.7717/PEERJ. 13503 10.3389/fvets.2022.1102836 1. Abdallah MF, Girgin G, Baydar T. Occurrence, prevention and limitation of mycotoxins in feeds. Animal Nutr Feed Technol. (2015) 15:471–90. doi: 10.5958/0974-181X.2015.00048.7 2. Abdallah MF, Van Hassel WHR, Andjelkovic M, Wilmotte A, Rajkovic A. Cyanotoxins and food contamination in developing countries: review of their types, toxicity, analysis, occurrence and mitigation strategies. Toxins. (2021) 13:786. doi: 10.3390/TOXINS13110786 3. Mahmood I, Imadi SR, Shazadi K, Gul A, Hakeem KR. Eﬀects of pesticides on environment. Plant, Soil 5. Zolotova N, Kosyreva A, Dzhalilova D, Fokichev N, Makarova O. Harmful eﬀects of the microplastic pollution on animal health: a literature review. PeerJ. (2022) 10:e13503. doi: 10.7717/PEERJ. 13503 1. Abdallah MF, Girgin G, Baydar T. Occurrence, prevention and limitation of mycotoxins in feeds. Animal Nutr Feed Technol. (2015) 15:471–90. doi: 10.5958/0974-181X.2015.00048.7 3. Mahmood I, Imadi SR, Shazadi K, Gul A, Hakeem KR. Eﬀects of pesticides on environment. Plant, Soil References 1. Abdallah MF, Girgin G, Baydar T. Occurrence, prevention and limitation of mycotoxins in feeds. Animal Nutr Feed Technol. (2015) 15:471–90. doi: 10.5958/0974-181X.2015.00048.7 2. Abdallah MF, Van Hassel WHR, Andjelkovic M, Wilmotte A, Rajkovic A. Cyanotoxins and food contamination in developing countries: review of their types, toxicity, analysis, occurrence and mitigation strategies. Toxins. (2021) 13:786. doi: 10.3390/TOXINS13110786 5. Zolotova N, Kosyreva A, Dzhalilova D, Fokichev N, Makarova O. Harmful eﬀects of the microplastic pollution on animal health: a literature review. PeerJ. (2022) 10:e13503. doi: 10.7717/PEERJ. 13503 3. Mahmood I, Imadi SR, Shazadi K, Gul A, Hakeem KR. Eﬀects of pesticides on environment. Plant, Soil 03 Frontiers in Veterinary Science frontiersin.org Frontiers in Veterinary Science
https://openalex.org/W1981498071	https://hal.science/hal-01004232/document	English	null	Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states	Proceedings of the National Academy of Sciences of the United States of America	2,011	cc-by	8,460	Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states Matthew L. M. L. Ferguson, Dominique D. Le Coq, Matthieu Jules, Stephane S. Aymerich, Ovidiu O. Radulescu, Nathalie N. Declerck, Catherine A. C. A. Royer Matthew L. M. L. Ferguson, Dominique D. Le Coq, Matthieu Jules, Stephane S. Aymerich, Ovidiu O. Radulescu, Nathalie N. Declerck, Catherine A. C. A. Royer To cite this version: Matthew L. M. L. Ferguson, Dominique D. Le Coq, Matthieu Jules, Stephane S. Aymerich, Ovidiu O. Radulescu, et al.. Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109 (1), pp.155 - 160. 10.1073/pnas.1110541108. hal- 01004232 To cite this version: Matthew L. M. L. Ferguson, Dominique D. Le Coq, Matthieu Jules, Stephane S. Aymerich, Ovidiu O. Radulescu, et al.. Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109 (1), pp.155 - 160. 10.1073/pnas.1110541108. hal- 01004232 Distributed under a Creative Commons Attribution 4.0 International License Matthew L. Fergusona, Dominique Le Coqb,c,d, Matthieu Julesb,d, Stéphane Aymerichb,d, Ovidiu Radulescue, Nathalie Declercka,f,1, and Catherine A. Royera,1 Noise pattern para- meters of the two antagonistic promoters controlling the nutrient switch were differentially affected on glycolytic and gluconeogenic carbon sources, discriminating between the different mechanisms that control their activity. Our stochastic model for the transcription events reproduced the observed noise patterns and identified the critical parameters responsible for the differences in expression profiles of the promoters. The model also resolved apparent contra- dictions between in vitro operator affinity and in vivo repressor activity at these promoters. Finally, our results demonstrate that negative feedback is not noise-reducing in the case of strong tran- scriptional bursting. p g In the present work we investigated gene expression noise patterns in a natural coherent genetic system in bacteria and a simple adaptation process—namely, the central carbon metabo- lism (CCM) in the model Gram positive bacterium Bacillus subtilis and a switch in carbon source. At the molecular level, we focused on the transcription initiation step of the genetic expres- sion process. Within the CCM network, we characterized pro- moter activity at main control points in the physiological switch between glycolysis and its reverse pathway, gluconeogenesis, which allows for growth on noncarbohydrate carbon sources. In B. subtilis, this switch involves two glyceraldehyde 3-phosphate dehydrogenases, GapA and GapB, that catalyze opposite reac- tions, and the phosphoenolcarboxykinase, PckA, that catalyses another irreversible gluconeogenic reaction under physiological conditions (Fig. 1A) (15, 16). The auto-repressed gapA operon, encoding its repressor CggR, GapA as well as four other central glycolytic enzymes, is induced under glycolytic conditions upon binding of fructose-1,6-bis-phosphate (FBP), a metabolite of glucose, to CggR (17). The operator site for CggR is located downstream of the transcription start site and upstream of the translation initiation region (RBS), and hence CggR is thought to function as a roadblock to the transcribing RNA Polymerase (RNAP) (17, 18). The GapB and PckA enzymes are both re- quired for the utilization of gluconeogenic carbon sources, such as malate, but their expression is deleterious under glycolytic regimes (15, 16, 19). CcpN is the repressor responsible for the very strong catabolite repression of the gapB and pckA promoters in the presence of glucose or other glycolytic substrates (16). It plays a dominant role in the control of carbon fluxes through central metabolic pathways in B. subtilis and is obligate for opti- B. Matthew L. Fergusona, Dominique Le Coqb,c,d, Matthieu Julesb,d, Stéphane Aymerichb,d, Ovidiu Radulescue, Nathalie Declercka,f,1, and Catherine A. Royera,1 aCentre de Biochimie Structurale, Institut National pour la Santé et la Recherche Médicale U554, Centre National pour la Recherche Scientifique Unité Mixte de Recherche 5048, Université Montpellier 1 and 2, F-34090 Montpellier, France; bInstitut National pour la Recherche Agronomique, Unité Mixte de Recherche 1319 Micalis, F-78350 Jouy-en-Josas, France; cCentre National pour la Recherche Scientifique, F-78350 Jouy-en-Josas, France; dAgroParisTech, Unité Mixte de Recherche Micalis, F-78350 Jouy-en-Josas, France ; eDynamique et Interactions des Membranes Normales et Pathologiques-Unité Mixte de Recherche 5235 Centre National pour la Recherche Scientifique, Université Montpellier 1 and 2, F-34095 Montpellier, France; and fInstitut National pour la Recherche Agronomique Département de Microbiologie, F-75338 Paris, France Edited by Eric Dean Siggia, The Rockefeller University, New York, NY, and approved October 21, 2011 (received for review July 7, 2011) Edited by Eric Dean Siggia, The Rockefeller University, New York, NY, and approved October 21, 2011 (received for review July 7, 2011) Edited by Eric Dean Siggia, The Rockefeller University, New York, NY, and approved October 21, 2011 (received for revie ean Siggia, The Rockefeller University, New York, NY, and approved October 21, 2011 (received for review July 7, 2011) been recently proposed (9). However, elevated expression noise is advantageous in the context of particular biological processes such as development (10) or for specific classes of genes (11). In the context of environmental changes, expression noise enabling stochastic phenotype switching can be used by cells or cell popu- lations as effective adaptation strategies (12, 13). Expression noise can also provide insights into gene function (14) or critical aspects of the molecular mechanism that control the expression of a particular gene. Assessing gene expression noise in order to obtain mechanistic insights requires accurate quantification of gene expression on many individual cells over a large dynamic range. We used a unique method based on 2-photon fluorescence fluctuation microscopy to measure directly, at the single cell level and with single-molecule sensitivity, the absolute concentration of fluorescent proteins pro- duced from the two Bacillus subtilis promoters that control the switch between glycolysis and gluconeogenesis. We quantified cell- to-cell variations in GFP concentrations in reporter strains grown on glucose or malate, including very weakly transcribed genes under strong catabolite repression. Results revealed strong transcriptional bursting, particularly for the glycolytic promoter. Author contributions: S.A., N.D., and C.A.R. designed research; M.L.F., D.L.C., M.J., and N.D. performed research; M.L.F., D.L.C., M.J., O.R., N.D., and C.A.R. analyzed data; S.A., O.R., N.D., and C.A.R. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1To whom correspondence may be addressed. E-mail: catherine.royer@cbs.cnrs.fr or nathalie.declerck@cbs.cnrs.fr. This article contains supporting information online at www.pnas.org/lookup/suppl/ doi:10.1073/pnas.1110541108/-/DCSupplemental. HAL Id: hal-01004232 https://hal.science/hal-01004232v1 Submitted on 29 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License www.pnas.org/cgi/doi/10.1073/pnas.1110541108 Matthew L. Fergusona, Dominique Le Coqb,c,d, Matthieu Julesb,d, Stéphane Aymerichb,d, Ovidiu Radulescue, Nathalie Declercka,f,1, and Catherine A. Royera,1 subtilis expressing gfpmut3 are recorded using infrared (930 nm) laser excitation and a dwell time of 50 μs at each pixel (faster than GFP diffusion); full scale of fluorescence intensity (F) is 10 photon counts∕pixel∕50 μs laser dwell time. The fluorescence fluctuations relative to the mean at each pixel are used to calculate the pixel-based maps of the true (shot noise corrected) molecular brightness (ϵ, full scale 1 photon∕molecule∕50 μs dwell time) and the number (npix) of the fluorescent particles detected in the 2-photon excitation volume (volex ¼ 0.07 fL inside B. subtilis); a 3D surface plot of npix is shown for the white-delineated area of the above intensity panel. Bottom right: Cartoon representation of the individual cells auto-detected using PaTrack (40) and showing the 50% central pixels used for averaging the particles number in each cell (ncell); the full scale for the npix and ncell maps is 180 molecules∕volex. Fig. 1. (A) Schematic of the central carbon metabolism showing the sw between glycolysis and gluconeogenesis controlled by the repressors C and CcpN. Important metabolites are in squares, regulatory proteins ellipses and the genes coding for the enzymes are in small italic let Rather, CcpN is thought to act on transcription initiation by pre- venting promoter escape by RNAP (20, 21). Assessing noise in gene expression in order to obtain molecu- lar mechanistic insights concerning its physical origins requires accurate quantification of expression levels in hundreds of indi- vidual cells over a large dynamic range, under repressing as well as inducing conditions, and in particular at very low levels of ex- pression where stochastic effects are expected to be most promi- nent. The fluorescent protein intensity measurements provided by the standard approaches typically used in such studies yield only fluorescence intensity values which are related to concentra- tion by an unknown scaling factor. Moreover, the sensitivity of such measurements is restricted by the relatively high auto-fluor- escence of the bacterial cell cytoplasm. Hence, measuring the activity of weakly transcribed genes using fluorescent protein reporter systems is usually not possible (2). Although recent approaches extend the range of the intensity-based methods to the single-molecule level (22–24), they remain indirect and suffer from uncertainty in molecular brightness and background fluor- escence. Matthew L. Fergusona, Dominique Le Coqb,c,d, Matthieu Julesb,d, Stéphane Aymerichb,d, Ovidiu Radulescue, Nathalie Declercka,f,1, and Catherine A. Royera,1 Bottom right: Cartoon representation of the individual cells auto-detected using PaTrack (40) and showing the 50% central pixels used for averaging the particles number in each cell (ncell); the full scale for the npix and ncell maps is 180 molecules∕volex. Using this approach, our primary objective was to focus on promoter activity. We sought to directly measure in single cells transcriptional activity of key regulated promoters in their re- pressed and derepressed states to characterize any heterogeneity among the cell population and to understand how cells are prepared for environmental changes. Moreover we sought to mea- sure transcriptional bursting and to reveal how control mechanisms at these different promoters affect the frequency and the size of the transcriptional bursts during the switch between inactive and active states. Finally, we asked whether, using stochastic models of the molecular control mechanisms of the studied promoters, we could correlate the operative mechanistic physical features with specific characteristics of the noise patterns. Fig. 1. (A) Schematic of the central carbon metabolism showing the switch between glycolysis and gluconeogenesis controlled by the repressors CggR and CcpN. Important metabolites are in squares, regulatory proteins are ellipses, and the genes coding for the enzymes are in small italic letters. When glucose is available for cell growth, fructose-1,6-biphosphate (FBP) accumulates and blocks the repressive action that CggR exerts on the tran- scription of gapA and four other central glycolytic genes (pgk, pgm, eno, and tpi). Because CggR is transcribed from the same gapA operon that it represses, it is also an autorepressor. Inversely, when cells are grown on malate or other nonglycolytic carbon sources, the CcpN repressor is inhibited by an unknown mechanism involving YqfL, allowing expression of the essen- tial gluconeogenic genes gapB and pckA. (B) Schematic of 2psN&B experi- ments. A stack of 50 raster scans of agarose immobilized live cells of B. subtilis expressing gfpmut3 are recorded using infrared (930 nm) laser excitation and a dwell time of 50 μs at each pixel (faster than GFP diffusion); full scale of fluorescence intensity (F) is 10 photon counts∕pixel∕50 μs laser dwell time. The fluorescence fluctuations relative to the mean at each pixel are used to calculate the pixel-based maps of the true (shot noise corrected) molecular brightness (ϵ, full scale 1 photon∕molecule∕50 μs dwell time) and the number (npix) of the fluorescent particles detected in the 2-photon excitation volume (volex ¼ 0.07 fL inside B. Matthew L. Fergusona, Dominique Le Coqb,c,d, Matthieu Julesb,d, Stéphane Aymerichb,d, Ovidiu Radulescue, Nathalie Declercka,f,1, and Catherine A. Royera,1 subtilis); a 3D surface plot of npix is shown for the white-delineated area of the above intensity panel. Bottom right: Cartoon representation of the individual cells auto-detected using PaTrack (40) and showing the 50% central pixels used for averaging the particles number in each cell (ncell); the full scale for the npix and ncell maps is 180 molecules∕volex. Fig. 1. (A) Schematic of the central carbon metabolism showing the switch between glycolysis and gluconeogenesis controlled by the repressors CggR and CcpN. Important metabolites are in squares, regulatory proteins are ellipses, and the genes coding for the enzymes are in small italic letters. When glucose is available for cell growth, fructose-1,6-biphosphate (FBP) accumulates and blocks the repressive action that CggR exerts on the tran- scription of gapA and four other central glycolytic genes (pgk, pgm, eno, and tpi). Because CggR is transcribed from the same gapA operon that it represses, it is also an autorepressor. Inversely, when cells are grown on malate or other nonglycolytic carbon sources, the CcpN repressor is inhibited by an unknown mechanism involving YqfL, allowing expression of the essen- tial gluconeogenic genes gapB and pckA. (B) Schematic of 2psN&B experi- ments. A stack of 50 raster scans of agarose immobilized live cells of B. subtilis expressing gfpmut3 are recorded using infrared (930 nm) laser excitation and a dwell time of 50 μs at each pixel (faster than GFP diffusion); full scale of fluorescence intensity (F) is 10 photon counts∕pixel∕50 μs laser dwell time. The fluorescence fluctuations relative to the mean at each pixel are used to calculate the pixel-based maps of the true (shot noise corrected) molecular brightness (ϵ, full scale 1 photon∕molecule∕50 μs dwell time) and the number (npix) of the fluorescent particles detected in the 2-photon excitation volume (volex ¼ 0.07 fL inside B. subtilis); a 3D surface plot of npix is shown for the white-delineated area of the above intensity panel. Bottom right: Cartoon representation of the individual cells auto-detected using PaTrack (40) and showing the 50% central pixels used for averaging the particles number in each cell (ncell); the full scale for the npix and ncell maps is 180 molecules∕volex. Matthew L. Fergusona, Dominique Le Coqb,c,d, Matthieu Julesb,d, Stéphane Aymerichb,d, Ovidiu Radulescue, Nathalie Declercka,f,1, and Catherine A. Royera,1 subtilis ∣central carbon metabolism ∣promoter activity ∣ stochastic gene expression ∣gene expression control G ene expression and regulation exhibit a high degree of sto- chasticity when studied at the level of individual cells. Even in genetically identical cell populations exposed to a uniform environment, gene activity levels and their phenotypic conse- quences are subject to random fluctuations that generate cell- to-cell variations and eventually lead to alternative cell fates. This stochastic “noise” in gene expression is thought to be a critical, biologically relevant property of genetic circuits in both microbial and eukaryotic cells (1–3). Noise in gene expression primarily originates from bursting of mRNA production and mRNA trans- lation into proteins. Gene promoters stochastically switch between “off” states with no mRNA produced and sharp produc- tion during “on” states. The burst size and frequency are thus two key parameters of stochastic protein expression at the single cell level (4–6). At the molecular level, the specific mechanisms of transcription and translation and, if relevant, the associated reg- ulatory mechanisms, generate different patterns and levels of noise. Gene expression noise patterns are thus expected to be under selection by evolution. Several indirect and direct lines of evidence for counterselection of noise in expression of genes important for cell growth have been reported (7, 8), and a quan- titative estimation of the general deleterious effect of noise has G e c PNAS ∣January 3, 2012 ∣vol. 109 ∣no. 1 ∣155–160 Fig. 1. (A) Schematic of the central carbon metabolism showing the switch between glycolysis and gluconeogenesis controlled by the repressors CggR and CcpN. Important metabolites are in squares, regulatory proteins are ellipses, and the genes coding for the enzymes are in small italic letters. When glucose is available for cell growth, fructose-1,6-biphosphate (FBP) accumulates and blocks the repressive action that CggR exerts on the tran- scription of gapA and four other central glycolytic genes (pgk, pgm, eno, and tpi). Because CggR is transcribed from the same gapA operon that it represses, it is also an autorepressor. Inversely, when cells are grown on malate or other nonglycolytic carbon sources, the CcpN repressor is inhibited by an unknown mechanism involving YqfL, allowing expression of the essen- tial gluconeogenic genes gapB and pckA. (B) Schematic of 2psN&B experi- ments. A stack of 50 raster scans of agarose immobilized live cells of B. 156 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1110541108 Matthew L. Fergusona, Dominique Le Coqb,c,d, Matthieu Julesb,d, Stéphane Aymerichb,d, Ovidiu Radulescue, Nathalie Declercka,f,1, and Catherine A. Royera,1 Here we applied a modified version of two-photon scan- ning (true) number and brightness (2psN&B) (25, 26), in which the intensity fluctuations at each pixel in a series of rapid raster scanned images of bacteria are used to deconvolve the average intensity (counts∕s) into the molecular brightness (counts/s/ molecule) and absolute number (molecules) of fluorescent pro- teins diffusing inside individual bacterial cells (Fig. 1B) (27). Thus unlike standard imaging techniques, which yield fluorescence intensity, sN&B provides absolute concentrations and can be carried out using confocal microscopy, although with more auto- fluorescence and photo-bleaching (Fig. S1) than with the 2-photon excitation used here. Fig. 1. (A) Schematic of the central carbon metabolism showing the switch between glycolysis and gluconeogenesis controlled by the repressors CggR and CcpN. Important metabolites are in squares, regulatory proteins are ellipses, and the genes coding for the enzymes are in small italic letters. When glucose is available for cell growth, fructose-1,6-biphosphate (FBP) accumulates and blocks the repressive action that CggR exerts on the tran- scription of gapA and four other central glycolytic genes (pgk, pgm, eno, and tpi). Because CggR is transcribed from the same gapA operon that it represses, it is also an autorepressor. Inversely, when cells are grown on malate or other nonglycolytic carbon sources, the CcpN repressor is inhibited by an unknown mechanism involving YqfL, allowing expression of the essen- tial gluconeogenic genes gapB and pckA. (B) Schematic of 2psN&B experi- ments. A stack of 50 raster scans of agarose immobilized live cells of B. subtilis expressing gfpmut3 are recorded using infrared (930 nm) laser excitation and a dwell time of 50 μs at each pixel (faster than GFP diffusion); full scale of fluorescence intensity (F) is 10 photon counts∕pixel∕50 μs laser dwell time. The fluorescence fluctuations relative to the mean at each pixel are used to calculate the pixel-based maps of the true (shot noise corrected) molecular brightness (ϵ, full scale 1 photon∕molecule∕50 μs dwell time) and the number (npix) of the fluorescent particles detected in the 2-photon excitation volume (volex ¼ 0.07 fL inside B. subtilis); a 3D surface plot of npix is shown for the white-delineated area of the above intensity panel. Results Absolute Quantification of Expression Levels Under Permissive and Repressing Conditions. Using N&B analysis we measured the absolute concentration of green fluorescent protein (GFP) in individual cells expressing gfpmut3 (encoding a very bright, fast- maturing and stable GFP variant) (28) from four promoters of interest, PcggR (gapA operon), PgapB, PpckA, and PccpN, (Fig. S2) grown at steady state under glycolytic (glucose) or gluconeogenic (malate) conditions. Fluorescent particles number maps and histograms of the distributions in the bacterial populations (Fig. 2 A and B) were calculated as described in detail elsewhere (27) (SI Text). Large differences in the expression levels and cell- to-cell variations were observed for the different strains depend- ing on the carbon source used for growth. The PcggRgfp fusion was strongly expressed on glucose but poorly repressed on malate, ex- hibiting an induction level of only sevenfold, in good agreement with bulk population measurements using gfpmut3 (Fig. S3) or other reporter systems (17). Expression from the PcggR promoter was visibly heterogeneous for the cell population grown on ma- late (Fig. 2A). The number of fluorescent particles detected in our 2p excitation volume inside the bacterial cell, volex (approxi- mately 0.07 fL) ranged from 30 to over 250 (Fig. 2B). In contrast, transcription from both PgapB and PpckA was very strongly re- mal growth under glycolytic conditions (16, 19). CcpN activity is linked to the energy charge of the cell (20) and is also negatively controlled by the coexpressed regulatory protein, YqfL (16). The CcpN operator site overlaps the promoter region of the gapB gene, but the bound repressor does not hamper RNAP binding. Ferguson et al. Fig. 2. Cell-by-cell quantification of catabolite regulation in B. subtilis by 2psN&B. (A) Pixel-based fluorescent particles number maps of B. subtilis cells ex- pressing gfpmut3 transcriptional fusion from PccgR, PgapB (results are similar for PpckA; not shown), and PccpN. Cells harvested from liquid cultures containing 0.5% glucose (G) or 0.5% malate (M) as the sole carbon source were immobilized on agarose pads for 2psN&B analysis as described in Fig. 1B. The full scale is 360 molecules∕volex. (B) Cell-based particles number (ncell) distributions for the indicated promoter-gfpmut3 fusion strains grown on glucose (black) or malate (gray). Inset in the first panel shows the expanded histogram of the probability density function PðncellÞ measured in malate for PcggR. Results Large bursts under repression by CggR are thus respon- sible for the heterogeneity observed in this population (Fig. 2A). Our findings reveal two distinct noise signatures associated with the glycolytic as opposed to gluconeogenic promoters. pressed on glucose and induced over 40-fold on malate (Fig. 2 A and B, middle panels). For these promoters under repressing condition, we were able to determine a population average of ap- proximately 3.5 GFP molecules (per volex) above the background of dim fluorescent particles observed in the reference BSB168 receiver strain (Fig. 2B, insets of panel 2 and 3). This corresponds to a population average concentration of approximately 80 nM or around 30–50 total GFP molecules in the bacterial cells, depending upon their size. For the strain carrying the gfpmut3 fusion with the weak and constitutive PccpN promoter we mea- sured rather low GFP concentrations, averaging approximately 1 μM on both carbon sources. Changes in Noise Pattern Parameters During Nutrient Shifts. The coefficient of variation (COV), one of the metrics used for quan- tifying noise in gene expression, is the ratio of the standard de- viation in the number of proteins (∕volex) over the mean, σn∕hni, for distributions in a population of bacteria (i.e., Fig. 2B). For all three regulated promoters (PcggR, PgapB, and PpckA), the COV is very large (approximately 60–100%) for the repressed promoters. The increase in the average number of GFP molecules under permissive conditions is accompanied by a drastic decrease in σn∕hni (Fig. 3A) but remains above the experimental uncertainty inherent to our method (approximately 0.05) (27). For the unre- gulated PccpN promoter, we observed no significant change in the σn∕hni value upon the shift of carbon source. We note that a small coefficient of variation does not necessarily mean that the noise is small; this is particularly true for skewed and lepto- kurtic histograms, such as that observed for PcggR, and which are usually associated with bursting (2, 5, 29). Modeling the Expression Patterns of PcggR and PgapB During the Nu- trient Shift. We modeled the kinetics of the transcription events from the PcggR and PgapB promoters with a unique realistic scheme inspired from a generic model for prokaryotic gene expression (30) (Fig. 4 A–B and Table S1). An in-depth justification for fixed parameter values and fitting procedure is given in SI Methods. Results Insets in panel 2 and 3 show the expanded histogram of the probability density function PðncellÞ observed in glucose for the PgapB and PpckA promoters in black, and that observed for the background BSB168 strain in gray. Fig. 2. Cell-by-cell quantification of catabolite regulation in B. subtilis by 2psN&B. (A) Pixel-based fluorescent particles number maps of B. subtilis cells ex- pressing gfpmut3 transcriptional fusion from PccgR, PgapB (results are similar for PpckA; not shown), and PccpN. Cells harvested from liquid cultures containing 0.5% glucose (G) or 0.5% malate (M) as the sole carbon source were immobilized on agarose pads for 2psN&B analysis as described in Fig. 1B. The full scale is 360 molecules∕volex. (B) Cell-based particles number (ncell) distributions for the indicated promoter-gfpmut3 fusion strains grown on glucose (black) or malate (gray). Inset in the first panel shows the expanded histogram of the probability density function PðncellÞ measured in malate for PcggR. Insets in panel 2 and 3 show the expanded histogram of the probability density function PðncellÞ observed in glucose for the PgapB and PpckA promoters in black, and that observed for the background BSB168 strain in gray. moter, the mean expression level, hni ¼ ab, can be decreased by reducing a or b or both. We note that in our system, differences in bursting characteristics from one promoter fusion to another and from one condition to another only emanate from transcrip- tional bursting. Indeed, in all our constructions, the gfpmut3 gene is under identical translational signal, insensitive to the different physiological conditions examined here. As expected for the constitutive promoter, PccpN, little variation is observed in either the values of a or b upon a switch in carbon source. In contrast, examination of the variations in these parameters for the glyco- lytic and gluconeogenic promoters on different carbon sources (Fig. 3B) reveals dramatic differences between their noise pat- terns. Indeed the parameters, a and b, are very dynamic and discriminative of the behavior of the two types of promoters, providing strong evidence that the major contribution to the observed cell-to-cell fluctuations is intrinsic to the regulatory circuitry. The gluconeogenic promoters (PgapB and PpckA) show a strong relative decrease of both burst size and frequency upon repression under glucose, whereas upon repression of PcggR under malate, a decreases significantly while b actually increases slightly. Ferguson et al. Results Our model included repressor binding and dissociation from the operator DNA, k1 on and k1 off, respectively. The main contri- bution to the fluctuations of mRNA production and of the other downstream variables arose from dissociation of the repressor, which justifies a two-state operator model. Transcription initia- Two other key parameters of stochastic gene expression can be extracted from the average and variance of GFP molecules in the cell population histograms: the apparent frequency of protein production burst per cell cycle (a ¼ hni2∕σn2) and the average number of protein molecules produced per burst, related to the Fano factor (b ¼ σn2∕hni) (5). Thus, for a repressible pro- PNAS ∣ January 3, 2012 ∣ vol. 109 ∣ no. 1 ∣ 157 Fig. 3. Changes in promoter activity levels and noise patterns upon a switch of carbon source. (A) The average number of GFPmut3 molecules per volex (hnigfp expressed in micromolar concentration) and its coefficient of variation (the standard deviation over the mean, σgfp∕hnigfp) in the cell populations grown on glucose (black bars) or malate (gray bars), estimated from the cell-based particles number distributions shown in Fig. 2B and considering a fixed auto-fluorescence background contribution as determined in the BSB168 receiver strain under identical experimental conditions. (B) Effect of nutrient switch on promoter activity noise patterns. The parameter of stochastic gene expression, the Fano factor b ðσ2 gfp∕hnigfpÞ, related to GFP production burst size is plotted against a ðhni2 gfp∕σ2 gfpÞ related to the GFP production burst frequency for the activity of the indicated promoters on glucose (black square) or malate (gray diamonds). The single arrows indicate the sense of repression for the regulated promoters. at two levels: affinity of the repressor for the DNA (k1 off) in the case of PcggR and concentration of active repressor and hence the burst duration and size (1∕k1 on½R) in the case of PgapB. Good fits for these two promoters were constrained by the modification of these two parameters, respectively. At PgapB, a decrease in the concentration of active repressor is consistent with the pro- posed sequestration of CcpN by YqfL under malate, although the total CcpN concentration is not thought to change (16). The change of k1 off is minimal upon induction, and hence KD values are constant and in the range of 0.8 nM, in reasonable agreement with the 8 nM affinity measured in vitro (33, 34). Results For CggR, operator affinity is high under repression, KD ¼ 0.1 nM, and decreases about 10-fold upon induction (k1 off increases), in rea- sonable agreement with the in vitro operator affinity (<0.5 and 10 nM) of CggR in absence and presence of inducer (35, 36). The model reconciles as well this high in vitro affinity with moderate repression by CggR in vivo (15). The position of the CggR opera- tor site between the promoter and the RBS allows unhindered RNAP binding and initiation and hence passage of multiple poly- merase molecules while the repressor is dissociated, leading to large amplitude bursts. Indeed, the large bursts for PcggR under repression cannot be modeled otherwise. Moreover, the enzymes of the CCM must be produced in large quantities when needed, and hence we must assume strong promoters in order to insure this strong production in the derepressed state. The price to pay is that strong promoters favor transcriptional bursting. In addition, strong bursting under repression for PcggR is favored by the fact that CggR is a self-repressed promoter, and hence the concentra- tion of repressor is lower under repressive, relative to permissive, conditions. Fig. 3. Changes in promoter activity levels and noise patterns upon a switch of carbon source. (A) The average number of GFPmut3 molecules per volex (hnigfp expressed in micromolar concentration) and its coefficient of variation (the standard deviation over the mean, σgfp∕hnigfp) in the cell populations grown on glucose (black bars) or malate (gray bars), estimated from the cell-based particles number distributions shown in Fig. 2B and considering a fixed auto-fluorescence background contribution as determined in the BSB168 receiver strain under identical experimental conditions. (B) Effect of nutrient switch on promoter activity noise patterns. The parameter of stochastic gene expression, the Fano factor b ðσ2 gfp∕hnigfpÞ, related to GFP production burst size is plotted against a ðhni2 gfp∕σ2 gfpÞ related to the GFP production burst frequency for the activity of the indicated promoters on glucose (black square) or malate (gray diamonds). The single arrows indicate the sense of repression for the regulated promoters. Discussion Our N&B data provide reliable measurements of absolute gene expression levels and fluctuations from metabolic promoters implicated in catabolite repression under both permissive and repressive conditions. The activity of the strongly repressed glu- coneogenic promoters has been undetectable at the single cell level by other methods. Indeed, combined two-photon excitation and raster scanning considerably reduce both the auto-fluores- cence from the bacterial cytoplasm and photo-bleaching of the GFP molecules, thereby achieving the single-molecule detection limits required for single cell quantification of gene expression levels from these strongly repressed promoters. Moreover, fluor- escence fluctuation-based methods such as N&B analysis do not require calibration of the light intensity signal and therefore allow for the direct counting of fluorescent reporter proteins diffusing in the excitation volume. tion was assigned rate constants k2 or k0 2, and the transcribing polymerase (tRNAP) was considered to synthesize the mRNA leader region containing the RBS with rate constants k3 (30, 31) or k0 3, depending on whether the operator DNA is free or bound by repressor. A switched, first order reaction of rate constant k4 was used to model dissociation of the elongation complex, thus reducing jamming of stalled polymerase molecules in the repres- sor-bound operator state for PcggR. We pooled several states (29, 31, 32) related by rapid transitions and consider that RBS gives rise directly to the ribosome elongating the protein chain (ElRib) then to the matured GFP (MdGFP). All reactions past RBS synthesis are considered to be identical for the promoters under study. We adjusted the parameters of the model to fit the experimental expression histograms. tion was assigned rate constants k2 or k0 2, and the transcribing polymerase (tRNAP) was considered to synthesize the mRNA leader region containing the RBS with rate constants k3 (30, 31) or k0 3, depending on whether the operator DNA is free or bound by repressor. A switched, first order reaction of rate constant k4 was used to model dissociation of the elongation complex, thus reducing jamming of stalled polymerase molecules in the repres- sor-bound operator state for PcggR. We pooled several states (29, 31, 32) related by rapid transitions and consider that RBS gives rise directly to the ribosome elongating the protein chain (ElRib) then to the matured GFP (MdGFP). All reactions past RBS synthesis are considered to be identical for the promoters under study. 158 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1110541108 Discussion We adjusted the parameters of the model to fit the experimental expression histograms. Our measurements, due to their absolute nature, reveal dis- tinct noise patterns for the glycolytic and gluconeogenic promo- ters, which inform on the adaptive strategy selected by evolution. For PcggR, 2psN&B allowed us to demonstrate significant cell-to- cell heterogeneity, in particular the existence of a subpopulation of cells expressing GFP under the repressed state at levels as high as half the average level of induced cells. This heterogeneity does not correspond to bistability that would provoke a bimodal distribution of the expression levels as reported for other bacter- ial regulation systems (37), instead of the skewed unimodal dis- tribution observed here. Rather it arises from strong trans- criptional bursting from this promoter under repression, which itself has two origins. First, PcggR is intrinsically a strong promoter, a required feature for the enzymes of the central carbon meta- bolism which must be produced in large quantity in the induced state. Secondly, the repressive mechanism of CggR, rather than competing with RNA polymerase for promoter binding, as in the case of the lactose repressor for example, acts as a roadblock to elongation. Hence multiple RNAP molecules can accomplish transcription during repressor dissociation episodes. While the affinity of CggR for its operator is quite high in vitro (35, 36), it The model reproduces well the observed behavior of PgapB and PcggR (Fig. 4C and Table S1). Testing key parameters for their uniqueness revealed significant constraints on their values (SI Methods and Fig. S4). The promoters function in a regime where k1 off∕k1 on½R is small for all conditions and promoters; the prob- ability, 1 −p, that the repressor, R, is bound to the DNA remains large, and repression does not vanish even under permissive conditions, in good agreement with genetic data showing consti- tutive overexpression of these promoters in repressor knockout strains (15, 16). The nutrient switch affects repression mainly Ferguson et al. Fig. 4. Model of gene regulation by CggR and CcpN. (A) Scheme describing the architecture of the B. subtilis PccgR and PgapB promoter region (boxed −10 and −35 RNAP recognition sequences) and tandem operator sites (black or gray upward triangles) for the CggR or CcpN repressors. Discussion Under glycolytic conditions, CcpN is thought to prevent promoter clearance by the RNA polymerase whereas under gluconeogenic conditions CggR acts as a roadblock to the transcribing polymerase when bound as a compact tetramer. (B) General model of prokaryotic gene expression and regulation applied to both repressors. RNAP-D is the RNAP-bound DNA, R the active repressor, tRNAP the elongating transcription complex, RBS the ribosome binding site on the transcribed mRNA, ElRib the elongating translation complex, and MdGFP the folded and matured green fluorescent protein. According to the above mechanistic models of regulation, besides changes in DNA affinity constants (Kd1 ¼ k1 off∕k1 on), CcpN repression affects primarily k2, the rate at which the elongation complex is formed, whereas CggR would affect the transcription rate in the mRNA leader region (k3), thereby increasing the dissociation rate of the (paused) polymerase (k4). In the gfpmut3 reporter system used in this study, all steps past RBS production are identical for all promoter constructs and all conditions. The GFPmut3 variant has been shown to be fast-maturing (within a few minutes) and slow degrading (stable for several hours) in B. subtilis (3), therefore the degradation rate kdeg corresponds to slow dilution whereas the lifetime of the mRNA is much shorter (i.e., k5 ≫kdeg). (C) Results of the model compared to the experimental data for the stochastic expression of PgapBgfp and PcggRgfp transcriptional fusions under glucose (red) or malate (blue). Lines correspond to the continuous distributions obtained from the model parameters reported in Table S1. The histogram from the PgapBgfp promoter fusion data was not corrected for the BSB168 back- ground contribution, as the deconvolution cannot be done reliably for experimentally reasonable dataset sizes; instead, a Gamma random variable having the same first two moments as the background contribution has been added to the model predictions. BIOPHYSICS AND COMPUTATIONAL BIOLO exhibits moderate repressor activity in vivo (17), as it is subject to strong transcriptional bursting. Although transcriptional bursting has been observed, albeit rarely, in prokaryotes (2, 38), quantita- tive and mechanistic insights into the process, such as those pre- sented here, have been lacking. Significant activity for the PcggR promoter in a subpopulation of cells grown on malate enables them to respond more rapidly when glucose becomes available, thereby conferring a competitive advantage to the clonal popula- tion. The high level of noise for this promoter may have been selected for implementing such a bet-hedging adaptation strategy. Discussion Under glycolytic conditions, CcpN is thought to prevent promoter clearance by the RNA polymerase whereas under gluconeogenic conditions CggR acts as a roadblock to the transcribing polymerase when bound as a compact tetramer. (B) General model of prokaryotic gene expression and regulation applied to both repressors. RNAP-D is the RNAP-bound DNA, R the active repressor, tRNAP the elongating transcription complex, RBS the ribosome binding site on the transcribed mRNA, ElRib the elongating translation complex, and MdGFP the folded and matured green fluorescent protein. According to the above mechanistic models of regulation, besides changes in DNA affinity constants (Kd1 ¼ k1 off∕k1 on), CcpN repression affects primarily k2, the rate at which the elongation complex is formed, whereas CggR would affect the transcription rate in the mRNA leader region (k3), thereby increasing the dissociation rate of the (paused) polymerase (k4). In the gfpmut3 reporter system used in this study, all steps past RBS production are identical for all promoter constructs and all conditions. The GFPmut3 variant has been shown to be fast-maturing (within a few minutes) and slow degrading (stable for several hours) in B. subtilis (3), therefore the degradation rate kdeg corresponds to slow dilution whereas the lifetime of the mRNA is much shorter (i.e., k5 ≫kdeg). (C) Results of the model compared to the experimental data for the stochastic expression of PgapBgfp and PcggRgfp transcriptional fusions under glucose (red) or malate (blue). Lines correspond to the continuous distributions obtained from the model parameters reported in Table S1. The histogram from the PgapBgfp promoter fusion data was not corrected for the BSB168 back- ground contribution, as the deconvolution cannot be done reliably for experimentally reasonable dataset sizes; instead, a Gamma random variable having the same first two moments as the background contribution has been added to the model predictions. Model of gene regulation by CggR and CcpN (A) Scheme describing the architecture of the B subtilis P and P promoter region (boxed Fig. 4. Model of gene regulation by CggR and CcpN. (A) Scheme describing the architecture of the B. subtilis PccgR and PgapB promoter region (boxed −10 and −35 RNAP recognition sequences) and tandem operator sites (black or gray upward triangles) for the CggR or CcpN repressors. Ferguson et al. Materials and Methods brightness (hεigfp) for a population of cells under a particular set of conditions were obtained by correcting the average values obtained from the distribu- tion for background fluorescence from the BSB168 receiver strain bearing no gfp. More details on our 2psN&B method are given in SI Text and ref. 27. Bacterial Strains, Cultures, and Sample Preparation. Strains of Bacillus subtilis carrying transcriptional fusions with the gfpmut3 reporter gene were con- structed using the pBaSysBioII plasmid designed for high throughput analysis of promoter activities by Live Cell Arrays (3) (http://www.basysbio.eu). (See Fig. S2 and SI Text for more details.) Microscopy samples were prepared with cells from exponentially growing cultures and immobilized on 1.5% agarose pads as described in SI Text and in ref. 27. Modeling. The model of stochastic gene expression is described by the set of biochemical reactions given in Fig. 4B and Table S1. We used Gillespie direct simulation accelerated by cycle averaging (29) to compute the stationary so- lutions of the chemical master equation. The model predictions were com- pared to the experimental histograms for the cggR and gapB promoters, under two nutritional conditions, glycolytic (glucose) and gluconeogenic (malate). Parameter values were approximated using analytical approxima- tions for the first two moments of the predicted expression distribution and then refined by minimizing the distance between predicted and observed histograms. Further description of the genetic switch modelling is available in SI Text. Two-Photon Scanning Microscopy. In scanning N&B analysis the number and molecular brightness of the diffusing fluorescent molecules are calculated from the fluctuations in fluorescence intensity at each pixel in a series of ras- ter scanned images, in which the laser dwell time is small with respect to the diffusion time of the molecule. Fluorescence fluctuations from the average intensity arise from Brownian diffusion into and out of the very small two- photon excitation volume (volex ¼ 0.07 fL) focused inside the bacterial cells. The pixel-based temporal average and variance images were calculated from 50 raster scans, then the true (shot noise corrected) molecular brightness, ε, was determined at each pixel. The average true molecular brightness hεi was determined for all of the cells in the field of view using only those central pixels for which the volex is encompassed within the cells. Materials and Methods Cai L, Friedman N, Xie XS (2006) Stochastic protein expression in individual cells at the single molecule level. Nature 440:358–362. 25. Digman MA, et al. (2008) Mapping the number of molecules and brightness in the laser scanning microscope. Biophys J 94:2320–2332. 5. Friedman N, Cai L, Xie XS (2006) Linking stochastic dynamics to population distribu- tion: an analytical framework of gene expression. 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Science 309:2075–2078. 13. Schultz D, et al. (2009) Deciding fate in adverse times: sporulation and competence in Bacillus subtilis. Proc Natl Acad Sci USA 106:21027–21034. 32. Radulescu O, et al. (2008) Robust simplifications of multiscale biochemical networks. Materials and Methods BMC Syst Biol 2:1–25. Bacillus subtilis. Proc Natl Acad Sci USA 106:21027–21034. 14. Rinott R, Jaimovich A, Friedman N (2011) Exploring transcription regulation through cell-to-cell variability. Proc Natl Acad Sci USA 108:6329–6334. 33. Zorrilla S, et al. (2008) Characterization of the control catabolite protein of gluconeo- genic genes repressor by fluorescence cross-correlation spectroscopy and other biophysical approaches. Biophys J 95:4403–4415. cell-to-cell variability. Proc Natl Acad Sci USA 108:6329–6334. p g genic genes repressor by fluorescence cross-correlation spectroscopy and other biophysical approaches. Biophys J 95:4403–4415. 15. Fillinger S, et al. (2000) Two glyceraldehyde-3-phosphate dehydrogenases with opposite physiological roles in a nonphotosynthetic bacterium. J Biol Chem 275: 14031–14073. 34. Licht A, Brantl S (2006) Transcriptional repressor CcpN from Bacillus subtilis compen- sates asymmetric contact distribution by cooperative binding. J Mol Biol 364:434–448. 16. Servant P, Le Coq D, Aymerich S (2005) CcpN (YqzB), a novel regulator for CcpA-inde- pendent catabolite repression of Bacillus subtilis gluconeogenic genes. Mol Microbiol 55:1435–1451. 35. Zorrilla S, et al. (2007) Fructose-1,6-bisphosphate acts both as an inducer and as a structural cofactor of the central glycolytic genes repressor (CggR). Biochemistry 46:14996–15008. p p structural cofactor of the central glycolytic genes repressor (CggR). Biochemistry 46:14996–15008. 17. Doan T, Aymerich S (2003) Regulation of the central glycolytic genes in Bacillus subtilis: Binding of the repressor CggR to its single DNA target sequence is modulated by fructose-1,6-bisphosphate. Mol Microbiol 47:1709–1721. 36. Zorrilla S, et al. (2007) Inducer-modulated cooperative binding of the tetrameric CggR repressor to operator DNA. Biophys J 92:3215–3227. 37. Dubnau D, Losick R (2006) Bistability in bacteria. Mol Microbiol 61:564–572. 18. Chaix D, et al. (2010) Physical basis of the inducer-dependent cooperativity of the Central glycolytic genes Repressor/DNA complex. Nucleic Acids Res 38:5944–5957. 38. Golding I, et al. (2005) Real-time kinetics of gene activity in individual bacteria. Cell 123:1025–1036. 19. Tännler S, et al. (2008) CcpN controls central carbon fluxes in Bacillus subtilis. J Bacter- iol 190:6178–6187. 39. Becskei A, Serrano L (2000) Engineering stability in gene networks by autoregulation. Nature 405:590–593. 40. Espenel C, et al. (2008) Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web. J Cell Biol 182:765–776. 40. Espenel C, et al. (2008) Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web. J Cell Biol 182:765–776. 20. Discussion case of the gluconeogenic promoters, because derepression under glycolytic conditions greatly impairs growth (16, 19), strict control by CcpN and limited noise are essential. Hence, the re- pressive mechanism of CcpN has evolved to limit bursting in the repressed state by impairing promoter escape by the RNAP, leading to very strong catabolite repression. In conclusion, our study demonstrates that N&B analysis can be applied for direct and absolute quantification of promoter activity and noise in bacterial populations upon changes in con- ditions. The highly quantitative nature of our approach, coupled with its sensitivity and broad dynamic range render 2psN&B particularly well suited to the study of promoter activity. Here it allowed us, in a natural bacterial system, to associate expression noise patterns with specific features of molecular mechanisms of transcriptional repression, to demonstrate transcriptional burst- ing and to show that negative feedback control does not neces- sarily reduce noise. Hence, 2psN&B offers broad perspectives for the quantitative characterization of a wide range of gene reg- ulatory networks in both prokaryotes and eukaryotes. As often invoked (39) one is inclined to expect that negative feedback reduces noise. Our results provide the experimental de- monstration that this property is not valid in the strong bursting regime of the self-repressed PcggR promoter. Indeed, the burst size scales with the lifetime of the active state of the operator, which for a self-repressed promoter, scales inversely with the concentration of repressor and is smaller under permissive con- ditions when the feedback is weak. In contrast to the strong tran- scriptional bursting observed for the glycolytic promoter, in the PNAS ∣ January 3, 2012 ∣ vol. 109 ∣ no. 1 ∣ 159 159 1. Eldar A, Elowitz MB (2010) Functional roles for noise in genetic circuits. Nature 467:167 173. Materials and Methods This spatially aver- aged true molecular brightness for each field of view allowed for the calcu- lation of the number of molecules, npixð∕volexÞ, at each pixel. Then for each cell in the field of view, npix was averaged over the pixels situated in an ellipse representing the central 50% of the cell area, yielding ncell. The averaged intracellular concentration of GFPmut3 molecules (hnigfp) and their intrinsic ACKNOWLEDGMENTS. The authors would like to thank Caroline Clerté for her assistance in student training on the microscope and Nicole Lautredou and Julien Cau of Montpellier Rio Imaging for their assistance with the confocal microscope. The work was supported by a grant from the Agence Nationale pour la Recherche (ANR-09-BLAN-0285) and by European Union BaSysBio Grant LSHG-CT-2006-037469. M.L.F was supported by postdoctoral fellow- ships from the National Science Foundation (OISE:IRFP #0710816), European Molecular Biology Organization (ALTF 660-2008), and the European Commis- sion (Marie Curie IIF#237835-InVivoTrnsReg). 1. Eldar A, Elowitz MB (2010) Functional roles for noise in genetic circuits. Nature 467:167 173. 1. Eldar A, Elowitz MB (2010) Functional roles for noise in genetic circuits. Nature 467:167 173. 21. Licht A, Brantl S (2009) The transcriptional repressor CcpN from Bacillus subtilis uses different repression mechanisms at different promoters. J Biol Chem 284: 30032–30038. 21. 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Elf J, Li GW, Xie XS (2007) Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316:1191–1194. 24. Taniguchi Y, et al. (2010) Quantifying E. coli proteome and transcriptome with single- molecule sensitivity in single cells. Science 329:533–538. 4. 160 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1110541108 2. Kaufmann BB, Van Oudenaarden A (2007) Stochastic gene expression: from single molecules to the proteome. Curr Opin Genet Dev 17:107–112. Materials and Methods Licht A, Golbik R, Brantl S (2008) Identification of ligands affecting the activity of the transcriptional repressor CcpN from Bacillus subtilis. J Mol Biol 380:17–30. ∣ www.pnas.org/cgi/doi/10.1073/pnas.1110541108 Ferguson et al. 160
https://openalex.org/W2986742642	https://research-repository.st-andrews.ac.uk/bitstream/10023/18962/1/Arnold_2019_NC_Antibody_CC.pdf	English	null	A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension	Nature communications	2,019	cc-by	20,584	1 Department of Infection, Immunity and Cardiovascular Disease, University of Shefﬁeld, Shefﬁeld S10 2RX, UK. 2 Kymab Ltd, Babraham Research Campus, Cambridge CB22 3AT, UK. 3 Cardiovascular Pulmonary Research Laboratories, Departments of Pediatrics and Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA. 4 Department of Chemistry, University of Shefﬁeld, Shefﬁeld S3 7HF, UK. 5 Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospital, Cambridge CB2 0QQ, UK. 6 School of Medicine, University of St. Andrews, St, Andrews KY16 9AJ, UK. 7 MRC/University of Edinburgh Centre for Inﬂammation Research, University of Edinburgh, The Queens Medical Research Institute, Edinburgh EH16 4TJ, UK. 8 Shefﬁeld Pulmonary Vascular Disease Unit, Shefﬁeld Teaching Hospitals Foundation Trust, Royal Hallamshire Hospital, Shefﬁeld S10 2JF, UK. 9These authors contributed equally: Nadine D. Arnold, Josephine A. Pickworth, Laura E. West, Sarah Dawson. email: a.lawrie@shefﬁeld.ac.uk NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications ARTICLE A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension We13,14 and others15 have reported that osteoprotegerin (OPG, Tnfrsf11b), a secreted gly- coprotein belonging to the TNF receptor superfamily capable of binding to TRAIL, is elevated in the lungs and sera from patients with idiopathic PAH (IPAH). OPG is a potent mitogen and migratory stimulus of PASMCs in vitro13. Jia et al. have demonstrated that mice lacking OPG display an attenuated PAH phenotype in the Sugen5416 plus hypoxia (SuHx) model15. p yp g p yp ( ) We report here that OPG expression is elevated in the mouse SuHx model, and in a different strain of OPG−/−mice, the PAH phenotype is similarly attenuated (Supplementary Figure 1). Levels of OPG also increase consequently with PAH development in the monocrotaline (Mct) rat (Supplementary Figure 2). Fur- thermore, we demonstrate in vitro that OPG binds to Fas receptor to activate cell proliferation, migration and survival pathways. Finally, using a human OPG antibody we demonstrate a robust therapeutic effect on established and severe PAH. Importantly, the efﬁcacy of our approach was mediated through both improved haemodynamics and pulmonary vascular remodelling. The haemodynamic efﬁcacy of our approach was at least equivalent to current standard of care PAH therapies (used in 10–50-fold excess in these rat models). Combination of current PAH therapies with our anti-OPG antibody demonstrated an improved response in both haemodynamics and pulmonary vascular remodelling over standard of care PAH therapies alone. Bone marrow-derived OPG drives PAH in the murine SuHx model. To determine if the source of OPG responsible for driving disease was originating from tissue resident, or bone marrow- derived cells we next examined the disease phenotype in chimeric mice generated by bone marrow transplantation (BMT). Mice lacking tissue OPG displayed signiﬁcantly reduced serum levels of OPG (Fig. 2a) but were not protected from developing PAH (Fig. 2b–f). In contrast, mice lacking OPG in bone marrow only (red dots, Fig. 2), were protected from developing PAH (Fig. 2b–f). The presence of OPG was noted within remodelled pulmonary arteries from mice that developed PAH (Fig. 2g) suggesting OPG expressing cells might be recruited from a bone marrow source. We next sought to investigate candidate bone marrow-derived cell-types that could release OPG and drive the PAH pathophy- siology. A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension Nadine D. Arnold 1,9, Josephine A. Pickworth 1,9, Laura E. West 1,9, Sarah Dawson1,9, Joana A. Carvalho2, Helen Casbolt1, Adam T. Braithwaite 1, James Iremonger 1, Lewis Renshall1, Volker Germaschewski2, Matthew McCourt2, Philip Bland-Ward2, Hager Kowash 1, Abdul G. Hameed 1, Alexander M.K. Rothman 1, Maria G. Frid3, A.A. Roger Thompson 1, Holly R. Evans 4, Mark Southwood5, Nicholas W. Morrell 5, David C. Crossman 6, Moira K.B. Whyte 7, Kurt R. Stenmark 3, Christopher M. Newman1, David G. Kiely1,8, Sheila E. Francis 1 & Allan Lawrie 1 Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase life expectancy but have limited impact on the progressive pulmonary vascular remodelling that drives PAH. Osteoprotegerin (OPG) is increased within serum and lesions of patients with idiopathic PAH and is a mitogen and migratory stimulus for pulmonary artery smooth muscle cells (PASMCs). Here, we report that the pro-proliferative and migratory phenotype in PASMCs stimulated with OPG is mediated via the Fas receptor and that treatment with a human antibody targeting OPG can attenuate pulmonary vascular remo- delling associated with PAH in multiple rodent models of early and late treatment. We also demonstrate that the therapeutic efﬁcacy of the anti-OPG antibody approach in the presence of standard of care vasodilator therapy is mediated by a reduction in pulmonary vascular remodelling. Targeting OPG with a therapeutic antibody is a potential treatment strategy in PAH. 1 TURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/ ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 P u maintain mice double deﬁcient for ApoE and opg. We subse- quently generated heterozygous ApoE mice (ApoE+/−), and mice heterozygous for ApoE but homozygous deﬁcient for OPG (ApoE+/−/OPG−/−). ApoE+/−mice developed PAH in response to 8 weeks of feeding HFD, consistent with our previously pub- lished data12,16. HFD-fed ApoE+/−/OPG−/−were protected from developing increased RVSP (Fig. 1a) with no signiﬁcant difference in left ventricular end-systolic (LVESP) or end-diastolic (LVEDP) pressure, in either strain (Fig. 1b–c). There was no statistically signiﬁcant difference in cardiac index (CI) between HFD-fed ApoE+/−and ApoE+/−/OPG−/−(Fig. 1d). Analysis of pulmonary vascular remodelling conﬁrmed that the reduced RVSP in the ApoE+/−/OPG−/−was associated with a signiﬁcantly lower media/CSA of small pulmonary arteries (Fig. 1e–f). We next examined whether treatment of established PAH with a poly- clonal anti-OPG antibody could stabilise or induce disease regression in the HFD-ApoE−/−model. In a separate group of animals, phenotype was conﬁrmed after 8 weeks feeding of ApoE−/−mice with HFD. A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension The remaining mice were then ran- domly assigned to receive blinded treatment with either a poly- clonal anti-OPG antibody or IgG control for 4 weeks (Fig. 1g). Compared to HFD-fed ApoE−/−mice phenotyped after 8 weeks, the mice treated with the IgG control antibody displayed an increase in disease severity (Fig. 1h–i). In contrast, mice treated with the anti-OPG antibody demonstrated a signiﬁcant increase in pulmonary artery acceleration time (PAAT) (Fig. 1h) and reduction of RVSP (Fig. 1i). There was no signiﬁcant effect of disease, or OPG antibody treatment on LVESP (Fig. 1j). The beneﬁcial haemodynamic response achieved by anti-OPG anti- body treatment was associated with a reduction in media/CSA (Fig. 1k–l) that was associated with fewer proliferating and more apoptotic cells (Fig. 1l). Since OPG is linked with bone remo- delling20 we examined whether antibody blockade of OPG would induce an osteoporotic phenotype but no detrimental effect of the anti-OPG antibody treatment was observed on either bone volume, trabecular number or trabecular thickness as assessed by microCT analysis (Fig. 1m–o). P ulmonary arterial hypertension (PAH) is a devastating disease driven by a sustained pulmonary-speciﬁc vasocon- striction which triggers a progressive pulmonary vasculo- pathy that leads to right heart failure1. Early endothelial cell dysfunction is thought to be an initiating event in the develop- ment of PAH. The subsequent proliferation of multiple resident cell types including pulmonary artery smooth muscle cells (PASMC), endothelial cells (PAEC) and ﬁbroblasts is critical to the vascular remodelling. The inﬁltration of circulating inﬂam- matory and mesenchymal cells has been shownt to play an important role in regulating disease pathogenesis2–5. Current therapies for PAH are effective in relieving symptoms and improve survival6; however, their effects are often transient and importantly do not stop the progressive pathological changes7. PAH remains an orphan disease with no cure other than transplantation. The molecular and cellular mechanisms involved in the pathogenesis of PAH are complex and involve cross-talk between several signalling pathways including the transforming growth factor beta (TGF-β)/bone morphogenetic protein (BMP) axis8, growth factors (e.g. PDGF)9 and vasoactive proteins (e.g. vasoactive intestinal peptide (VIP)10 and endothelin-1 (ET-1)11 (reviewed with respect to anti-remodelling therapies in ref. 5). We previously reported that tumour necrosis factor (TNF) related apoptosis inducing-ligand (TRAIL) is also a critical mediator of PAH in experimental models12. A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension Since both endothelial21 and mesenchymal22 progenitors have been implicated in PAH, and are present in remodelled arteries we investigated the expression of OPG in PASMCs (SMC), pulmonary artery ﬁbroblasts (PA-Fib) and ﬁbrocytes isolated from the hypoxic neonatal calf model of PAH23 and blood outgrowth endothelial cells (BOEC)24. OPG expression was 2-fold higher in both PA-Fibs and SMCs, but dramatically higher in ﬁbrocytes isolated from hypoxic calves with PAH compared to controls (Fig. 2h). We subsequently performed immunohisto- chemical analysis of the remodelled pulmonary arterioles from the hypoxic neonatal calf model and observed a marked increase in diffuse OPG staining throughout the lesions and in the number NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 ARTICLE OPG positive cells within the vessel wall, particularly in the ventitial outward remodelled parts of the artery (Fig. 2i). In OECs, whereas vascular endothelial growth factor (VEGF) hanced proliferation of BOEC obtained from healthy and AH donors, OPG only induced proliferation in BOECs derived m IPAH patients (Fig. 2j). Results OPG antibody treatment reverses PAH in HFD-ApoE−/−mice. Studies by Jia et al15, and conﬁrmed by us, demonstrate the requirement for OPG expression to develop the full PAH phe- notype in the mouse SuHx model (Supplementary Figure 1), we also demonstrated the increase of OPG expression with devel- opment of PAH in the monocrotaline rat model (Supplementary Figure 2). We next sought to determine whether OPG was a tractable therapeutic target in PAH models of established disease. We investigated the effect of genetic deletion of OPG in the Paigen high fat, high cholate containing diet (HFD) fed ApoE−/− mouse as a model with severe and progressive (non-resolving) pulmonary vascular remodelling12,16. Despite previous reports from other groups17–19, we were unable to successfully breed and TURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Since OPG is naturally secreted we OPG regulates genes important in PH/PAH pathoge gain mechanistic insight into how OPG might regulate proliferative PASMC phenotype, we examined the tran and intracellular signalling mediated by OPG in human Microarray analysis of PASMC mRNA identiﬁed 190 from the microarray that were signiﬁcantly regulated a d e f h i g j k l m n o b c 80 110 10 8 6 4 2 0 100 90 80 70 60 Chow Paigen Chow Paigen Chow Paigen Chow Paigen Chow Paigen 60 RVSP (mmHg) Cardiac index (μl min–1 g–1) Media/CSA PA AT (ms) RVSP (mmHg) LVESP (mmHg) Bone volume per tissue volume (%) Trabecular thickness (mm) Trabecular number per mm Media/CSA LVESP (mmHg) LVEDP (mmHg) 40 20 250 1.0 0.8 ABEVG SMA 0.6 0.4 0.2 0.0 20 120 100 80 60 40 20 0 120 0.75 0.50 0.25 0.00 100 80 60 40 20 0 12 0.040 4.0 3.5 3.0 2.5 2.0 0.038 0.036 0.034 0.032 0.030 0.028 11 10 9 8 IgG OPG Ab IgG OPG Ab IgG OPG Ab 19 18 17 16 15 8 week >100 <50 51–100 12 week 8 week ABEVG IgG OPG Ab SMA TUNEL PUNA 12 week 8 week 12 week HFD Chow Treatment Treatment Treatment HFD IgG HFD OPG Ab HFD Chow HFD IgG HFD OPG Ab HFD Chow HFD IgG HFD OPG Ab 200 150 100 50 0 Echo 0 8 12 Week Echo High fat diet 0.8 ng g–1 h–1 Ab or lgG Catheter /harvest Echo IgG OPG Ab 0 OPG–/– ApoE+/– ApoE+/–/OPG–/– OPG–/– ApoE+/– ApoE–/– mice ApoE+/–/OPG–/– OPG–/– ApoE+/– ApoE+/– ApoE+/–/OPG–/– OPG–/– ApoE+/– ApoE+/–/OPG–/– OPG–/– ApoE+/– ApoE+/–/OPG–/– ApoE+/–/OPG–/– ATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 AR a 80 Chow Paigen 60 RVSP (mmHg) 40 20 0 OPG–/– ApoE+/– ApoE+/–/OPG–/– b 110 100 90 80 70 60 Chow Paigen LVESP (mmHg) OPG–/– ApoE+/– ApoE+/–/OPG–/– c 10 8 6 4 2 0 Chow Paigen LVEDP (mmHg) OPG–/– ApoE+/– ApoE+/–/OPG–/– b a c d Chow Paigen Cardiac index (μl min–1 g–1) 250 200 150 100 50 0 OPG–/– ApoE+/– ApoE+/–/OPG–/– e Chow Paigen Media/CSA 1.0 0.8 0.6 0.4 0.2 0.0 OPG–/– ApoE+/– ApoE+/–/OPG–/– f ABEVG SMA ApoE+/– ApoE+/–/OPG–/– f f d e g Echo 0 8 12 Week Echo High fat diet 0.8 ng g–1 h–1 Ab or lgG Catheter /harvest Echo ApoE–/– mice h PA AT (ms) 20 19 18 17 16 15 8 week 12 week HFD Chow Treatment HFD IgG HFD OPG Ab i RVSP (mmHg) 120 100 80 60 40 20 0 8 week 12 week Treatment HFD Chow HFD IgG HFD OPG Ab h i g j LVESP (mmHg) 120 100 80 60 40 20 0 8 week 12 week Treatment HFD Chow HFD IgG HFD OPG Ab j k Media/CSA 0.75 0.50 0.25 0.00 >100 <50 51–100 IgG OPG Ab k l l ABEVG IgG OPG Ab SMA TUNEL PUNA n Trabecular thickness (mm) 0.040 0.038 0.036 0.034 0.032 0.030 0.028 IgG OPG Ab m Bone volume per tissue volume (%) 12 11 10 9 8 IgG OPG Ab o Trabecular number per mm 4.0 3.5 3.0 2.5 2.0 IgG OPG Ab o m of OPG positive cells within the vessel wall, particularly in the adventitial outward remodelled parts of the artery (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Further analysis of a selection of these differentially regulated genes with TaqMan PCR validated several genes previously described as important in the pathogenesis of PH/PAH, speciﬁcally TRAIL, PDGFRA, tenascin- C, VEGFA, and caveolin-1, as all being signiﬁcantly up-regulated by OPG, and the VIP receptor as being signiﬁcantly down- regulated by OPG (Fig. 3c). To examine the intracellular signal- ling pathways we performed a KinexTM antibody microarray (KAM) and identiﬁed 63 from 800 phosphorylation and pan- speciﬁc antibodies that were signiﬁcantly regulated by OPG at either 10, 60 min, or both (Supplementary Figure 3). Signiﬁcantly regulated proteins included a number of pro-survival, anti- apoptotic and cell cycle (Fig. 3d) proteins and members of the NF-5β pathway (Fig. 3e). Several proteins were validated by western immunoblotting, further emphasising activation of MAPK signalling (pERK1/2), anti-apoptotic proteins (pHsp27, CDK5) and mammalian target of rapamycin (mTOR) and cell cycle (CDK4) (Fig. 3f). OPG immunoblotting (Fig. 4d). Furthermore, Fas immunor- eactivity strongly associated with both remodelled pulmonary arteries, and the right ventricle of patients with IPAH (Fig. 4e) compared to controls. Investigation of rat lung isolated from control (saline) and moncrotaline rats, as well as control (nor- moxic) and SuHx rats also demonstrate a signiﬁcant increase in expression of both Fas gene expression (Fig. 4f) and protein expression within remodelled pulmonary arterioles (Fig. 4g). Fas regulates OPG signalling and phenotype in PASMCs. To determine the functional and signalling consequences of the OPG-Fas interaction, PASMCs were stimulated with OPG after pre-incubation with an anti-human Fas neutralising antibody. Blockade of the Fas receptor prevented OPG induction of PDGFRA, TNC, VEGFA and CAV1 gene expression (Fig. 5a–d) but interestingly not TRAIL (Fig. 5e). To validate the functional role of the OPG-Fas interaction, we used the well-described model of FasL/TRAIL-induced apoptosis of HT1080 cells27. Pre- incubation of HT1080 cells with OPG signiﬁcantly blocked both TRAIL but also FasL-induced apoptosis, as measured by Cas- pase3/7 activation (Fig. 5f) indicating that OPG can antagonise FasL–Fas binding. To further examine this in a disease-relevant cell type, we examined the effect of Fas neutralisation on OPG stimulated human PASMC. Fas neutralisation signiﬁcantly reduced OPG-induced transwell PASMC migration (Fig. 5g) and suppressed OPG-induced proliferation (Fig. 5h). However, Fas neutralisation had no effect on PDGF-induced proliferation (Fig. 5h). The observed increase in TRAIL expression following ligation of Fas receptor with either the Fas neutralising antibody, or OPG itself (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Fig. 1 Genetic deletion of OPG prevents and antibody treatment reverses PAH. Panels (a–f) are data obtained from high fat diet (HFD) fed OPG−/−, ApoE+/−, and ApoE+/−/OPG−/−mice to determine the requirement of OPG for the development of PAH. Panels (g–o) are data obtained high fat diet (HFD) fed ApoE−/−mice treated with IgG or OPG antibody to determine if OPG antibody treatment can reverse established PAH. Bar graphs (a, i) show right ventricular systolic pressure (RVSP), (b, j) left ventricular end-systolic pressure (LVESP), (c) left ventricular end-diastolic pressure, (d) cardiac index, (e&k) the degree of medial wall thickness as a ratio of total vessel size (Media/CSA), (f) representative photomicrographs of serial lung sections stained with Alcian Blue Elastic van Gieson (ABEVG) or immunostained for α-smooth muscle actin (α-SMA). Panel (g) demonstrates a schema from the therapeutic intervention with polyclonal mouse OPG antibody. (h) pulmonary artery acceleration time (PA AT). l Representative photomicrographs of serial lung sections from ApoE−/−mice fed on Paigen diet for 12 weeks. Sections were stained with ABEVG or α-SMA, proliferating cell nuclear antigen (PCNA) or Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Bar graphs show femoral trabecular bone volume (%) (m), trabecular thickness (mm) (n), trabecular number (mm−1) (o), bars represent mean with error bars showing the standard error of the mean. Box and Whisker plots represent the interquartile range (box) with the line representing the median and whisker the full range of the data, each animal is represented by a dot in each graph; panels (a–f) OPG−/−n = 3 per group, ApoE+/−n = 4 per group, ApoE+/−/OPG−/−n = 5 per group. * p < 0.05, p < 0.01, * p < 0.001 compared to OPG−/−or chow-fed mice following a two-way ANOVA followed by Bonferroni’s multiple comparisons test, or were only two groups, unpaired t-tests. All images are presented at their original magniﬁcation ×400, scale bar represent 50 µm stimulation with OPG, most notably TGFβ signalling, cytoskeletal organisation, motility and survival pathways (Fig. 3a). To ﬁlter the data we ﬁrst applied gene enrichment utilising a previously curated PAH gene list26. This highlighted 57 genes either pre- viously associated with PH/PAH, or in key cellular mechanisms important in disease pathogenesis (Fig. 3b). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 2i). In BOECs, whereas vascular endothelial growth factor (VEGF) enhanced proliferation of BOEC obtained from healthy and IPAH donors, OPG only induced proliferation in BOECs derived from IPAH patients (Fig. 2j). Since OPG is naturally secreted we postulated from these data that BM-derived cells may be secreting and in turn responding to OPG alongside resident PASMCs to drive pulmonary vascular remodelling. of OPG positive cells within the vessel wall, particularly in the adventitial outward remodelled parts of the artery (Fig. 2i). In BOECs, whereas vascular endothelial growth factor (VEGF) enhanced proliferation of BOEC obtained from healthy and IPAH donors, OPG only induced proliferation in BOECs derived from IPAH patients (Fig. 2j). Since OPG is naturally secreted we postulated from these data that BM-derived cells may be secreting and in turn responding to OPG alongside resident PASMCs to drive pulmonary vascular remodelling. OPG regulates genes important in PH/PAH pathogenesis. To gain mechanistic insight into how OPG might regulate the pro- proliferative PASMC phenotype, we examined the transcriptome and intracellular signalling mediated by OPG in human PASMCs. Microarray analysis of PASMC mRNA identiﬁed 1900 probes from the microarray that were signiﬁcantly regulated by OPG. Utilising the full transcriptomic analysis we performed pathway analysis using Signalling Pathway Impact Analysis (SPIA)25 and identiﬁed 13 KEGG pathways as being signiﬁcantly perturbed by NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications 3 TURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Our data indicate that OPG is a likely therapeutic candidate for PAH. Using the KyMouse™system28 we generated a diverse panel of high afﬁnity anti-human OPG monoclonal antibodies with cross- reactivity to rat and cynomolgus monkey, displaying distinct neutralisation proﬁles and varying ability to block the interaction of OPG with TRAIL and RANKL (Supplementary Figure 4). Selected antibodies were chosen to cover a spectrum of partial and full inhibition of OPG-TRAIL and OPG-RANKL bindin OPG-FAS signalling was examined later (see below). Four can didate anti-OPG antibodies (Supplementary Figure 4e) wer tested for their ability to attenuate the development o monocrotaline-induced PAH (Fig. 6a). Weekly delivery of 3 m kg−1 antibody or IgG control resulted in the expected levels o circulating plasma antibody (Fig. 6b). Analysis of the complet dataset identiﬁed the Ky3 antibody as having a signiﬁcan e g j i h f n/s n/s * * * # # # # # # ** * * **** 50 0 80 60 40 20 0 400 300 200 6 4 2 4 3 2 1 0 SFM OPG VEGF Control 0 RVH (RV/LV+ Media/CSA Fold lnc. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 cell number LVESP (mmH % Muscular arteries OPG/1000 hprt copies 0.6 0.4 0.2 0.0 0.8 0.6 0.4 0.2 0.0 Normoxic SuHx Normoxic ABEVG OPG–/– to OPG–/– OPG–/– to C57BL/6 C57BL/6 to OPG–/– C57BL/6 to C57/BL6 SMA vWF OPG TRAIL SuHx Normoxic Fib Healthy BOEC IPAH BOEC OPG/DAPI SMC OPG 50 μm 50 μm 50 μm 50 μm Fibrocytes SuHx Normoxic SuHx C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– Control Hypoxic Hypoxic C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications e n/s * * # # Media/CSA 0.8 0.6 0.4 0.2 0.0 Normoxic SuHx C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– f n/s # # ** * 80 60 40 20 0 % Muscular arteries Normoxic SuHx C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– f e g i h * 400 300 200 6 4 2 Control 0 OPG/1000 hprt copies ABEVG OPG–/– to OPG–/– OPG–/– to C57BL/6 C57BL/6 to OPG–/– C57BL/6 to C57/BL6 SMA vWF OPG TRAIL Fib OPG/DAPI SMC OPG 50 μm 50 μm 50 μm 50 μm Fibrocytes Control Hypoxic Hypoxic h * 400 300 200 6 4 2 0 OPG/1000 hprt copies Fib SMC Fibrocytes Control Hypoxic h h g Control Hypoxic i i j **** 4 3 2 1 0 SFM OPG VEGF Fold lnc. cell number Healthy BOEC IPAH BOEC j Identiﬁcation of a lead therapeutic anti-OPG antibody. Our data indicate that OPG is a likely therapeutic candidate for PAH. Using the KyMouse™system28 we generated a diverse panel of high afﬁnity anti-human OPG monoclonal antibodies with cross- reactivity to rat and cynomolgus monkey, displaying distinct neutralisation proﬁles and varying ability to block the interaction of OPG with TRAIL and RANKL (Supplementary Figure 4). Selected antibodies were chosen to cover a spectrum of partial and full inhibition of OPG-TRAIL and OPG-RANKL binding. OPG-FAS signalling was examined later (see below). Four can- didate anti-OPG antibodies (Supplementary Figure 4e) were tested for their ability to attenuate the development of monocrotaline-induced PAH (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 In both PASMC lysates and recombinant protein pre- parations, immunoprecipitation with a Fas monoclonal antibody pulled down a 50 kDa band that stained positive following anti- 4 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 5e), led us to hypothesise that the remaining proliferation in response to OPG where Fas is neutralised may be mediated by TRAIL (since we have previously described TRAIL as a PASMC mitogen12). Pre-incubation with both an anti- TRAIL antibody and anti-Fas antibody signiﬁcantly reduced OPG-induced PASMC proliferation to near baseline levels (Fig. 5h) suggesting a direct activation of TRAIL-induced pro- liferation in PASMCs following Fas binding. Based on these and earlier data (Fig. 3), we therefore propose that OPG binding to Fas causes intracellular kinase signalling, including phosphor- ylation of ERK1/2, CDK4/5 leading to the activation of multiple genes associated with PAH, notably TRAIL. This induces a pro- survival, migratory and proliferative phenotype promoting pul- monary vascular remodelling and PAH (Fig. 5i). Furthermore, we propose that inhibition of OPG, e.g. via antibody blockade, will prevent this signalling and subsequent alteration in pro-PAH gene expression leading to a reversal of pulmonary vascular remodelling, normalisation of pulmonary vascular resistance and inhibition of PAH via alteration in the proliferation, migration and apoptosis of pulmonary vascular cells (Fig. 5j). OPG binds to Fas receptor on PASMCs. Given the effect of OPG on cell phenotype and the intracellular signalling identiﬁed, we felt that OPG may be acting as a ligand and signalling through a previously undescribed receptor. To identify the signalling receptor for OPG, we conducted a reverse transfection membrane protein array (Retrogenix, UK). Primary and secondary screens identiﬁed six twice-validated OPG-protein interacting partners, RANKL (tnfsf11), syndecan-1 (SDC-1), Fas, IL1-receptor acces- sory protein (IL-1RAcP), growth associated protein 43 (GAP43) and TMPRSS11D (Fig. 4a). We have previously reported that levels of SDC-1 were undetectable, and RANKL was only detected at low level in IPAH tissues13. Therefore we focused on investi- gating the four OPG-interacting proteins identiﬁed (Fas, IL- 1RAcP, Gap43 and TMPRSS11D). Expression of TMPRSS11D was undetectable in mRNA isolated from PASMCs. The RNA expression of Fas, IL-RAcP and GAP43 was conﬁrmed in PASMCs, with Fas being the most abundantly expressed, and further induced by OPG (Fig. 4b). Similarly, Fas mRNA was more highly expressed in PASMCs from patients with IPAH compared to healthy controls (Fig. 4c). Since Fas was the most abundantly expressed putative receptor we performed immunoprecipitation on lysates from PASMCs stimulated with OPG to validate binding. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 a c d e g j i h f b 5000 125 n/s n/s n/s n/s n/s ## ## * * * * * * * * # # # # # # * * **** * * 100 75 50 25 0 100 50 0 80 60 40 20 0 400 300 200 6 4 2 4 3 2 1 0 SFM OPG VEGF Control 0 4000 3000 Serum OPG (pg/ml) RVH (RV/LV+S) Media/CSA Fold lnc. cell number RVSP (mmHg) LVESP (mmHg) % Muscular arteries OPG/1000 hprt copies 2000 1000 0 0.8 0.6 0.4 0.2 0.0 0.8 0.6 0.4 0.2 0.0 Normoxic SuHx Normoxic SuHx Normoxic ABEVG OPG–/– to OPG–/– OPG–/– to C57BL/6 C57BL/6 to OPG–/– C57BL/6 to C57/BL6 SMA vWF OPG TRAIL SuHx Normoxic Fib Healthy BOEC IPAH BOEC OPG/DAPI SMC OPG 50 μm 50 μm 50 μm 50 μm Fibrocytes SuHx Normoxic SuHx Normoxic SuHx C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– Control Hypoxic Hypoxic C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– ATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467 019 13139 9 ARTICL a 5000 n/s *** * * * * 4000 3000 Serum OPG (pg/ml) 2000 1000 0 Normoxic SuHx C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– b 125 n/s ## ## * * ** 100 75 50 25 0 RVSP (mmHg) Normoxic SuHx C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– b a d 100 50 0 LVESP (mmHg) Normoxic SuHx C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– c n/s * # # ** RVH (RV/LV+S) 0.8 0.6 0.4 0.2 0.0 Normoxic SuHx C57 BM to C57 C57 BM to OPG–/– OPG–/– BM to C57 OPG–/– BM to OPG–/– d c Identiﬁcation of a lead therapeutic anti-OPG antibody. NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 6j), possibly due to retention of antibody bound OPG in the circulation rather than allowing it to access the vessel wall. vessels (Fig. 8j–k). To try and elucidate the different mechanisms of action of sildenaﬁl and Ky3 we performed Caspase 3 and PCNA staining to examine the relationship between treatment and apoptosis and proliferation on serial sections within the small remodelled pulmonary arterioles (Fig. 8l). In the sildenaﬁl and IgG4 treated groups there was evidence of apoptosis, pre- dominantly in endothelial cells, and medial proliferation. By contrast, Ky3 treated rats appeared to have apoptosis in both endothelial and medial layers and reduced medial cell proliferation. Plasma levels of OPG were signiﬁcantly elevated in all SuHx rats compared to controls at week 6 (Fig. 8m). Consistent with previous experiments, rats treated with Ky3 displayed a signiﬁcant increase in circulating OPG from week 7 through to week 9 compared to other groups (Fig. 8m). To assess any potential detrimental side-effect of anti-OPG treatment on bone turnover, microCT studies were performed on the tibia. Treatment with Ky3 had no signiﬁcant effect on bone volume (Fig. 8n) or trabecular thickness (Fig. 8o) compared to IgG4 treated rats; however, there was a small but signiﬁcant decrease in trabecular number (Fig. 8p) in IgG4 treated rats compared to Ky3, although Ky3 treated rats were not signiﬁcantly different compared to control or SuHx rats. Ky3 inhibits OPG-induced phenotype and NF-κβ activation. Once Ky3 was identiﬁed as the lead candidate antibody for fur- ther development we conﬁrmed that Ky3 inhibited OPG-induced proliferation (Fig. 7a) and migration (Fig. 7b) in human PASMC in vitro. Having previously identiﬁed an NF-κβ response to OPG (Fig. 3e) we investigated, and show that Ky3 inhibits this acti- vation (Fig. 7c). In the Mct model we also observed a signiﬁcant reduction in pulmonary vascular remodelling with only 2 weeks of Ky3 treatment but this did not alter the haemodynamic proﬁle (Supplementary Figure 5). We proposed that this was due to the shorter treatment duration and, particularly advanced/severe phenotype in this instance of the model. Ky3 attenuates severe PAH. Antibody Ky3 was tested ther- apeutically in two rat models with severe established PAH, Mct and SuHx. Rats were exposed to Sugen5416 and hypobaric hypoxia (18,000 ft, equivalent to 10.8% O2) for 3 weeks before returning to room air for 3 weeks to allow the progression of pulmonary vascular remodelling. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Fig. 2 Bone marrow cell derived OPG is required to initiate PAH in the mouse SuHx model. Bar graphs show (a) serum levels of OPG, (b) right ventricular systolic pressure (RVSP), (c) right ventricular hypertrophy (RVH), (d) left ventricular end-systolic pressure (LVESP), (e) the degree of medial wall thickness as a ratio of total vessel size (Media/CSA) in small pulmonary arteries pulmonary arteries less than 50 µm, (f) the relative percentage of muscularised pulmonary arteries less than 50 µm (<50 µm) in diameter. Representative photomicrographs (g) of serial lung sections from bone marrow- transplanted (BMT) mice. Sections were stained with Alcian Blue Elastic van Gieson (ABEVG), or immunostained for α-smooth muscle actin (α-SMA), von Willebrand factor (vWF), OPG, or TRAIL. Panel (h) shows OPG gene expression from RNA-seq performed on control and PAH-derived pulmonary artery smooth muscle cells (SMC), pulmonary artery ﬁbroblasts (Fib) and ﬁbrocytes obtained from the hypoxic neonatal calf model of PAH. Representative photomicrographs of lung sections from the hypoxic neonatal calf stained with OPG (i). Proliferation of blood outgrowth endothelial cells (BOEC) from patients with IPAH and healthy controls (j). Box and Whisker plots represent the interquartile range (box) with the line representing the median and whisker the full range of the data, each animal is represented by a dot in each graph. C57-C57 BMT n = 6 for each group, OPG−/–OPG−/−n = 3 for each group, C57-OPG−/−n = 3 and OPG−/−-C57 n = 5 for each group. * p < 0.05, p < 0.01,* p < 0.001 compared to C57-C57 BMT Normoxic mice unless otherwise stated, # p < 0.05, ## p < 0.01 compared to C57–C57 SuHx mice following one-way ANOVA with Bonferroni’s multiple comparisons post hoc test. All images are presented at their original magniﬁcation x400, scale bars represent 50 µm attenuation on markers of PAH including RVSP (Fig. 6c), RVH (Fig. 6d) and ePVRi (Fig. 6e). There was no signiﬁcant effect of either treatment on LVESP (Fig. 6f). Immunohistochemical analysis of the lung demonstrated a signiﬁcant reduction in the media/CSA area (Fig. 6g) and percentage of thickened sub-50 μm pulmonary arterioles (Fig. 6h–i) in rats treated with the Ky3 antibody. Interestingly rats treated with either the commercial polyclonal anti-mouse OPG antibody (AF459), which demon- strated partial efﬁcacy in the SuHx and efﬁcacy in the ApoE−/− mouse (Fig. 1), or Ky3 resulted in a signiﬁcant increase in serum levels of OPG (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Rats were then randomised into groups to receive either sildenaﬁl (50 mg kg−1 per day), Ky3 (3 mg kg−1 per week) or IgG (3 mg kg−1 per week) control antibody from week 6 for 3 weeks (Fig. 8a). Sustained levels of Ky3 and IgG were maintained throughout the study (Fig. 8b). PA AT decreased from week 0 to week 6 as disease progressed. There was a trend for increased PA AT in sildenaﬁl vs SuHx and Ky3 vs IgG4 treated animals but this did not reach signiﬁcance (Fig. 8c). Sildenaﬁl treated rats showed an increase in cardiac output (CO) (Fig. 8d). Treatment with sildenaﬁl and Ky3 signiﬁcantly reduced RVSP (Fig. 8e) compared to untreated and IgG4 controls, respectively. RV arterial elastance (RV Ea) and ePVRi were sig- niﬁcantly reduced only by Ky3 (Fig. 8f–g), treatment with silde- naﬁl and Ky 3 signiﬁcantly reduced RVH (Fig. 8h). There was no signiﬁcant effect of any treatment on LVESP (Fig. 8i) indicating speciﬁc effects on the pulmonary circulation. Immunohisto- chemical analysis of the lung demonstrated that the haemody- namic changes induced by anti-OPG treatment were associated with a reduction in both the media/CSA (Fig. 8j) and percentage of muscularised pulmonary arterioles sub-50 μm in diameter (Fig. 8k). In contrast there was no signiﬁcant effect of sildenaﬁl on either the degree of remodelling, or the percentage of remodelled Ky3 reduces tissue expression of IL-6, OPG and TRAIL. To demonstrate that the therapeutic effects of Ky3 treatment in the SuHx rat model were associated with reduced OPG signalling, we examined the expression of OPG and identiﬁed downstream mediators in the lung tissue. Despite the increase in circulating levels of OPG (Fig. 8m), Ky3 treatment resulted in a signiﬁcant reduction in OPG RNA (Fig. 9a) and protein within whole lung lysates (Fig. 9b), Similarly, levels of TRAIL were also decreased at RNA (Fig. 9c) and protein level (Fig. 9d). Treatment with Ky3 was also associated with a reduction in inﬂammation within the lung as shown by IL-6 RNA expression (Fig. 9e) although there was no effect on total circulating levels of IL-6 (Fig. 9f). These changes were also consistent with those observed within remodelled pulmonary arterioles by IHC (Fig. 9g). Ky3 and standard of care vasodilator therapy combination. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 6a). Weekly delivery of 3 mg kg−1 antibody or IgG control resulted in the expected levels of circulating plasma antibody (Fig. 6b). Analysis of the complete dataset identiﬁed the Ky3 antibody as having a signiﬁcant 5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 As previ demonstrated rats treated with Ky3 had increased circu levels of OPG (Fig 10i) Caspase 3 and PCNA staining iden a b c d e f SPIA two-way evidence plot 8 6 4 –log (P PERT) 2 0 KEGG ID Pathway name Status Inhibited Inhibited Inhibited Inhibited Inhibited Activated Activated Pathogenic escherichia coli infection Gap junction TGF-beta signaling pathway ECM-receptor interaction Focal adhesion Pathways in cancer Arrhythmogenic right ventricular cardiomyopathy (ARVC) 05130 04540 04350 04512 04510 05412 05200 0 2 4 –log (P NDE) 6 8 04540 05200 04512 04510 04350 05130 05412 Color key –1 0 Value OPG Control HSPB7 BMP6 ATG9A S100A4 FKBP1B IL1RN PFKFB3 SOCS2 LDHA DAG1 CFL1 KLF2 NFKBIA HMOX1 IRAK1 BNPR1A ID3 FKBP2 CCL3 CCND1 ID2 ENG GREM1 FKBP1A CDC25C HIF1A APLN ICAM1 SOD2 NPPB TGFBR1 IL8 MMP3 CCL2 IL6 ACVRL1 RUNX1 VIPR1 ATG4C IL1RAPL1 CDKN1A CXCL12 PDE1A VEGFA HDAC4 LRP1 TGFBR2 PPARG DAPK2 PTGIS SMAD1 TNFSF10 CAV1 FKBP9 TNC PDGFRB PDGFRA 1 8 6 4 TRAIL RQ to 18S PDGFRA RQ to 18S 2 0 8 6 4 VEGFA (RQ/18S) TNC (RQ/18S) Fold increase pERK to total ERK Fold increase pHSP27 to β-actin Fold increase mTOR phospho to total 2 0 8 10 6 4 CAV1 (RQ/18S) VIPR (RQ/18S) 2 0 Un OPG Un OPG Pro-survival/anti apoptotic NF-κβ activation Un OPG Un OPG Un OPG Un OPG 6 * ** * * * * ** * * * 4 2 0 6 5 4 3 2 1 0 3 2 1 0 Fold increase pCDK4 to β-actin 3 2 1 0 Fold increase CDK5 to GAPDH 3 2 1 0 4 2 0 Un 10 60 Un 10 60 Un 10 60 Un 10 60 Un 10 60 6 4 2 0 4 3 2 1 0 ASK1 p50 p65 IKBa IKK Phospho IKK 2 4 2 0 –2 –4 1 0 –1 –2 Phospho p38 Phospho p53 p21 DAXX CDK4 CDK6 HSP90 Phospho CDK1/2 pERK1/2 pHSP27 pmTOR pCDK4 GAPDH CDK5 b-actin mTOR β-actin 42/44 kDa 27 kDa 289 kDa 30 kDa 37 kDa 30 kDa 45 kDa 289 kDa 45 kDa 42/44 kDa ERK1/2 OPG 10′ OPG 60′ OPG 10′ OPG 60′ a SPIA two-way evidence plot 8 6 4 –log (P PERT) 2 0 0 2 4 –log (P NDE) 6 8 04540 05200 04512 04510 04350 05130 05412 b Color key –1 0 Value 1 b Color key –1 0 Value OPG Control HSPB7 BMP6 ATG9A S100A4 FKBP1B IL1RN PFKFB3 SOCS2 LDHA DAG1 CFL1 KLF2 NFKBIA HMOX1 IRAK1 BNPR1A ID3 FKBP2 CCL3 CCND1 ID2 ENG GREM1 FKBP1A CDC25C HIF1A APLN ICAM1 SOD2 NPPB TGFBR1 IL8 MMP3 CCL2 IL6 ACVRL1 RUNX1 VIPR1 ATG4C IL1RAPL1 CDKN1A CXCL12 PDE1A VEGFA HDAC4 LRP1 TGFBR2 PPARG DAPK2 PTGIS SMAD1 TNFSF10 CAV1 FKBP9 TNC PDGFRB PDGFRA 1 b b Color key –1 0 Value OPG Control HSPB7 BMP6 ATG9A S100A4 FKBP1B IL1RN PFKFB3 SOCS2 LDHA DAG1 CFL1 KLF2 NFKBIA HMOX1 IRAK1 BNPR1A ID3 FKBP2 CCL3 CCND1 ID2 ENG GREM1 FKBP1A CDC25C HIF1A APLN ICAM1 SOD2 NPPB TGFBR1 IL8 MMP3 CCL2 IL6 ACVRL1 RUNX1 VIPR1 ATG4C IL1RAPL1 CDKN1A CXCL12 PDE1A VEGFA HDAC4 LRP1 TGFBR2 PPARG DAPK2 PTGIS SMAD1 TNFSF10 CAV1 FKBP9 TNC PDGFRB PDGFRA 1 a 6 4 –log (P PERT) 2 0 KEGG ID Pathway name Status Inhibited Inhibited Inhibited Inhibited Inhibited Activated Activated Pathogenic escherichia coli infection Gap junction TGF-beta signaling pathway ECM-receptor interaction Focal adhesion Pathways in cancer Arrhythmogenic right ventricular cardiomyopathy (ARVC) 05130 04540 04350 04512 04510 05412 05200 0 2 4 –log (P NDE) 6 8 04540 05200 04512 04510 04350 05130 05412 c 8 6 4 TRAIL RQ to 18S PDGFRA RQ to 18S 2 0 8 6 4 VEGFA (RQ/18S) TNC (RQ/18S) 2 0 8 10 6 4 CAV1 (RQ/18S) VIPR (RQ/18S) 2 0 Un OPG Un OPG Un OPG Un OPG Un OPG Un OPG 6 * * ** * * * 4 2 0 6 4 2 0 4 3 2 1 0 c S e NF-κβ activation p50 p65 IKBa IKK Phospho IKK 4 2 0 –2 –4 OPG 10′ OPG 60′ d d Pro-survival/anti apoptotic ASK1 2 1 0 –1 –2 Phospho p38 Phospho p53 p21 DAXX CDK4 CDK6 HSP90 Phospho CDK1/2 OPG 10′ OPG 60′ e f Fold increase pERK to total ERK Fold increase pHSP27 to β-actin Fold increase mTOR phospho to total ** * * * 6 5 4 3 2 1 0 3 2 1 0 Fold increase pCDK4 to β-actin 3 2 1 0 Fold increase CDK5 to GAPDH 3 2 1 0 4 2 0 Un 10 60 Un 10 60 Un 10 60 Un 10 60 Un 10 60 pERK1/2 pHSP27 pmTOR pCDK4 GAPDH CDK5 b-actin mTOR β-actin 42/44 kDa 27 kDa 289 kDa 30 kDa 37 kDa 30 kDa 45 kDa 289 kDa 45 kDa 42/44 kDa ERK1/2 f Fold increase pERK to total ERK ** * 6 4 2 0 Un 10 60 pERK1/2 42/44 42/44 ERK1/2 * Fold increase pCDK4 to β-actin 3 2 1 0 Fold increase CDK5 to GAPDH 3 2 1 0 Un 10 60 Un 10 60 pCDK4 GAPDH CDK5 b-actin 9 kDa 30 kDa 37 kDa 30 kDa 45 kDa 9 kDa Fold increase pHSP27 to β-actin 5 4 3 2 1 0 Un 10 60 pHSP27 β-actin Da 27 kDa 45 kDa Da f Fold increase mTOR phospho to total * 3 2 1 0 Un 10 60 pmTOR mTOR rats only demonstrated a reduction in pulmonary vascular remodelling when also receiving Ky3 (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 day) or bosentan (60 mg kg−1 per day) treated rats exposed to x, with IgG4 treatment as a control (Fig. 10a). There was no ct of either sildenaﬁl or bosentan on the levels of circulating as measured by IgG4 luminex assay (Fig 10b) Treatment of rats only demonstrated a reduction in pulmonary vas remodelling when also receiving Ky3 (Fig. 10h). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Finally, Ky3 antibody (3 mg kg−1 per week) was then tested in comparison, and combination with, sildenaﬁl (50 mg kg−1 NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 ARTICLE Discussion We report that OPG promotes cell survival, pro-migratory and pro-proliferative signalling in PASMCs through binding to Fas receptor. Furthermore, we demonstrate that OPG is required for full development of PAH in multiple rodent models. PAH was substantially attenuated and reversed in these models by administration of a human anti-OPG therapeutic antibody (Ky3). The mechanism for this effect was due to a reduction in pul- monary vascular remodelling indices through the modulation of proliferation and apoptosis within small pulmonary arterioles due to alterations in downstream signalling via NF-κβ, ERK, CDKs. This effect was in contrast to rats treated with sildenaﬁl (a vasodilator and ﬁrst line treatment), which displayed a similar haemodynamic response but was without effect upon pulmonary vascular remodelling. Ky3 in combination with bosentan further reduced RVSP compared to bosentan alone suggesting that anti- OPG treatment may have a beneﬁt in addition to existing vaso- dilator therapy (even when used in relative excess in these rodent models compared to human use). Although new drugs29,30 have recently been added to the treatment options available to PAH physicians, these therapies continue to target sustained pulmon- ary vasoconstriction. While this is a common pathophysiological feature of all forms of PAH, there is little evidence that drugs targeting the endothelin, nitric oxide or prostacyclin pathways31 have a direct or lasting effect on pulmonary vascular cell pro- liferation. Indeed, they do not reverse the proliferative changes observed in PAH, emphasising the need for anti-proliferative therapies9. Our data demonstrate an unequivocal role for OPG in the pathogenesis of PAH via the modulation of proliferative and apoptotic changes observed in PAH. OPG has also been shown to block TRAIL binding to its receptors, a key regulator of apoptosis in sensitive cells32, immunoregulation and immune surveillance33,34 and in both neutrophil35,36 and macrophage37,38 clearance in the lung. Of particular relevance, we have previously described an important role for TRAIL in PAH12,39 and have described how both TRAIL and OPG can be separately regulated by a number of pathways associated with PAH including BMPs, 5-HT and inﬂammatory cytokines12,13. The wider implications of the identiﬁed interaction between OPG and IL-1RAcP have not yet been fully examined. We16 and others46 have previously highlighted the importance of IL-1 in the pathogenesis of PAH but the direct effect of OPG on IL- 1/IL-1R1, or IL-33/ST2 to complex with IL-1RAcP remains unclear. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Fig. 3 OPG activates pro-proliferative signalling and a disease-relevant transcriptome. Panel (a) Signalling Pathway Impact Analysis (SPIA) with each pathway represented by one dot. The pathways to the right of the red diagonal line are signiﬁcant after Bonferroni correction of the global p-values obtained using Fisher’s methods from the combination of pPERT and pNDE values, the pathways to the right of the blue line are signiﬁcant after FDR correction. (b) shows a heat map of signiﬁcant differentially regulated genes after gene enrichment against PAH-associated genes in OPG stimulated PASMCs, (c) TaqMan validation of gene expression microarray, TaqMan expression data normalised using ΔΔCT with 18 s rRNA as the endogenous control gene. Panel (d) shows a heat map of cell cycle/CDK proteins signiﬁcantly regulated by OPG at 10 and 60 min expressed as a ratio to unstimulated controls from the same time point from Kinex phospho-arrays identiﬁed, with (e) showing those speciﬁcally related to NF-κβ. f Western blot validation of Kinex array data in unstimulated (0.2% FCS, Un) or OPG-stimulated (50 ng ml−1) PASMCs at 10 min (10) and 60 min (60) with relative band densities of phospho-ERK1/2, phospho-HSP27, phospho-mTOR, phospho-CDK4 and total CDK5 are shown by the bar graphs and representative western blot images shown above the graph. Heat maps show Z-ratio gene or protein expression. Bars represent mean with error bars showing the standard error of the mean, n = 3 for pooled triplicate samples (a, b), n = 12 (c), n = 4 (d, e), n = 5 (f) from three donors of PASMCs, dots represent experimental repeats. Bars from unstimulated cells are white, OPG stimulated blue. p < 0.05, * p < 0.01, *** p < 0.001 compared OPG-stimulated to unstimulated PASMCs using one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test. When there were only two groups, unpaired t-tests were used unbiased screen of around 60% of known transmembrane pro- teins and identiﬁed OPG binding to RANKL, syndecan-1 but also with Fas, IL-1RAcP, GAP43 and TMPRSS11D (Fig. 4). Having previously examined RANKL and syndecan-113, we assessed the expression of the other binders within PASMCs, and subse- quently focused on Fas due its relatively high expression levels within diseased tissue and its close relationship to OPG and TRAIL (all belong to the TNF superfamily). Our data suggest that neutralisation of Fas, either by anti-Fas antibody or binding to OPG, up-regulates TRAIL expression. ARTICLE This may reﬂect a redun- dancy mechanism between the two death-receptor signalling pathways. FasL has been reported to induce PASMC apoptosis43, so our data highlight another potential mechanism by which increased OPG (via Fas) may drive PAH pathology. Inhibiting FasL/Fas binding with endogenous OPG may limit the ability of FasL to cause apoptosis44. Indeed, we clearly show that OPG induces a pro-survival/anti-apoptotic phenotype, and activates many genes previously associated with PAH, including TRAIL, suggesting a pivotal role in the disease process. The implication that OPG can regulate the local expression of TRAIL within the vessel wall ﬁts with our reports demonstrating that TRAIL39, and speciﬁcally tissue-derived TRAIL12 is required for mice to develop PAH. Of note, TRAIL was also recently described to be an important member of an immune cluster of circulating pro- teins that deﬁned poor prognosis in patients with mixed aetiology PAH45. The relationship between cell expressed, and circulating TRAIL is however complex. TRAIL is widely expressed, including by immune cells and circulating “soluble” TRAIL requires pro- teolytic cleavage of the C-terminal extracellular domain of the transmembrane TRAIL protein. Whether disease is mediated by locally expressed and retained TRAIL or by released circulating TRAIL remains unclear. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 10h). As previously demonstrated rats treated with Ky3 had increased circulating levels of OPG (Fig. 10i). Caspase 3 and PCNA staining identiﬁed an increase in apoptosis and decrease in proliferation within the small remodelled pulmonary arterioles in the lungs of rats treated with Ky3 when compared to either sildenaﬁl or bosentan alone (Fig. 10j). per day) or bosentan (60 mg kg−1 per day) treated rats exposed to SuHx, with IgG4 treatment as a control (Fig. 10a). There was no effect of either sildenaﬁl or bosentan on the levels of circulating Ky3 as measured by IgG4 luminex assay (Fig. 10b). Treatment of SuHx rats with sildenaﬁl, bosentan or Ky3 resulted in a com- parable reduction PAH phenotype (Fig. 10c–h). Ky3 in combi- nation with bosentan resulted in a signiﬁcant further reduced RVSP compared to bosentan alone (Fig. 10c). Sildenaﬁl treated per day) or bosentan (60 mg kg−1 per day) treated rats exposed to SuHx, with IgG4 treatment as a control (Fig. 10a). There was no effect of either sildenaﬁl or bosentan on the levels of circulating Ky3 as measured by IgG4 luminex assay (Fig. 10b). Treatment of SuHx rats with sildenaﬁl, bosentan or Ky3 resulted in a com- parable reduction PAH phenotype (Fig. 10c–h). Ky3 in combi- nation with bosentan resulted in a signiﬁcant further reduced RVSP compared to bosentan alone (Fig. 10c). Sildenaﬁl treated rats only demonstrated a reduction in pulmonary vascular remodelling when also receiving Ky3 (Fig. 10h). As previously demonstrated rats treated with Ky3 had increased circulating levels of OPG (Fig. 10i). Caspase 3 and PCNA staining identiﬁed an increase in apoptosis and decrease in proliferation within the small remodelled pulmonary arterioles in the lungs of rats treated with Ky3 when compared to either sildenaﬁl or bosentan alone (Fig. 10j). 7 TURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 TaqMan expression of Fas in whole lung RNA (f) and protein expression in lung sections (g) isolated from control (saline), monocrotaline (d28), control (normoxia) and SuHx (wk9) rats. TaqMan expression data normalised using ΔΔCT with 18 s rRNA as the endogenous control gene. Bars represent the mean with error bars showing the standard error of the mean. Panel (c) n = 4 and panel (d) n = 3 from three individual donors, dots represent experimental repeats. * p < 0.05, p < 0.01, * p < 0.001 following one-way ANOVA with Bonferroni’s multiple comparisons post hoc test. When there were only two groups, unpaired t-tests were used. Scale bar represents 25 µm Fig. 4 OPG binds to Fas, which is increased in IPAH lung and right ventricle. Panel (a) demonstrates conﬁrmed protein binding between OPG and syndecan-1 (SDC-1), RANKL (TNFSF11), Growth Associated Protein 43 (GAP43), Fas, IL1-receptor accessory protein (IL-1RAcP) and transmembrane protease, serine 11D. b TaqMan expression of Fas, IL-1RAcP and GAP43 in control (white bars, 0.2% FCS) and OPG-stimulated (blue bars, 50 ng ml−1) purchased PASMCs, and (c) PASMCs from patients with IPAH (grey bars) and healthy controls (white bars). d Anti-Fas co-immunoprecipitation of OPG in endogenous primary human PASMC lysates or recombinant protein replicated 3 times. e OPG and Fas are expressed within remodelled pulmonary arteries and the right ventricle of patients with IPAH. TaqMan expression of Fas in whole lung RNA (f) and protein expression in lung sections (g) isolated from control (saline), monocrotaline (d28), control (normoxia) and SuHx (wk9) rats. TaqMan expression data normalised using ΔΔCT with 18 s rRNA as the endogenous control gene. Bars represent the mean with error bars showing the standard error of the mean. Panel (c) n = 4 and panel (d) n = 3 from three individual donors, dots represent experimental repeats. * p < 0.05, p < 0.01, * p < 0.001 following one-way ANOVA with Bonferroni’s multiple comparisons post hoc test. When there were only two groups, unpaired t-tests were used. Scale bar represents 25 µm included Ky3. Although there are limitations of each rodent model of PH used in this study, the utilisation of multiple models, each with different characteristics, combined with human data circumvent these concerns. Furthermore, the efﬁ- cacy demonstrated here may not reﬂect the full potential effect in humans due to incomplete homology between human and rat proteins. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 * p < 0.05, p < 0.01, * p < 0.001 following one-way ANOVA with Bonferroni’s mult i t h t t Wh th l t i d t t t d S l b t 25 a c b anti-OPG 1° anti-goat lgG 2° anti-OPG 1° anti-goat lgG 2° anti-goat lgG 2° rhOPG 3 Ctrl OPG 2 1 0 4 2.5 2.0 1.5 1.0 0.5 0.0 3 2 1 0 FAS Control IPAH Control IPAH Control IPAH IL-1RAcP Gap43 FAS RQ to 18S 3 2 1 0 GAP43 RQ to 18S FAS RQ to 18S lL-1RAcP RQ to 18S FCGR1A SLC13A GAP43 FCGR2B TNFSF11 FCGR2A FAS lL-1RAcP SDC1 TMPRSS11D rhOPG b 3 Ctrl OPG 2 1 0 FAS IL-1RAcP Gap43 FAS RQ to 18S b a c 4 2.5 2.0 1.5 1.0 0.5 0.0 3 2 1 0 Control IPAH Control IPAH Control IPAH 3 2 1 0 GAP43 RQ to 18S FAS RQ to 18S lL-1RAcP RQ to 18S c 4 3 2 1 0 Control IPAH FAS RQ to 18S c d f g e PASMC FAS OPG FAS OPG Pulmonary artery Right ventricle rOPG FAS IP 80 kDa Control Control Control Mct SuHx IPAH FAS 50 kDa OPG 30 kDa 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 + + – – Control Mct Control SuHx FAS (RQ/18S) FAS (RQ/18S) d f e PASMC rOPG FAS IP 80 kDa C 50 kDa OPG 30 kDa 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 + + – – Control Mct Control SuHx FAS (RQ/18S) FAS (RQ/18S) d g e FAS OPG FAS OPG Pulmonary artery Right ventricle Control Control Control Mct SuHx IPAH FAS e f g Fig. 4 OPG binds to Fas, which is increased in IPAH lung and right ventricle. Panel (a) demonstrates conﬁrmed protein binding between OPG and syndecan-1 (SDC-1), RANKL (TNFSF11), Growth Associated Protein 43 (GAP43), Fas, IL1-receptor accessory protein (IL-1RAcP) and transmembrane protease, serine 11D. b TaqMan expression of Fas, IL-1RAcP and GAP43 in control (white bars, 0.2% FCS) and OPG-stimulated (blue bars, 50 ng ml−1) purchased PASMCs, and (c) PASMCs from patients with IPAH (grey bars) and healthy controls (white bars). d Anti-Fas co-immunoprecipitation of OPG in endogenous primary human PASMC lysates or recombinant protein replicated 3 times. e OPG and Fas are expressed within remodelled pulmonary arteries and the right ventricle of patients with IPAH. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 a c d f g e b anti-OPG 1° anti-goat lgG 2° anti-OPG 1° anti-goat lgG 2° anti-goat lgG 2° rhOPG 3 Ctrl OPG 2 1 0 4 2.5 2.0 1.5 1.0 0.5 0.0 3 2 1 0 FAS Control PASMC FAS OPG FAS OPG Pulmonary artery Right ventricle rOPG FAS IP 80 kDa Control Control Control Mct SuHx IPAH FAS 50 kDa OPG 30 kDa 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 + + – – IPAH Control Mct Control SuHx Control IPAH Control IPAH IL-1RAcP Gap43 FAS RQ to 18S 3 2 1 0 GAP43 RQ to 18S FAS RQ to 18S FAS (RQ/18S) FAS (RQ/18S) lL-1RAcP RQ to 18S FCGR1A SLC13A GAP43 FCGR2B TNFSF11 FCGR2A FAS lL-1RAcP SDC1 TMPRSS11D rhOPG g. 4 OPG binds to Fas, which is increased in IPAH lung and right ventricle. Panel (a) demonstrates conﬁrmed protein binding between OPG ndecan-1 (SDC-1), RANKL (TNFSF11), Growth Associated Protein 43 (GAP43), Fas, IL1-receptor accessory protein (IL-1RAcP) and transmem otease, serine 11D. b TaqMan expression of Fas, IL-1RAcP and GAP43 in control (white bars, 0.2% FCS) and OPG-stimulated (blue bars, 50 rchased PASMCs, and (c) PASMCs from patients with IPAH (grey bars) and healthy controls (white bars). d Anti-Fas co-immunoprecipitation dogenous primary human PASMC lysates or recombinant protein replicated 3 times. e OPG and Fas are expressed within remodelled pulmona d the right ventricle of patients with IPAH. TaqMan expression of Fas in whole lung RNA (f) and protein expression in lung sections (g) isol ntrol (saline), monocrotaline (d28), control (normoxia) and SuHx (wk9) rats. TaqMan expression data normalised using ΔΔCT with 18 s rR dogenous control gene. Bars represent the mean with error bars showing the standard error of the mean. Panel (c) n = 4 and panel (d) n = 3 f dividual donors, dots represent experimental repeats. Discussion Similarly, the binding of OPG to GAP43 and TMPRSS11D has not been further pursued at this stage due to their low expression in diseased cells. GAP43 is reported to be a neuron-speciﬁc protein47 and TMPRSS11D (human airway trypsin-like protease, HAT) is a type-II transmembrane trypsin-like serine protease that is largely found in sputum and expressed by bronchial ciliated endothelial cells48. Further work is clearly required to determine the inﬂuence of OPG in other biological processes and diseases where IL-1RAcP, GAP43 and TMPRSS11D play an important role. Our study was initially limited by the lack of availability of monoclonal anti-human OPG antibodies with cross-reactivity to rat but we overcame this by generating a suite of human monoclonal antibodies that Previous data suggest that the predominant function of OPG is to regulate osteoclastogenesis, with data from mice demonstrating that reduced OPG expression results in osteoporosis40 and over- expression of OPG causes osteopetrosis41 via binding to RANKL. These data perhaps suggest that therapeutic strategies targeting OPG might have detrimental effects on bone remodelling; how- ever, encouragingly in our studies we demonstrate a positive therapeutic effect on pulmonary vascular remodelling with no signiﬁcant effect on bone phenotype. OPG also binds proteins other than RANKL and TRAIL, e.g. syndecan-1, glycosami- noglycans (GAGs), von Willebrand factor and factor VIII-von Willebrand factor complex42. We therefore performed an 8 ARTICLE NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications Methods A i l l treatments with the human monoclonal anti-OPG antibody or control IgG4 isotype were performed (3 mg kg−1 per week, i.p.) alone or in combination with sildenaﬁl (50 mg kg−1 per day) or bosentan (60 mg kg−1 per day) in chow from weeks 6 with animals sacriﬁced at week 9. / ( ) d / ( ) f Animals. All animal experiments were approved by the University of Shefﬁeld Project Review Committee and conformed to the UK Home Ofﬁce ethical guide- lines. A sample size of at least four animals was used to provide greater than 95% power to detect a difference in RVSP of 10 mmHg with a SD of 3 mmHg with 95% conﬁdence. Additional animals were studied in large group comparisons and to obtain sufﬁcient tissue for analysis. Animals used for antibody intervention studies were randomised blindly based on weights to achieve a similar distribution of weights across all groups where possible. ApoE−/−(JAX 2052) and OPG−/−(JAX 010672) mice from a C57BL/6 J background were purchased from Jackson Labs. ApoE+/−/OPG−/−were subsequently bred in-house. Male C57BL/6, ApoE+/−, OPG−/−and ApoE+/−/OPG−/−aged 10–12 week were fed normal chow (4.3% fat, 0.02% cholesterol, 0.28% sodium) or Paigen diet (18.5% fat, 0.9% cholesterol, 0.5% cholate, 0.269% sodium) for 8 weeks8,11. Where stated BMT was performed on male mice aged 6–8 weeks old, where each received a sub-lethal dose of whole- body irradiation (1100 rads, split into two doses, 4 h apart). Irradiated recipients then received 3–4 million cells isolated from 4 to 6 week old mice, in Hanks’ balanced salt solution, by tail-vein injection12,49. Mice were allowed to recover for 6 weeks after bone marrow transfer prior to induction of PAH. Where stated, neutralising goat polyclonal anti-OPG antibody (AF459, R&D Systems) or control IgG isotype antibody (AF6775, R&D systems) was used. Antibodies were delivered via an Alzet 1004 micro pump (100 μl reservoir, 0.1 μl h−1 for 4 weeks at 20 ng h−1 (0.8 ng g−1 h−1). For the Sugen hypoxic model (SuHx), C57BL/6 and OPG−/− male mice were exposed to hypoxia (10% v/v O2) for 3 weeks with weekly injections of 20 mg kg−1 Sugen5416 (Tocris) during exposure to hypoxia50. Male Sprague Dawley rats were purchased from Charles River UK. PAH was induced by a single subcutaneous injection of monocrotaline (MCT, Sigma Aldrich, St. Louis, MO, USA) at 60 mg kg−1 in rats 200–210 g, alongside saline injected control animals. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Dots represent experimental repeats, Panels (a–e) (n = 4), panel (f) (n = 3), panel (g) (n = 4), panel (h) (n = 4 for SFM, 10 for PDGF & OPG stimulations) * p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001 following one-way ANOVA with Bonferroni’s multiple comparisons post hoc test Un Ctrl FAS MAb 0 1 2 3 4 5 CAV1 (RQ/18S) OPG * * Un Ctrl FAS MAb 0 1 2 3 4 5 TNC (RQ/18S) OPG ** * b d c Un Ctrl FAS MAb 0 1 2 3 4 5 CAV1 (RQ/18S) OPG * * Un Ctrl FAS MAb 0 1 2 3 4 5 TNC (RQ/18S) OPG ** * Un Ctrl FAS MAb 0.0 0.5 1.0 1.5 2.0 PDGFRA (RQ/18S) OPG d c b Un Ctrl FAS MAb 0 2 4 6 8 VEGFA (RQ/18S) OPG * * a b a Un Ctrl FAS MAb 0 2 4 6 8 TRAIL (RQ/18S) OPG * * Un Ctrl FAS MAb 0 1 2 3 4 CAV1 (RQ/18S) OPG Un Ctrl FAS MAb 0 1 2 3 4 TNC (RQ/18S) OPG Un Ctrl FAS MAb 0 2 4 6 VEGFA (RQ/18S) OPG Un Ctrl FAS MAb 0.0 0.5 1.0 1.5 PDGFRA (RQ/18S) OPG f e 0.5 1.0 1.5 2.0 Fold activation caspase 3/7 OPG FasAb FasL FasL and OPG TRAIL TRAIL and OPG Un Ctrl FAS MAb 0 2 4 6 8 TRAIL (RQ/18S) OPG * * OPG e f 0.5 1.0 1.5 2.0 Fold activation caspase 3/7 OPG FasAb FasL FasL and OPG TRAIL TRAIL and OPG f 0 20 40 60 80 100 200 250 300 % migration to OPG PDGF OPG OPG + FasAb (0.5 nM) OPG + FasAb (1 nM) g SFM PDGF OPG 0 20 40 60 80 100 120 % Proliferation (normalised to PDGF) NoAb FAS Ab TRAIL Ab Fas and TRAIL Abs ** h h g Fig. 5 OPG-Fas interaction mediates the OPG-induced phenotypic response of PASMC. TaqMan expression of (a) VEGFA, (b) PDGFRA, (c) TNC, (d) Cav1 and (e) TRAIL in response to OPG in the presence (hash bars) or absence (Grey bars) of anti-Fas neutralising antibody (1500 ng ml−1). Panel (f) demonstrates OPG inhibition of FasL and TRAIL-induced apoptosis in HT1080 cells. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Un Ctrl FAS MAb 0 2 4 6 8 TRAIL (RQ/18S) OPG * * Un Ctrl FAS MAb 0 1 2 3 4 5 CAV1 (RQ/18S) OPG * * Un Ctrl FAS MAb 0 1 2 3 4 5 TNC (RQ/18S) OPG ** * Un Ctrl FAS MAb 0 2 4 6 8 VEGFA (RQ/18S) OPG * * Un Ctrl FAS MAb 0.0 0.5 1.0 1.5 2.0 PDGFRA (RQ/18S) OPG a f e 0.5 1.0 1.5 2.0 Fold activation caspase 3/7 OPG FasAb FasL FasL and OPG TRAIL TRAIL and OPG 0 20 40 60 80 100 200 250 300 % migration to OPG PDGF OPG OPG + FasAb (0.5 nM) OPG + FasAb (1 nM) g SFM PDGF OPG 0 20 40 60 80 100 120 % Proliferation (normalised to PDGF) NoAb FAS Ab TRAIL Ab Fas and TRAIL Abs h d c b ** Fig. 5 OPG-Fas interaction mediates the OPG-induced phenotypic response of PASMC. TaqMan expression of (a) VEGFA, (b) PDGFRA, (c) TNC, (d) Cav1 and (e) TRAIL in response to OPG in the presence (hash bars) or absence (Grey bars) of anti-Fas neutralising antibody (1500 ng ml−1). Panel (f) demonstrates OPG inhibition of FasL and TRAIL-induced apoptosis in HT1080 cells. g PASMC migration following 6 h stimulation with PDGF (20 ng ml−1), OPG (30 ng ml−1) or 0.2% FCS (serum-free media, SFM), in the presence or absence of Fas neutralising antibody. h Proliferation of PASMCs following stimulation with OPG for 72 h in the presence or absence of Fas neutralising antibody and/or TRAIL neutralising antibody (0.5 nM). Proliferation expressed as a percentage of proliferation to PDGF. Bars represent the mean with error bars showing the standard error of the mean. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 g PASMC migration following 6 h stimulation with PDGF (20 ng ml−1), OPG (30 ng ml−1) or 0.2% FCS (serum-free media, SFM), in the presence or absence of Fas neutralising antibody. h Proliferation of PASMCs following stimulation with OPG for 72 h in the presence or absence of Fas neutralising antibody and/or TRAIL neutralising antibody (0.5 nM). Proliferation expressed as a percentage of proliferation to PDGF. Bars represent the mean with error bars showing the standard error of the mean. Dots represent experimental repeats, Panels (a–e) (n = 4), panel (f) (n = 3), panel (g) (n = 4), panel (h) (n = 4 for SFM, 10 for PDGF & OPG stimulations) * p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001 following one-way ANOVA with Bonferroni’s multiple comparisons post hoc test s the OPG-induced phenotypic response of PASMC. TaqMan expression of (a) VEGFA, (b) PDGFRA, (c) TNC, (d) Cav1 ( ) p p ( ) ( y ) g y ( g ) ( ) demonstrates OPG inhibition of FasL and TRAIL-induced apoptosis in HT1080 cells. g PASMC migration following 6 h stimulation with PDGF (20 ng ml−1), OPG (30 ng ml−1) or 0.2% FCS (serum-free media, SFM), in the presence or absence of Fas neutralising antibody. h Proliferation of PASMCs following stimulation with OPG for 72 h in the presence or absence of Fas neutralising antibody and/or TRAIL neutralising antibody (0.5 nM). Proliferation expressed as a percentage of proliferation to PDGF. Bars represent the mean with error bars showing the standard error of the mean. Dots represent experimental repeats, Panels (a–e) (n = 4), panel (f) (n = 3), panel (g) (n = 4), panel (h) (n = 4 for SFM, 10 for PDGF & OPG stimulations) * p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001 following one-way ANOVA with Bonferroni’s multiple comparisons post hoc test NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 We provide a strong body of evidence with concordant data that OPG is a key driver in the pulmonary vascular remodelling in PAH, thereby validating it as a ther- apeutic target. It seems likely that Ky3 might be useful as an adjunct therapy alongside existing treatments that target vasoconstriction and we are currently exploring the potential for translation of this human therapeutic anti-OPG antibody to clinical studies in PAH. 9 ARTICLE treatments with the human monoclonal anti-OPG antibody or control IgG4 isotype were performed (3 mg kg−1 per week, i.p.) alone or in combination with sildenaﬁl (50 mg kg−1 per day) or bosentan (60 mg kg−1 per day) in chow from weeks 6 with animals sacriﬁced at week 9. / / NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 ion of a diverse panel of high afﬁnity anti-human OPG monoclonal anti- Various immunisation regimens, including conventional intraperitoneal ns as well as a rapid immunisation at multiple sites (RIMMS) regimes were using recombinant human or rat OPG mature peptide sequences fused to IgG-Fc domains expressed in CHO cells (Supplementary Figure 4). At the each regime, secondary lymphoid tissue such as the spleen, and in some he lymph nodes were removed Tissues were prepared into a single cell hybridoma cell lines. A number of human and mouse OPG cross-reacti bodies were grouped by their neutralisation proﬁles and varying ability to interaction of OPG with TRAIL and RANKL were identiﬁed following assessment of hybridoma supernatants in a sequential primary and seco screen cascade using HTRF® (Homogeneous Time-Resolved Fluorescenc see Supplementary Methods) and label-free surface plasmon resonance Selected leads were produced in larger quantity in suspension CHO cell a c e f d b Mct 60 mg/kg Week 60 40 lgG4-PE (μg ml–1) RVSP (mmHg) 20 100 80 60 40 20 0 0 3 mg kg–1 antibody Catheter /harvest 0 1 3 2 Week 0 Week 1 Week 2 Week 3 lgG4 Ky1 Ky2 Ky3 Ky4 Ctrl Mct AF IgG4 Ky1 Ky2 Ky3 Ky4 Ctrl Mct AF IgG4 Ky1 Ky2 Ky3 Ky4 Ctrl Mct AF IgG4 Ky1 Ky2 Ky3 Ky4 Ctrl Control Mct No Rx Mct Ctrl Ab Mct KymAb 3 α SMA Mct AF IgG4 vWF Ky1 Ky2 Ky3 Ky4 Ctrl Mct IgG4 Ky3 Ctrl Mct IgG4 Ky3 Week 0 Week 1 Week 2 Week 3 0.0 0.2 0.4 0.6 0.8 1.0 Media/CSA <50 μm 60 80 100 120 140 LVESP (mmHg) 100 0 200 300 400 ePVRi (mmHg RVU–1 min–1 g–1) 0.0 0.2 0.4 0.6 RVH (RV/LV+S) g h j i 0 20 40 60 80 100 10,000 8000 6000 4000 2000 0 120 % Muscularised Plasma OPG (pg ml–1) lgG4 Ky4 Ky3 Ky2 Ky1 AF459 Non-Musc. Musc. Methods A i l l For time course experiments animals were sacriﬁced at days 7, 14, 21 and 28. Preventative treatments with the neutralising goat polyclonal anti-OPG antibody (AF459, R&D Systems, Minneapolis, MN, USA) or control IgG isotype (AF6775, R&D systems) were administered via an Alzet 2002 mini-pump (200 μl reservoir, 0.5 μl h−1 for 2 weeks from day 0); preventative treatments with the human monoclonal anti-OPG antibody or control IgG4 isotype were performed (3 mg kg−1, i.p.) at day 0, 7 and 14 with the animals sacriﬁced at day 21. Therapeutic intervention was performed at day 21 and day 28 (3 mg kg−1, i.p.) and animals sacriﬁced at day 35. PAH was induced in male Wistar (Charles River, UK) rats of 200–220 g by a single subcutaneous injection of Sugen5416 (Tocris, Bristol, UK) at 20 mg kg−1 followed by housing in hypobaric chambers at an equivalent of 18,000 ft for 3 weeks, followed by normobaric pressures for remaining 6 weeks. Therapeutic Human antibody generation. KyMouse™system of genetically engineered mice containing a large number of human immunoglobulin genes28 was used for the 10 NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 RE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 ART a Mct 60 mg/kg Week 3 mg kg–1 antibody Catheter /harvest 0 1 3 2 b 60 40 lgG4-PE (μg ml–1) 20 0 Week 0 Week 1 Week 2 Week 3 lgG4 Ky1 Ky2 Ky3 Ky4 b c RVSP (mmHg) 100 80 60 40 20 0 Ctrl Mct AF IgG4 Ky1 Ky2 Ky3 Ky4 d Ctrl Mct AF IgG4 Ky1 Ky2 Ky3 Ky4 0.0 0.2 0.4 0.6 RVH (RV/LV+S) d d c c f Ctrl Mct AF IgG4 Ky1 Ky2 Ky3 Ky4 60 80 100 120 140 LVESP (mmHg) e Ctrl Mct AF IgG4 Ky1 Ky2 Ky3 Ky4 100 0 200 300 400 ePVRi (mmHg RVU–1 min–1 g–1) f e Ctrl Mct IgG4 Ky3 0.0 0.2 0.4 0.6 0.8 1.0 Media/CSA <50 μm g Control Mct No Rx Mct Ctrl Ab Mct KymAb 3 α SMA vWF i i g Ctrl Mct IgG4 Ky3 h 0 20 40 60 80 100 120 % Muscularised Non-Musc. Musc. h Week 0 Week 1 Week 2 Week 3 j 10,000 8000 6000 4000 2000 0 Plasma OPG (pg ml–1) lgG4 Ky4 Ky3 Ky2 Ky1 AF459 j Week 0 Week 1 Week 2 Week 3 hybridoma cell lines. A number of human and mouse OPG cross-reactive anti- bodies were grouped by their neutralisation proﬁles and varying ability to block the interaction of OPG with TRAIL and RANKL were identiﬁed following the assessment of hybridoma supernatants in a sequential primary and secondary screen cascade using HTRF® (Homogeneous Time-Resolved Fluorescence — see Supplementary Methods) and label-free surface plasmon resonance (SPR). Selected leads were produced in larger quantity in suspension CHO cells and puriﬁed as fully human IgG4 PE (human IgG4 Fc region with mutated to amino generation of a diverse panel of high afﬁnity anti-human OPG monoclonal anti- bodies. Various immunisation regimens, including conventional intraperitoneal injections as well as a rapid immunisation at multiple sites (RIMMS) regimes were set up using recombinant human or rat OPG mature peptide sequences fused to human IgG-Fc domains expressed in CHO cells (Supplementary Figure 4). At the end of each regime, secondary lymphoid tissue such as the spleen, and in some cases, the lymph nodes were removed. Tissues were prepared into a single cell suspension and fused with SP2/0 cells by electrofusion to generate stable hybridoma cell lines. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 EmPAP was derived from RVSP, by substituting systolic PAP for RVSP, to give [EmPAP = (0.61 x RVSP) + 2 mmHg]52. EmPAP was then used in place of mean PAP in the PVRi equation shown above 12. The animals were then humanely killed under anaesthesia and tissues harvested for analysis described below12,26. acids P and E at residues S228 and L235 (EU index) to stabilise the hinge region and remove residual antibody-dependent cell-mediated cytotoxicity) and assessed in in vitro and in vivo studies. The anti-OPG antibodies, KY1–KY4, described in this manuscript are corporate assets, protected by various patents and, as such, are only available through licensing or an MTA, the terms of which will be agreed on a case-by-case basis. right internal carotid artery and right external jugular vein. Pressure volume measurements were collected using the following catheters: PVR-1045 1F (mouse LV), PVR-1030 1F (mouse RV), SPR-838 2F (rat LV) and SPR-847 1.4F (rat RV; Millar Inc.), coupled to a Millar MPVS Ultra and PowerLab 8/30 data acquisition system (AD Instruments Ltd, Oxford, UK). Data were recorded using LabChart v7 software (AD Instruments Ltd) and analysed using PVAN v2.3 (Millar, Houston, TX, USA). Estimated pulmonary vascular resistance (ePVRi) was calculated using the equation (estimated mean pulmonary artery pressure(EmPAP) — left ventricular end — diastolic pressure (LVEDP)/cardiac index)51. EmPAP was derived from RVSP, by substituting systolic PAP for RVSP, to give [EmPAP = (0.61 x RVSP) + 2 mmHg]52. EmPAP was then used in place of mean PAP in the PVRi equation shown above 12. The animals were then humanely killed under anaesthesia and tissues harvested for analysis described below12,26. Pulmonary hypertension phenotyping. Operators were blinded to treatment groups through the collection and analysis of phenotype data. Echocardiography was performed using the Vevo 770 system (VisualSonics, Toronto, Canada) using either the RMV707B (mice) or RMV710B (rat) scan head. Rectal temperature, heart rate and respiratory rate were recorded continuously throughout the study. Anaesthesia was induced and maintained using isoﬂurane, sustaining heart rates at 450–500 (mice) and 325–350 (rats) beats per minute (bpm). Rodents were depi- lated and pre-heated ultrasound gel applied (Aquasonics 100 Gel, Parker Labs Inc., Fairﬁeld, NJ). Right ventricle free wall parameters were collected using M-mode from the right parasternal long axis view. Standard left ventricle parameters were determined using two-dimensional, M-mode and Doppler pulse wave in the short axis view at the level of the papillary muscles. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Fig. 6 Human anti-OPG antibody attenuates monocrotaline-induced PAH in rats. Panel (a) shows the schema for disease initiation and treatment time course. b Plasma concentrations of antibody and IgG. Bar graphs show (c) right ventricular systolic pressure (RVSP), (d) right ventricular hypertrophy (RVH), (e) estimated pulmonary vascular resistance (ePVRi), (f) left ventricular end-systolic pressure (LVESP), (g) the degree of medial wall thickness as a ratio of total vessel size (Media/CSA), (h) relative percentage of muscularised small pulmonary arteries and arterioles in <50 µm vessels. Panel (i) shows representative photomicrographs of serial lung sections. Sections were immunostained for α-smooth muscle actin (α-SMA), or von Willebrand factor (vWF). Panel (j) shows the circulating plasma levels of OPG. Box and Whisker plots represent the interquartile range (box) with the line representing the median and whisker the full range of the data, each animal is represented by a dot. Ctrl boxes (white, n = 4), Mct (blue n = 5), AF459 (purple, n = 6), IgG (grey, n = 8), Ky1 (yellow, n = 8), Ky2 (orange, n = 7), Ky3 (green, n = 8) and Ky4 (red, n = 7). * p < 0.05, p < 0.01, * p < 0.001 compared to IgG treated rats following one-way ANOVA followed by Bonferroni’s multiple comparisons test. All images are presented at their original magniﬁcation ×400, scale bar represents 100 µm 0.2%FCS OPG OPG and IgG4 OPG and Ky3 –2 0 2 4 6 NF-κβ activation * * 0 1 2 3 4 Fold Inc. migration OPG * NTsi FASsi IgG4 Ky3 – + – – – + + – – – + + + – – + + – + – + + – – + SFM PDGF OPG –25 0 25 50 75 100 125 % Proliferation (normalised to PDGF) IgG4 Ky3 * b a c * Fig. 7 Ky3 blocks OPG-induced proliferation, migration and NF-κβ activation. Box and whisker plots shows the inhibition of OPG-induced proliferation (a) and migration (b) in PASMC stimulated with serum-free media (SFM), PDGF or OPG in the presence of either IgG4 (grey) or Ky3 antibody (green), Fas siRNA (yellow) or non-targeting siRNA (NTsi) (white). Bar graph shows the mean with the error bars showing the standard error o the mean with (c) showing the activation of NF-κβ in response to OPG (blue) in the presence of either IgG4 (grey) or Ky3 antibody (green). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 A number of human and mouse OPG cross-reactive anti- bodies were grouped by their neutralisation proﬁles and varying ability to block the interaction of OPG with TRAIL and RANKL were identiﬁed following the assessment of hybridoma supernatants in a sequential primary and secondary screen cascade using HTRF® (Homogeneous Time-Resolved Fluorescence — see Supplementary Methods) and label-free surface plasmon resonance (SPR). Selected leads were produced in larger quantity in suspension CHO cells and puriﬁed as fully human IgG4 PE (human IgG4 Fc region with mutated to amino generation of a diverse panel of high afﬁnity anti-human OPG monoclonal anti- bodies. Various immunisation regimens, including conventional intraperitoneal injections as well as a rapid immunisation at multiple sites (RIMMS) regimes were set up using recombinant human or rat OPG mature peptide sequences fused to human IgG-Fc domains expressed in CHO cells (Supplementary Figure 4). At the end of each regime, secondary lymphoid tissue such as the spleen, and in some cases, the lymph nodes were removed. Tissues were prepared into a single cell suspension and fused with SP2/0 cells by electrofusion to generate stable 11 NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Box and Whisker plots represent the interquartile range (box) with the line representing the median and whisker the full range of the data, each dot represents an experimental repeat, n = 6 (a), n = 5 (b) and n = 4 (c), * p < 0.05 following two-way ANOVA followed by Sidak’s multiple comparisons test (a), or one-way ANOVA with Bonferroni’s multiple comparisons post hoc test (b&c) SFM PDGF OPG –25 0 25 50 75 100 125 % Proliferation (normalised to PDGF) IgG4 Ky3 * a 0 1 2 3 4 Fold Inc. migration * NTsi + + + + + SFM PDGF OPG –25 0 25 50 75 100 125 % Proliferation (normalised to PDGF) IgG4 Ky3 * b a * a 0.2%FCS OPG OPG and IgG4 OPG and Ky3 –2 0 2 4 6 NF-κβ activation * * c Fig. 7 Ky3 blocks OPG-induced proliferation, migration and NF-κβ activation. Box and whisker plots shows the inhibition of OPG-induced proliferation (a) and migration (b) in PASMC stimulated with serum-free media (SFM), PDGF or OPG in the presence of either IgG4 (grey) or Ky3 antibody (green), Fas siRNA (yellow) or non-targeting siRNA (NTsi) (white). Bar graph shows the mean with the error bars showing the standard error o the mean with (c) showing the activation of NF-κβ in response to OPG (blue) in the presence of either IgG4 (grey) or Ky3 antibody (green). Box and Whisker plots represent the interquartile range (box) with the line representing the median and whisker the full range of the data, each dot represents an experimental repeat, n = 6 (a), n = 5 (b) and n = 4 (c), * p < 0.05 following two-way ANOVA followed by Sidak’s multiple comparisons test (a), or one-way ANOVA with Bonferroni’s multiple comparisons post hoc test (b&c) right internal carotid artery and right external jugular vein. Pressure volume measurements were collected using the following catheters: PVR-1045 1F (mouse LV), PVR-1030 1F (mouse RV), SPR-838 2F (rat LV) and SPR-847 1.4F (rat RV; Millar Inc.), coupled to a Millar MPVS Ultra and PowerLab 8/30 data acquisition system (AD Instruments Ltd, Oxford, UK). Data were recorded using LabChart v7 software (AD Instruments Ltd) and analysed using PVAN v2.3 (Millar, Houston, TX, USA). Estimated pulmonary vascular resistance (ePVRi) was calculated using the equation (estimated mean pulmonary artery pressure(EmPAP) — left ventricular end — diastolic pressure (LVEDP)/cardiac index)51. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Non-Musc * n/s * Catheter /harvest 0 3 6 9 8 7 Su5416 20 mg kg–1 Echo Echo Hypoxia 3 mg kg–1 antibody Normoxia Week 10 15 20 25 30 Percentage (BV by TV) ** n/s Scale bar = 20 μm Week 6 0 1000 2000 3000 4000 Plasma OPG (pg ml–1) IgG4 Ky3 SuHx Sildenafil Control * * * # 0 2 4 6 8 10 Trabecular number (Tb.N) * n/s * * 0.025 0.030 0.035 0.040 0.045 Trabecular thickness (Tb.Th) * n/s * * Echo Week 9 Week 8 Week 7 Week 6 Week 9 Week 8 Week 7 a Catheter /harvest 0 3 6 9 8 7 Su5416 20 mg kg–1 Echo Echo Hypoxia 3 mg kg–1 antibody Normoxia Week Echo b 0 25 50 75 100 125 IgG4-PE (μg ml–1) IgG4 Ky3 Week 6 Week 9 Week 8 Week 7 b c Week 0 Week 6 SuHx Sil IgG4 Ky3 0 10 20 30 PA AT (s) Week 9 c Week 9 d Week 0 Week 6 SuHx Sil IgG4 Ky3 0 50 100 150 200 CO (ml min–1) Week 9 * f Ctrl SuHx Sil IgG4 Ky3 0 10 20 30 40 50 RV Ea (mmHg RVU–1) * n/s * e 0 25 50 75 100 125 RVSP (mmHg) * * Ctrl SuHx Sil IgG4 Ky3 d f ) d e h Ctrl SuHx Sil IgG4 Ky3 0.0 0.2 0.4 0.6 0.8 RVH (RV/LV+S) ** * i Ctrl SuHx Sil IgG4 Ky3 0 50 100 150 LVESP (mmHg) * i g Ctrl SuHx Sil IgG4 Ky3 0 200 400 600 800 1000 ePVRi (mmHg RVU–1 min–1 g–1) n/s h g 1 l Ctrl SuHx Sil IgG4 Ky3 Ctrl SuHx Sil IgG4 ABEVG Ctrl SuHx SuHx and lgG4 SuHx and KymAb3 SuHx and Sidenafil SMA vWF PCNA CASPASE 3 Ky3 Scale bar = 20 μm j j Ctrl SuHx Sil IgG4 Ky3 0.0 0.2 0.4 0.6 0.8 Media/CSA * * n/s l k Ctrl SuHx Sil IgG4 Ky3 0 20 40 60 80 100 120 % Muscularised Musc. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Cardiac output (CO) was derived from ﬂow and annulus diameter at the outﬂow tract and aortic valve junction, then normalised by body weight. Analysis was performed using Vevo 770 software (v3.0, VisualSonics). All measurements were made during the relevant cardiac cycle phase, avoiding inspiration artefact12,16. Right ventricular hypertrophy. Right ventricular hypertrophy (RVH) was mea- sured by calculating the ratio of the right ventricular free wall weight over left ventricle plus septum weight. Immunohistochemistry. Immediately after harvest, the left lung was perfusion ﬁxed via the trachea with 10% (v/v) formalin buffered saline by inﬂation to 20 cm of H2O. The lungs were then processed into parafﬁn blocks for sectioning. Parafﬁn embedded sections (5 μm) of mouse and rat lung were histologically stained for Alcian Blue Elastic van Gieson (ABEVG) and immunohistochemically stained for α-smooth muscle actin (α-SMA (1:150), M0851, Dako (Agilent), Santa Clara, CA, Following echocardiography and under isoﬂurane-induced anaesthesia, left and right ventricular catheterisation was performed using a closed chest method via the 12 NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Will b d f t ( WF (1 300) A0082 D k ) F4/80 ((1 100) tib d (1 200) dd d b f idi bi ti l a b c d e f g j l k m o p n h i 0 25 50 75 100 125 IgG4-PE (μg ml–1) IgG4 Ky3 Week 0 Week 6 SuHx Sil IgG4 Ky3 Week 0 Week 6 SuHx Sil IgG4 Ky3 0 10 20 30 PA AT (s) Week 9 0 25 50 75 100 125 RVSP (mmHg) * * 0 50 100 150 200 CO (ml min–1) Week 9 * Ctrl SuHx Sil IgG4 Ky3 Ctrl SuHx Sil IgG4 Ky3 Ctrl SuHx Sil IgG4 Ky3 Ctrl SuHx Sil IgG4 ABEVG Ctrl SuHx SuHx and lgG4 SuHx and KymAb3 SuHx and Sidenafil SMA vWF PCNA CASPASE 3 Ky3 Ctrl SuHx Sil IgG4 Ky3 Ctrl SuHx Sil IgG4 Ky3 Ctrl SuHx Sil IgG4 Ky3 Ctrl SuHx Sil IgG4 Ky3 Ctrl SuHx Sil IgG4 Ky3 Ctrl SuHx Sil IgG4 Ky3 0 10 20 30 40 50 RV Ea (mmHg RVU–1) * n/s * 0.0 0.2 0.4 0.6 0.8 RVH (RV/LV+S) * 0 50 100 150 LVESP (mmHg) * 0 200 400 600 800 1000 ePVRi (mmHg RVU–1 min–1 g–1) n/s 0.0 0.2 0.4 0.6 0.8 Media/CSA * *** n/s 0 20 40 60 80 100 120 % Muscularised Musc. ARTICLE The degree of muscularisation was also determined for each group, and given as the area of positive α-smooth muscle actin staining in the vessel media divided by the total vessel cross-sectional area (media/CSA)12. Retrogenix cell microarray. Identiﬁcation of OPG human protein binding part- ners was performed using the Retrogenix Cell Microarray (Shefﬁeld, UK). Optimal binding conditions were ﬁrst established using syndecan-1 (positive control) and TREM-1 (negative control). HEK293 cells were reverse transfected with expression vectors consisting of one of 2505 human plasma membrane proteins. Cells were treated with 0.5 μg ml−1 rhOPG (Peprotech, London, UK), 0.5 μg ml−1 anti-OPG (Peprotech) followed by Alexaﬂuor647 anti-goat antibody. Fluorescent images were analysed and quantiﬁed using the ImageQuant software (GE) (http://www. retrogenix.com/default.asp). Quantiﬁcation of bone structure by microCT. Femora were scanned on a Sky- scan microCT scanner (1172a, Bruker, Belgium) at 50 kV and 200 μA using a 0.5 mm aluminium ﬁlter and a detection pixel size of 4.3 μm. Images were captured every 0.7° through 180° rotation and 2x averaging of each bone. Scanned images were reconstructed using Skyscan NRecon software (v. 1.6.8.0) and datasets ana- lysed using Skyscan CT analysis software (v. 1.13.2.1). Trabecular bone was mea- sured over a 1 mm³ volume, 0.2 mm from the growth plate. Trabecular bone volume as a proportion of tissue volume (BV/TV, %), trabecular thickness (Tb. Th, mm), trabecular number (Tb. N, mm−1) and trabecular structure model index (SMI) were assessed in this area. Cortical bone was measured over a 1 mm³ volume, 1 mm from the growth plate, and cortical bone volume (C. BV, mm³) assessed in this area. Co-immunoprecipitation. PASMCs were stimulated with rhOPG (500 ng ml−1) for 30 min at 37 °C. After stimulation, cells were lysed and the protein lysate concentration determined by a Pierce 660 nm protein assay. Co- immunoprecipitation was then performed using an anti-Fas or Ky3 antibody with human PASMC lysate and recombinant proteins, alongside negative controls, where antibodies were not added. ProteinG sepharose 4 Fast Flow beads (50% slurry) were added to each Co-IP reaction and immune complexes were pre- cipitated. Each Co-IP reaction was then centrifuged and the pellet washed before re-suspending in sample reducing agent (NuPAGE, Life Sciences) with 5% v/v SDS and heating at 95°C. The supernatant was then analysed by western blotting. ARTICLE Phosphorylation targets were identiﬁed from protein lysates by Kinex antibody microarray (Kinexus, Vancouver, Canada). A Z-ratio of ± 1.5 was deemed signiﬁcant. Uniprot accession codes of proteins were analysed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation to generate fold enrichment pathway analysis through the KEGG Pathway Database. Immunoﬂuorescent staining. Lung tissue was obtained from chronically hypoxic neonatal calves and normoxic age-matched controls. This neonatal calf model of severe hypoxic pulmonary hypertension has been described previously53 and includes the development of PA pressure equal to, or exceeding, systemic pressure as well as remarkable PA remodelling with medial and adventitial thickening, resembling that of human neonatal PH. Indirect immunostaining was performed with rabbit polyclonal anti-OPG antibodies ((1:500), Bioss Antibodies, Woburn, MA, USA) followed by biotin-conjugated anti-rabbit secondary antibody ((1:100), Vector Laboratories) and Streptavidin-Alexa-488 ((1:200), Invitrogen, Carlsbad, CA, USA). Western blotting. PASMCs were stimulated with rhOPG (50 ng ml−1) (R&D systems), alongside quiesced cells (negative control) for 10 and 60 min, before lysing. Cell lysates were mixed with sample buffer (Life Technologies, Carlsbad, CA, USA) and sample reducing agent (Life Technologies), denatured by heating and subjected to gel electrophoresis. The membranes were then incubated with primary antibodies against phospho-CDK4, phospho-HSP27, total mTOR, phospho-mTOR (1:500) and GAPDH (1:1000) (Cell Signalling Technology), CDK5 (1:500) (Abcam), or β-actin (1:1000) (Santa Cruz Biotechnology, Heidel- berg, Germany). Membranes were then incubated with anti-Rabbit IRDye 800CW and anti-Mouse IRDye 800CW (Li-COR, Lincoln, NE, USA) and signal detection and band density quantiﬁcation was performed using the LiCOR Odyssey SA system. Quantiﬁcation of pulmonary vascular remodelling. Images of stained sections were captured using a Zeiss Imager Z2 microscope with an Axiocam 506 colour (brightﬁeld) or MRm (ﬂuorescence) camera with HXP 120 V light source (Carl Zeiss, Oberkochen, Germany). Zen 2 software (Carl Zeiss) was used for image analysis. Pulmonary vascular remodelling was quantiﬁed by assessing the degree of muscularisation and the percentage of affected pulmonary arteries and arterioles. For each lung, pulmonary arteries were categorised as either muscularised (i.e. with crescent or complete rings of muscle) or non-muscularised (no apparent muscle) on ABEVG stained sections. Vessels were also divided into sub-groups determined by their external diameter: <50 μm for small arterioles and, additionally where stated, 51–100 and >100 μm for medium arteries. The proportion of muscularised vessels within each sub-group was calculated as a percentage of the total number of vessels. ARTICLE Fig. 8 Therapeutic delivery of Ky3 attenuates development of established severe SuHx PAH. Panel (a) shows the schema for disease initiation and treatment time course. b Plasma concentrations of antibody and IgG. Bar graphs show (c) Pulmonary Artery Acceleration Time (PA AT), (d) cardiac output, (e) right ventricular systolic pressure (RVSP), (f) right ventricular arterial elastance (RV Ea), (g) estimated pulmonary vascular resistance (ePVRi), (h) right ventricular hypertrophy (RVH), (i) left ventricular end-systolic pressure (LVESP). Bar graphs (j) show the degree of medial wall thickness as a ratio of total vessel size (Media/CSA) and (k) the relative percentage of muscularised small pulmonary arteries and arterioles in < 50 µm vessels. Panel (l) shows representative photomicrographs of serial lung sections. Sections were stained for Alcian Blue Elastic van Gieson (ABEVG), immunostained for α-smooth muscle actin (α-SMA), or von Willebrand factor (vWF), proliferating cell nuclear antigen (PCNA) or cleaved Caspase 3. Panel (m) shows the circulating level of OPG and quantiﬁcation of femoral trabecular bone volume (%) (n), trabecular thickness (mm) (o), trabecular number (mm−1) (p). Box and Whisker plots represent the interquartile range (box) with the line representing the median and whisker the full range of the data, each animal is represented by a dot, white boxes represent control (n = 8), blue (SuHx, n = 8), yellow (Sildenaﬁl treated, n = 7), grey (IgG4 treated, n = 8) and green (Ky3 treated, n = 8) rats. * p < 0.05, p < 0.01, * p < 0.001 compared to IgG treated rats following one-way ANOVA with Tukey’s multiple comparisons post hoc test. All images are presented at their original magniﬁcation ×400, scale bar represents 20 µm Proliferation was assessed after 72 h using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Southampton, UK). Proliferation was assessed after 72 h using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Southampton, UK). USA). Human pulmonary artery and right ventricle histology sections were obtained from patients with IPAH and control lung resection patients from Pap- worth Hospital (Cambridge, UK) tissue bank and immunohistochemically stained for Fas ((1:100), ADI-AMM-227-E, Enzo Life Sciences, Exeter, UK) and IL-1RAcP ((1:1000), ab8110, Abcam). Kinex antibody microarray (KAM). PASMCs were synchronised with growth arrest media (DMEM, 0.2% FBS, penicillin and streptomycin) for 48 h prior to stimulation. Cells were then stimulated with 0.2% (v/v) FBS (negative), rhOPG (50 ng ml−1) and PDGF (20 ng ml−1) for 10 and 60 min. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Non-Musc * n/s * k n Ctrl SuHx Sil IgG4 Ky3 10 15 20 25 30 Percentage (BV by TV) ** n/s m Week 6 0 1000 2000 3000 4000 Plasma OPG (pg ml–1) IgG4 Ky3 SuHx Sildenafil Control * * * # Week 9 Week 8 Week 7 n m p Ctrl SuHx Sil IgG4 Ky3 0 2 4 6 8 10 Trabecular number (Tb.N) * n/s * * o Ctrl SuHx Sil IgG4 Ky3 0.025 0.030 0.035 0.040 0.045 Trabecular thickness (Tb.Th) * n/s * * W p o USA); von Willebrand factor (vWF (1:300), A0082, Dako); F4/80 ((1:100), ab111101, Abcam, Cambridge, UK); interleukin-6 (IL-6 (1:15), ab6672, Abcam); OPG ((1:50), ab73400, Abcam); TRAIL ((1:100), ab231063, Abcam); Fas ((1:500), ab133619, Abcam) and IκBα ((1:100), ab32518, Abcam). To assess proliferation, slides were stained with a mouse anti-human proliferating cell nuclear antigen antibody (PCNA (1:125), M0879, Dako). In each case a biotinylated secondary USA); von Willebrand factor (vWF (1:300), A0082, Dako); F4/80 ((1:100), ab111101, Abcam, Cambridge, UK); interleukin-6 (IL-6 (1:15), ab6672, Abcam); OPG ((1:50), ab73400, Abcam); TRAIL ((1:100), ab231063, Abcam); Fas ((1:500), ab133619, Abcam) and IκBα ((1:100), ab32518, Abcam). To assess proliferation, slides were stained with a mouse anti-human proliferating cell nuclear antigen antibody (PCNA (1:125), M0879, Dako). In each case a biotinylated secondary USA); von Willebrand factor (vWF (1:300), A0082, Dako); F4/80 ((1:100), antibody (1:200) was added before an avidin-biotin enzyme complex (Vectastain® Kit, Vector Laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine tet- rahydrochloride (DAB) substrate. Apoptotic nuclei were detected with a TUNEL assay using a colorimetric DNA fragmentation detection kit (fragEL™, QIA33, Calbiochem®, Merck, Burlington, MA, USA)12,26, or stained immunohistochemi- cally for cleaved caspase 3 ((1:50), 9661, Cell Signalling Technology, Danvers, MA, TURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications 13 ARTICLE Boxplots demonstrate a signiﬁcant reduction in whole lung expression of OPG RNA (a), OPG Protein (b), TRAIL RNA (c), TRAIL protein (d) and IL-6 RNA (e) and plasma protein (f). Panel (g) shows representative photomicrographs of serial lung sections. Sections were stained for macrophages (F4/80), OPG, TRAIL, IL-6 and Iκβα. Box and Whisker plots represent the interquartile range (box) with the line representing the median and whisker the full range of the data, each animal is represented by a dot (Ctrl (white) n = 7, SuHx (blue) n = 5, IgG (grey) n = 7 & Ky3 (green) n = 12 animals per group). * p < 0.05, ** p < 0.01, compared to IgG treated rats using one-way ANOVA followed by Sidak’s multiple comparisons test. All images are presented at their original magniﬁcation ×400, scale bar represents 20 µm with a measurement of the perturbation in a pathway to identify signalling net- works that are relevant in a given dataset. Full gene expression data (not ﬁltered for PAH relevant genes) were analysed (BH adjusted p-value < 0.01) using the SPIA package25 in R to identify KEGG Pathways55–57 regulated by OPG. with a measurement of the perturbation in a pathway to identify signalling net- works that are relevant in a given dataset. Full gene expression data (not ﬁltered for PAH relevant genes) were analysed (BH adjusted p-value < 0.01) using the SPIA package25 in R to identify KEGG Pathways55–57 regulated by OPG. Agilent RNA microarray. mRNA expression proﬁling was performed using the SurePrint G3 Human Gene Expression 8 × 60 K v2 Microarray according to the manufacturer’s instructions (Agilent Technologies, UK). Human PASMCs (Lonza) were stimulated in triplicate with 0.2% FCS (control) or 50 ng ml−1 OPG (Peprotech). RNA samples (200 ng) from each condition were labelled and hybridised using standard Agilent protocols. Sample array matrices were scanned on an Agilent Technologies Scanner G2505C using Feature Extraction Software (Agilent Technologies). Loess normalisation and data analysis was performed using the Linear Models for Microarray Data (LIMMA) package54 in R (http://www.r- project.org/). Data were analysed by two means. (1) A Medline (PubMed) search using term ‘pulmonary hypertension’ was used to compile a curated list of disease- relevant genes (Supplemental table 5) (39). This list was used to identify PAH related genes differentially regulated in PASMCs between OPG and control sam- ples (BH adjusted p-value < 0.05 and log2 FC > 1.2). ARTICLE Membranes were incubated with goat polyclonal anti-OPG antibody (1:1000) (SC8468, Santa Cruz Biotechnology) or anti-Fas antibody (MA1–7622, Invitrogen) and IRDye 680LT Donkey anti-goat secondary antibody (1:15000) or IRDye 800CW donkey anti-mouse secondary antibody (1:15000) (Li-COR) to detect co- immunoprecipitated OPG. Membranes were scanned using the Li-COR Odyssey Sa system (LiCOR). Cell culture. Prior to experimentation, human PASMCs (CC2581; Lonza, Basel, Switzerland) were sub-cultured in SmBM containing SmGM-2 SingleQuot™Kit supplements and growth factors (Lonza) containing penicillin and streptomycin at 37 °C (5% CO2). Cells were synchronised with growth arrest media (DMEM, 0.2% FBS, penicillin and streptomycin) for 48 h prior to stimulation. All experimentation was conducted at 37 °C with 5% CO2 with cells aged between passage 4–7. Proliferation assay. PASMCs were seeded into 96 well plates (0.5 × 104 cells per well) and allowed to adhere for 24 h (37 °C, 5% CO2). Cells were then synchronised with growth arrest media (DMEM, 0.2% FBS, penicillin and streptomycin) for 48 h prior to stimulation. PASMCs were pre-incubated with Fas neutralising antibody (1500 ng ml−1, Clone ZB4, Merck) and/or TRAIL neutralising antibody (1500 ng ml−1, Clone 75411, R&D Systems), where indicated for 30 min before stimulation with PDGF (20 ng ml−1, R&D Systems) or OPG (30 ng ml−1, R&D Systems). HT1080 apoptosis assay. HT1080 cells (CCL121; ATCC, USA) were seeded at 5 × 104 cells per ml in 96 well white walled cell culture plates in EMEM (EBSS) with 2 mM glutamine, 1% non-essential amino acids (NEAA) and 10% foetal bovine serum (FBS) (Life Sciences Ltd, UK). After 24 h, cells were stimulated with OPG 30 ng ml−1 alone, or OPG 30 ng ml−1 with 1 or 5 ng cross-linked FasL (R&D Systems), 2 nM Fas neutralising Ab (05–338, Merck) or 5 ng ml−1 TRAIL (R&D Systems). Apoptosis was measured using a Caspase 3/7 assay (G8091, Promega). ARTICLE 14 NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 A ﬁl f d h h f h b h 0 2 4 6 8 10 IL-6 RQ to 18S * 0.0 0.2 0.4 0.6 0.8 TRAIL normalised to GAPDH * Ctrl SuHx IgG4 Ky3 Ctrl SuHx IgG4 Ky3 Ctrl SuHx IgG4 Ky3 0.0 0.5 1.0 1.5 2.0 OPG normalised to GAPDH 0 1 2 3 TRAIL RQ to 18S Ctrl SuHx IgG4 Ky3 Ctrl SuHx IgG4 Ky3 Ctrl SuHx IgG4 Ky3 0 1 2 3 OPG RQ to 18S * 0 200 400 600 800 IL-6 (pg ml–1) b a d c f e g Ctrl F4/80 OPG TRAIL IL-6 Scale bar = 20 μm IκBα SuHx SuHx and IgG4 SuHx and KymAb3 tissue expression of IL-6, OPG and TRAIL. Boxplots demonstrate a signiﬁcant reduction in whole lung expression o RNA (c), TRAIL protein (d) and IL-6 RNA (e) and plasma protein (f). Panel (g) shows representative photomicro were stained for macrophages (F4/80), OPG, TRAIL, IL-6 and Iκβα. Box and Whisker plots represent the interqua ng the median and whisker the full range of the data, each animal is represented by a dot (Ctrl (white) n = 7, Su 3 (green) n = 12 animals per group). * p < 0.05, p < 0.01, compared to IgG treated rats using one-way ANOVA ons test. All images are presented at their original magniﬁcation ×400, scale bar represents 20 µm NICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Ctrl SuHx IgG4 Ky3 0.0 0.5 1.0 1.5 2.0 OPG normalised to GAPDH b Ctrl SuHx IgG4 Ky3 0 1 2 3 OPG RQ to 18S * a 0.0 0.2 0.4 0.6 0.8 TRAIL normalised to GAPDH * Ctrl SuHx IgG4 Ky3 d 0 1 2 3 TRAIL RQ to 18S ** Ctrl SuHx IgG4 Ky3 c c 0 2 4 6 8 10 IL-6 RQ to 18S * Ctrl SuHx IgG4 Ky3 e Ctrl SuHx IgG4 Ky3 0 200 400 600 800 IL-6 (pg ml–1) f f e g Ctrl F4/80 OPG TRAIL IL-6 Scale bar = 20 μm IκBα SuHx SuHx and IgG4 SuHx and KymAb3 g Scale bar = 20 μm Fig. 9 Ky3 reduces tissue expression of IL-6, OPG and TRAIL. NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunicatio ARTICLE (2) Signalling Pathway Impact Analysis (SPIA) is an unbiased method that combines over-representation analysis Taqman PCR. PASMCs were stimulated with 0.2% (v/v) FCS (control) or OPG (50 ng ml−1) alone or in the presence of Fas antibody (1500 ng ml−1) following 30 minute pre-incubation with Fas antibody. After 6 h stimulation, total RNA was extracted using the Direct-zol™RNA kit (Zymo Research, Irvine, CA, USA). Puriﬁed RNA was reverse transcribed with the High Capacity RNA-to-cDNA Kit (Life Technologies). Gene expression was measured by performing TaqMan PCR using Gene Expression MasterMix (Applied Biosystems) for, Cav-1 (Hs00971716_m1), PDGFRa (Hs00998018_m1), TNC (Hs01115665_m1), TRAIL (Hs00921974_m1, Rn0059556_m1, Mn01182929_m1), VEGFA (Hs00900055_m1), VIPR1 15 TURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Ctrl SuHx IgG4 Ky3 Sil Sil and Ky3 Bos Ky3 and Bos 0 50 100 150 RVSP (mmHg) ### p < 0.05 ### * ### # * ### Ctrl SuHx IgG4 Ky3 Sil Sil and Ky3 Bos Ky3 and Bos 0.0 0.2 0.4 0.6 0.8 1.0 RVH (RV/LV+S) # # Ctrl SuHx IgG4 Ky3 Sil Sil and Ky3 Bos Ky3 and Bos 0 ABEVG Ctrl SuHx SuHx and lgG4 SuHx and KymAb3 SuHx and Sidenafil SuHx and Bosentan SuHx and KymAb3 + Sildenafil SuHx and KymAb3 + Bosentan SMA vWF PCNA CASPASE 3 50 100 150 LVESP (mmHg) Ctrl SuHx IgG4 Ky3 Sil Sil and Ky3 Bos Ky3 and Bos 0 20 40 60 80 RV Ea (mmHg RVU–1) # # ### Week 6 Week 7 Week 8 Week 9 0 25 50 75 100 125 IgG4-PE (μg ml–1) IgG4 Ky3 Sil and Ky3 Bos and Ky3 Ctrl SuHx IgG4 Ky3 Sil Ky3 and Sil Bos Ky3 and Bos 0 20 40 60 80 100 % Muscularised ### # # # * Ctrl SuHx IgG4 Ky3 Sil Sil and Ky3 Bos Ky3 and Bos 0.0 0.2 0.4 0.6 0.8 1.0 Media/CSA #* b a d c e g h i j f ek 6 ek 7 ek 8 ek 9 0 1000 2000 3000 4000 5000 Serum OPG (pg ml–1) IgG4 Ky3 SuHx Sildenafil Scale bar = 20 μm Control * * * # Bos Ky3 and Sil Ky3 and Bos * 0 3 9 8 7 6 Su5416 20 mg kg–1 Echo Echo Hypoxia 3 mg kg–1 Antibody Normoxia Wk Catheter /Harvest +/– sildenafil +/– bosentan Echo TICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019 Ctrl SuHx IgG4 Ky3 Sil Sil and Ky3 Bos Ky3 and Bos 0 50 100 150 LVESP (mmHg) Ctrl SuHx IgG4 Ky3 Sil Sil and Ky3 Bos Ky3 and Bos 0 20 40 60 80 RV Ea (mmHg RVU–1) # # ### Week 6 Week 7 Week 8 Week 9 0 25 50 75 100 125 IgG4-PE (μg ml–1) IgG4 Ky3 Sil and Ky3 Bos and Ky3 b d f b a d c S Ky Ctrl SuHx IgG4 Ky3 Sil Sil and Ky3 Bos Ky3 and Bos 0.0 0.2 0.4 0.6 0.8 1.0 RVH (RV/LV+S) # # e e Ctrl SuHx IgG4 Ky3 Sil Sil and Ky3 Bos Ky3 and Bos 0.0 0.2 0.4 0.6 0.8 1.0 Media/CSA #* g j j j g ABEVG Ctrl SuHx SuHx and lgG4 SuHx and KymAb3 SuHx and Sidenafil SuHx and Bosentan SuHx and KymAb3 + Sildenafil SuHx and KymAb3 + Bosentan SMA vWF PCNA CASPASE 3 j Scale bar = 20 μm Ctrl SuHx IgG4 Ky3 Sil Ky3 and Sil Bos Ky3 and Bos 0 20 40 60 80 100 % Muscularised ### # # # * Sil Ky3 h h Ky3 a Ky3 a i Week 6 Week 7 Week 8 Week 9 0 1000 2000 3000 4000 5000 Serum OPG (pg ml–1) IgG4 Ky3 SuHx Sildenafil Control * * * # Bos Ky3 and Sil Ky3 and Bos * i ) (Hs00270351_m1), Fas (Hs00236330_m1, Rn00685720_m1) and OPG (Mn01205928_m1, Rn00563499_m1) on the 7900HT fast real time PCR system (Applied Biosystems). References 25. Tarca, A. L. et al. A novel signaling pathway impact analysis. Bioinformatics 25, 75–82 (2009). 1. Hoeper, M. M. et al. Deﬁnitions and diagnosis of pulmonary hypertension. J. Am. Coll. Cardiol. 62, D42–D50 (2013). 26. Rothman, A. M. K. et al. MicroRNA-140-5p and SMURF1 regulate pulmonary arterial hypertension. J. Clin. Invest. 126, 2495–2508 (2016). 2. Tuder, R. M. et al. Relevant issues in the pathology and pathobiology of pulmonary hypertension. J. Am. Coll. Cardiol. 62, D4–D12 (2013). 27. Leaman, D. W. et al. Identiﬁcation of X-linked inhibitor of apoptosis- associated factor-1 as an interferon-stimulated gene that augments TRAIL Apo2L-induced apoptosis. J. Biol. Chem. 277, 28504–28511 (2002). 3. Schermuly, R. T., Ghofrani, H. A., Wilkins, M. R. & Grimminger, F. Mechanisms of disease: pulmonary arterial hypertension. Nat. Rev. Cardiol. 8, 443–455 (2011). 28. Lee, E.-C. et al. 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ARTICLE þ 5) and informed consent was obtained from all subjects Sections of formalin- ﬁxed lung and right ventricle from patients with IPAH or unused donors were þ 5) and informed consent was obtained from all subjects Sections of formalin- ﬁxed lung and right ventricle from patients with IPAH or unused donors were stained for Fas ((1:500), ab133619, Abcam) and OPG ((1:50), ab73400. In each case a biotinylated secondary antibody (1:200) was added before an avidin-biotin enzyme complex (Vectastain® Kit, Vector Laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine tetrahydrochloride (DAB) substrate. p y yp 14. Condliffe, R. et al. Serum osteoprotegerin is increased and predicts survival in idiopathic pulmonary arterial hypertension. Pulm. Circ. 2, 21–27 (2012). 15. Jia, D. et al. Osteoprotegerin disruption attenuates HySu-induced pulmonary hypertension through integrin αvβ3/FAK/AKT pathway suppression. Circ. Cardiovasc. Genet. 10, e001591 (2017). 16. Lawrie, A. et al. 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All animal experiments were approved by the University of Shefﬁeld Project Review Committee and conformed to the UK Home Ofﬁce ethical guidelines. 19. Moran, C. S., Jose, R. J., Biros, E. & Golledge, J. Osteoprotegerin deﬁciency limits angiotensin II-induced aortic dilatation and rupture in the apolipoprotein E-knockout mouse. Arterioscler. Thromb. Vasc. Biol. 34, 2609–2616 (2014). Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 20. Lacey, D. L. et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93, 165–176 (1998). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-13139-9 Gene expression was calculated using the ΔΔCT comparative quantiﬁcation method with 18 S rRNA (Hs03003631_g1) as an endogenous control. kit (Qiagen) and Lipofectamine 2000 transfection reagent (Invitrogen) and incu- bated for 24 h (37°C, 5% CO2). Media was then renewed in the presence or absence of stimulation with OPG (30 ng ml−1, R&D Systems) with or without 1500 ng ml−1 of Ky3 or control IgG4 antibodies. Luciferase activity was detected following 48 h stimulation using Dual-Glo luciferase assay system (Promega). kit (Qiagen) and Lipofectamine 2000 transfection reagent (Invitrogen) and incu- bated for 24 h (37°C, 5% CO2). Media was then renewed in the presence or absence of stimulation with OPG (30 ng ml−1, R&D Systems) with or without 1500 ng ml−1 of Ky3 or control IgG4 antibodies. Luciferase activity was detected following 48 h stimulation using Dual-Glo luciferase assay system (Promega). (Hs00270351_m1), Fas (Hs00236330_m1, Rn00685720_m1) and OPG (Mn01205928_m1, Rn00563499_m1) on the 7900HT fast real time PCR system (Applied Biosystems). Gene expression was calculated using the ΔΔCT comparative quantiﬁcation method with 18 S rRNA (Hs03003631_g1) as an endogenous control. NF-kB activation assay. PASMCs were seeded into 96 well plates (0.5 × 104 cells per well) and allowed to adhere for 24 h (37 °C, 5% CO2). Cells were then trans- fected with 100 ng per well inducible NFkB responsive ﬁreﬂy luciferase reporter and constitutively active Renilla construct mixture using the Cignal reporter assay NF-kB activation assay. PASMCs were seeded into 96 well plates (0.5 × 104 cells per well) and allowed to adhere for 24 h (37 °C, 5% CO2). Cells were then trans- fected with 100 ng per well inducible NFkB responsive ﬁreﬂy luciferase reporter and constitutively active Renilla construct mixture using the Cignal reporter assay Human tissue. Experimental procedures using human tissues or cells conformed to the principles outlined in the Declaration of Helsinki. Papworth Hospital ethical review committee approved the use of the human tissues (Ethics Ref 08 -H0304–56 NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunicatio 16 Data availability 21. Toshner, M. et al. Evidence of dysfunction of endothelial progenitors in pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 180, 780–787 (2009). The data that support the ﬁndings of this study are available from the correspondingauthor upon reasonable request. The source data underlying Fig. 3a is available from the Gene Expression Omnibus (GEO), GSE137886. The data for all other ﬁgures are provided as a Source Data ﬁle. 22. Yeager, M. E., Frid, M. G. & Stenmark, K. R. Progenitor cells in pulmonary vascular remodeling. Pulm. Circ. 1, 3–16 (2011). 23. Stenmark, K. R., Fagan, K. A. & Frid, M. G. 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Boxplots show (c) right ventricular systolic pressure (RVSP), (d) right ventricular arterial elastance (RV Ea), (e) right ventricular hypertrophy (RVH), (f) left ventricular end-systolic pressure (LVESP), (g) degree of medial wall thickness as a ratio of total vessel size (Media/CSA) and (h) the relative percentage of muscularised small pulmonary arteries and arterioles in < 50 µm vessels. Graph (i) shows the circulating level of OPG, and panel (j) shows representative photomicrographs of serial lung sections. Sections were stained for Alcian Blue Elastic van Gieson (ABEVG), immunostained for α-smooth muscle actin (α-SMA), or von Willebrand factor (vWF), proliferating cell nuclear antigen (PCNA) or cleaved Caspase 3. Box and Whisker plots represent the interquartile range (box) with the line representing the median and whisker the full range of the data, each animal is represented by a dot, white boxes represent control (n = 9), blue (SuHx, n = 10), grey (IgG4 treated, n = 9) and green (Ky3 treated, n = 11), yellow (sildenaﬁl treated, n = 7), purple (sildenaﬁl & Ky3 treated, n = 8), orange (bosentan treated, n = 6) and red (bosentan & Ky3 treated, n = 10) rats. # p < 0.05, ## p < 0.01, ### p < 0.001 compared to IgG, p < 0.05, * p < 0.01, *** p < 0.001 compared to SuHx treated rats using one-way ANOVA followed by Sidak’s multiple comparisons test. All images are presented at their original magniﬁcation ×400, scale bar represents 20 µm 13. Lawrie, A. et al. Evidence of a role for osteoprotegerin in the pathogenesis of pulmonary arterial hypertension. Am. J. Pathol. 172, 256–264 (2008). 13. Lawrie, A. et al. 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S.D. helped design and performed experiments, and helped write the manuscript; J.A.P. helped design and performed experiments, and helped write the manuscript; J.C. helped design and performed experiments, and helped write the manuscript; A.T.B. helped design and performed experiments, and help prepare the manuscript; L.E.W. helped design and performed experiments, analysed data and help write the manuscript; J.I. helped design and performed experiments, performed statistical analysis of microarray data, and helped write the manuscript; H.C. helped performed experiments; L.R. per- formed experiments; V.G. helped design and performed experiments; M.M. helped design and performed experiments; A.M.K.R. helped design and performed experiments, performed statistical analysis of microarray data, and helped write the manuscript. H.K. Help perform experiments; A.G.H. helped performed experiments, and helped write the manuscript. 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Med. 209, 1919–1935 (2012). 17 TURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications Reprints and permission information is available at http://www.nature.com/reprints metabolism in KEGG. Nucleic Acids Res. 42, D199–D205 ( h k l d f © The Author(s) 2019 Acknowledgements Funding for this study was provided by a British Heart Foundation Senior Basic Science Research Fellow (FS/13/48/30453 and FS/18/52/33808, AL), Medical Research Council Career Development Award (G0800318, AL); Medical Research Council Conﬁdence In Concepts (MC/PC12022), Medical Research Council Developmental Pathway Funding Scheme (MR/L023040/1), British Heart Foundation Clinical Research Training Fellow- ship (FS/08/061/25740, AGH); Medical Research Council Clinical Research Training Fellowship (MR/K002406/1) and Wellcome Trust Clinical Research Career Development Fellowship (206632/Z/17/Z), AMKR, British Heart Foundation Intermediate Clinical Fellowship (FS/18/13/33281, AART), National Institute for Health Research Shefﬁeld Cardiovascular Biomedical Research Unit (NA/JP/DC); Cambridge National Institute of Health Research Biomedical Research Centre. © The Author(s) 2019 18 NATURE COMMUNICATIONS \| (2019) 10:5183 \| https://doi.org/10.1038/s41467-019-13139-9 \| www.nature.com/naturecommunications
https://openalex.org/W4245960973	https://www.qeios.com/read/BKHEQQ/pdf	English	null	Subcutis	Definitions	2,020	cc-by	86	Subcutis National Cancer Institute National Cancer Institute Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Qeios ID: BKHEQQ · https://doi.org/10.32388/BKHEQQ Source National Cancer Institute. Subcutis. NCI Thesaurus. Code C33645. Adipose tissue located under the dermis. It binds underlying structures with the skin. The subcutis is important in the regulation of temperature of the skin itself and the body. The size of this layer varies throughout the body and from person to person. Qeios ID: BKHEQQ · https://doi.org/10.32388/BKHEQQ 1/1
https://openalex.org/W2804219376	https://www.biorxiv.org/content/biorxiv/early/2018/05/18/325670.full.pdf	English	null	Reproduction costs can drive the evolution of groups	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	21,505	Abstract A fascinating wealth of life cycles is observed in biology, from unicellularity to the 8 concerted fragmentation of multi-cellular units. However, the understanding of factors 9 driving the evolution of life cycles is still limited. We investigate how reproduction costs 10 inﬂuence this process. We consider a basic model of a group structured population of 11 undifferentiated cells, where groups reproduce by fragmentation. Fragmentation events 12 are associated with a cost expressed by either a fragmentation delay, a fragmentation risk, 13 or a fragmentation loss. The introduction of such fragmentation costs vastly increases 14 the set of potentially optimal life cycles. Based on these ﬁndings, we suggest that the 15 evolution of life cycles and the splitting into multiple offspring can be directly associated 16 with the fragmentation cost. Moreover, the impact of this cost alone is strong enough to 17 drive the emergence of multicellular groups, even under scenarios that strongly disfavour 18 groups compared to solitary individuals. 19 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint 1 Introduction 20 Another example are cells constituting the outer layer 40 of a Volvox carteri colonies – these cells die upon the colony reproduction [Smith, 1944]. 41 Combined, this evidence shows that reproduction can be associated with a conspicuous cost. 42 There are only a few studies of the evolution of reproductive modes which explicitly 43 take into account the fragmentation cost. Libby et al. [2014] modelled the evolution of life 44 cycles of colonial forms of Saccharomyces cerevisiae. In their model, the fragmentation of 45 tree-structured cell clusters was attributed to the death of cells. These cells become weak 46 links and loose connections with neighbouring cells causing fragmentation of the cluster. 47 However, while Libby et al. considered a detailed model of binary fragmentations of cell 48 clusters, they did not investigate the whole range of fragmentation outcomes. In previous 49 work, we have extensively analysed all possible ways of group fragmentation and found 50 One such property which can limit the possible life cycles is the group fragmentation 34 cost. There is substantial evidence that reproduction is costly in natural populations. For 35 example, during the fragmentation of a simple multicellular organisms, the release of cells 36 requires the break of the cell matrix, which takes time and resources [Birkendal-Hansen, 37 One such property which can limit the possible life cycles is the group fragmentation 34 cost. There is substantial evidence that reproduction is costly in natural populations. For 35 example, during the fragmentation of a simple multicellular organisms, the release of cells 36 requires the break of the cell matrix, which takes time and resources [Birkendal-Hansen, 37 1995, Basbaum and Zena, 1996]. Also, not every cell may pass to the next generation of 38 groups, for instance in slime molds cells forming the stalk of the colony die shortly after the 39 spores are released [Bonner, 1959]. Another example are cells constituting the outer layer 40 of a Volvox carteri colonies – these cells die upon the colony reproduction [Smith, 1944]. 41 Combined, this evidence shows that reproduction can be associated with a conspicuous cost. 42 There are only a few studies of the evolution of reproductive modes which explicitly 43 take into account the fragmentation cost. Libby et al. [2014] modelled the evolution of life 44 cycles of colonial forms of Saccharomyces cerevisiae. 1 Introduction 20 All living and evolving organisms are born, grow and reproduce, giving birth to new organ- 21 isms [van Gestel and Tarnita, 2017, Stearns, 1992, Maynard Smith and Szathm´ary, 1995, 22 1 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint Bonner, 1998, Roze and Michod, 2001, Pfeiffer and Bonhoeffer, 2003, Rainey and Kerr, 23 2010, Ratcliff et al., 2012, Hammerschmidt et al., 2014, De Monte and Rainey, 2014, Kaveh 24 et al., 2016]. Natural selection promotes those organisms that perform this cycle in a more 25 efﬁcient way than others, as these produce more offspring per time. Surprisingly, even the 26 simplest organisms demonstrate a great variety of reproduction modes: Staphylococcus au- 27 reus produces independent propagule cells [Koyama et al., 1977], cyanobacteria ﬁlaments 28 fragment into multicellular threads [Rippka et al., 1979] while Gonium pectorale disperses 29 into independent cells [Stein, 1958]. These instances show that there is no universally opti- 30 mal reproduction mode. Instead, the way how cell groups produce offspring is an adaptation 31 to the environmental conditions and constrained by the biological properties of the organism 32 [van Gestel and Tarnita, 2017]. 33 [van Gestel and Tarnita, 2017]. 33 One such property which can limit the possible life cycles is the group fragmentation 34 cost. There is substantial evidence that reproduction is costly in natural populations. For 35 example, during the fragmentation of a simple multicellular organisms, the release of cells 36 requires the break of the cell matrix, which takes time and resources [Birkendal-Hansen, 37 1995, Basbaum and Zena, 1996]. Also, not every cell may pass to the next generation of 38 groups, for instance in slime molds cells forming the stalk of the colony die shortly after the 39 spores are released [Bonner, 1959]. 1 Introduction 20 In their model, the fragmentation of 45 tree-structured cell clusters was attributed to the death of cells. These cells become weak 46 links and loose connections with neighbouring cells causing fragmentation of the cluster. 47 However, while Libby et al. considered a detailed model of binary fragmentations of cell 48 clusters, they did not investigate the whole range of fragmentation outcomes. In previous 49 work, we have extensively analysed all possible ways of group fragmentation and found 50 2 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint evolutionary optimal life cycles under various ﬁtness landscapes [Pichugin et al., 2017]. For 51 costless group reproduction, only binary fragmentation, where a larger group splits into two 52 parts, can be evolutionary optimal in terms of maximising population growth. The same holds 53 for the case of proportional cost, where upon division into s parts, s −1 cells die. However, 54 for fragmentation with a ﬁxed cost in a form of a single cell loss, fragmentation modes with 55 multiple offspring can become evolutionary optimal. 56 In this study, we investigate the inﬂuence of the fragmentation cost on the evolution of 57 “staying together” life cycles [Tarnita et al., 2013]. We explicitly incorporate fragmentation 58 costs arising from three scenarios: fragmentation delay, fragmentation risk and cell loss. We 59 discuss the set of life cycles which can be evolutionary optimal for costly fragmentation. 60 Then, we investigate how the distribution of optimal life cycles on a set of random ﬁtness 61 landscapes depends on the value of the fragmentation cost. Finally, we consider in detail 62 those ﬁtness landscapes in which the increase in a group size always reduces the perfor- 63 mance of the group, i.e. the fastest growth and the best protection is achieved by independent 64 cells. 1 Introduction 20 We show that even in these ﬁtness landscapes that strongly disfavour multicellular 65 groups, fragmentation costs can promote the evolution of life cycles involving the emergence 66 of multicellular groups. 67 2.1 Growth and death of groups 69 We consider a population composed of unstructured groups (or complexes) of cells, which 70 emerge, grow and fragment into offspring groups, thus completing the life cycle. Groups 71 grow by dividing cells staying together after reproduction [Tarnita et al., 2013]. Due to the 72 absence of any structure, the properties of a group are determined by its size i alone. We 73 denote the abundance of groups of i cells in a population as xi. We additionally assume that 74 the size of groups in a population is bounded by n. Groups of size i have a death rate di and 75 cells in a group have the division rate bi, thus the growth rate of a group is ibi. The vectors of 76 3 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint birth rates b = (b1, . . . , bn) and of death rates d = (d1, . . . , bn) deﬁne the ﬁtness landscape 77 of the model, see Fig. 1a. 78 2.2 Group fragmentation 79 New groups are produced by the fragmentation of existing groups. We further assume that 80 the fragmentation occurs immediately after the growth of the group. Thus, upon each cell 81 division, a group grows in size by one and either remains in this state until the next cell 82 division, or splits into two or more smaller groups. As any group can be characterized by 83 the number of cells comprising it, any fragmentation or growth can be characterized by a 84 partition of this integer number. A partition is a way of decomposing an integer m into a sum 85 of integers without regard to order, summands are called parts [Andrews, 1998]. We use the 86 notation κ ⊢m to indicate that κ is a partition of m, for example 2 + 2 ⊢4, see Fig. 1b. The 87 number of partitions of m grows fast with m. In the current study, we use n = 19 and thus 88 m does not exceed 20. For m = 20, there are in total 2693 non-trivial partitions (with more 89 than one part). 90 As example of using partitions to characterize fragmentation modes, consider a group 91 of 2 cells in which the 3rd cell is born. If the group fragments without any cell dying, the 92 product is either three independent cells (partition 1 + 1 + 1 ⊢3) or a group of two cells 93 and an independent cell (partition 2 + 1 ⊢3). If a cell is lost upon fragmentation, the only 94 possible result is two independent cells (partition 1+1 ⊢2). In the absence of fragmentation, 95 the product is the single group of three cells (the trivial partition 3 ⊢3). 96 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint 2.3.1 Fragmentation delay 100 In the case of the fragmentation delay, the process of fragmentation is not immediate and 101 takes time T. This scenario covers situations where the fragmentation of the group requires 102 the investment of resources, which otherwise would be spent on the further growth of the 103 group. The transition time is inverse to the transition rate, thus we deﬁne the rate of fragmen- 104 tation of a clusters of size m by 105 1 mb′ m = 1 mbm + T, such that b′ m = bm 1 + mbmT ≤bm, (1) b′ m = bm 1 + mbmT ≤bm, (1) (1) where T it the fragmentation delay. Consequently, this scenario can be captured by changing 106 the ﬁtness landscape in terms of the birth rate at the size prior to fragmentation. 107 2.3.2 Fragmentation with risk of death 108 In the case of the fragmentation with risk, the organism expresses risky behavior prior to the 109 fragmentation. For example, an organism could leave the shelter or break its shell in order to 110 reproduce. Under this scenario, the risky behaviour increases the death rate at the ﬁnal stage 111 of the organism life cycle by R 112 d′ m = dm + R. (2) d′ m = dm + R. d′ m = dm + R. (2) (2) Again, this scenario corresponds to a change of the ﬁtness landscape. 113 Again, this scenario corresponds to a change of the ﬁtness landscape. 113 2.3 Three way of implementing fragmentation costs 97 It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint 2.3 Three way of implementing fragmentation costs 97 We consider three qualitatively different scenarios that capture the fragmentation cost: frag- 98 mentation delay, fragmentation risk, and fragmentation loss. 99 4 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint a) growth and death growth death ... b1 2b2 3b3 4b4 d1 d2 d3 d4 b) examples of fragmentation partitions b) examples of fragmentation partitions 1 +1 2+1 1 +1 +1 3+1 2+2 2+1 +1 1 +1 +1 +1 κ ˫ 2 κ ˫ 3 κ ˫ 4 c) a pure life cycle with costly fragmentation b1 2b2 3b'3 d1 d2 d'3 Figure 1: Model of life cycles. (a) The ﬁtness landscape is deﬁned by vectors of growth and death rates. Cells in a group of size i grow at rate bi and groups die at rate di. (b) The fragmentation of groups is described by a partition of an integer number into a sum of integers. All possible fragmentations of groups of size 2, 3, and 4 are presented here. (c) In a deterministic life cycle, all groups follow the same partition at the fragmentation. For costly fragmentation, the growth rate at the maturity size may be smaller than prescribed by the ﬁtness landscape b′ m ≤bm, the death rate at the maturity size may be larger than prescribed by the ﬁtness landscape d′ m ≥dm and some cells may be lost upon the fragmentation (one cell in the illustrated case). 5 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 2.3.3 Fragmentation with loss 114 For fragmentation with loss, L cells die as upon the group fragmentation, thus the combined 115 size of offspring groups is by L smaller than the size of the fragmented cell cluster. Under 116 this scenario, the fragmentation followed by the growth from size m to m+1 is characterized 117 by a partition κ ⊢m+1−L. We assume L to be constant, i.e. clusters loose the same number 118 of cells independently on the partition of the parent group into offspring. 119 6 6 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint The three considered scenarios are not mutually exclusive, all three types of cost may be 120 present simultaneously. However, for simplicity of the presentation of results, we illustrate 121 each scenario of the fragmentation cost independently. 122 2.4 Population dynamics under a deterministic life cycle 123 ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint next term −dixi describes the death of groups. The last term πi(κ)mb′ mxm describes the 144 emergence of new groups of size i resulting from the fragmentation of mature groups. The 145 integer πi(κ) is the number of groups of size i that emerge in a single act of fragmentation 146 according to the partition κ, and mb′ m is the growth rate prior to fragmentation (see Eq. (1)). 147 Eq. (3c) describes the dynamics of groups of maturity size m, which will inevitably frag- 148 ment according to the partition κ upon the next cell division. For fragmentation with de- 149 lay, the rate of transition to the next state (fragmentation) is smaller than the cell birth rate 150 (b′ m < bm) implied by the ﬁtness landscape birth vector b (see Eq. (1)). For fragmentation 151 with risk, the death rate is larger (d′ m > dm) than implied by the ﬁtness landscape death vector 152 d (see Eq. (2)). 153 The equation system (3) is linear with respect to xi. Thus, it can be written in a form of 154 matrix differential equation 155 d dtx = Ax, (4) d dtx = Ax, (4) d dtx = Ax, (4) where x = (x1, x2, · · · , xm−1)T, and matrix A is 156 where x = (x1, x2, · · · , xm−1)T, and matrix A is 156 where x = (x1, x2, · · · , xm−1)T, and matrix A is 156 A =               −b1 −d1 0 0 · · · π1(κ)mb′ m b1 −2b2 −d2 0 · · · π2(κ)mb′ m 0 2b2 −3b3 −d3 · · · π3(κ)mb′ m 0 0 3b3 · · · π4(κ)mb′ m ... ... ... ... ... 0 0 0 · · · πm(κ)mb′ m −mb′ m −d′ m               (5) (5) In the long run, the solution of Eq. 2.4 Population dynamics under a deterministic life cycle 123 For costless fragmentation, natural selection favours a narrow subset of life cycles, called 124 deterministic life cycles in [Pichugin et al., 2017], see Fig. 1c. In these life cycles, groups al- 125 ways grow up to some maturity size m ≤n, always fragment immediately after the m + 1-st 126 cell is born, and the fragmentation always follow the same pattern, given by a single parti- 127 tion. Also for costly fragmentation, natural selection promotes only deterministic life cycles, 128 see Appendix A.1. Thus, here we do not consider any life cycles other than deterministic 129 ones, where a life cycle would follow several paths, sometimes fragmenting in one way and 130 sometimes in another one. 131 Under a given deterministic life cycle, the state of a population can be described by abun- 132 dances of groups xi of each possible size i from one cell to m cells given by the vector 133 (x1, x2, · · · , xm). There are no groups of size m + 1 or larger, because under determinis- 134 tic life cycle, any group fragments immediately after the next cell is born in a group of the 135 maturity size m. 136 The dynamics of the population state can be expressed in a form of the system of m 137 differential equations: one equation for each particular size of groups. The change in the 138 number of groups of a given size is inﬂuenced by growth, death and fragmentation. This 139 leads to the set of equations 140 dx1 dt = −b1x1 −d1x1 + π1(κ)mb′ mxm (3a) dxi dt = −ibixi + (i −1)bi−1xi−1 −dixi + πi(κ)mb′ mxm if 1 < i < m (3b) dxm dt = −mb′ mxm + (m −1)bm−1xm−1 −d′ mxm + πm(κ)mb′ mxm, (3c) (3a) (3b) (3c) Here, Eqs. (3a) and (3b) describe the dynamics of the abundances of groups xi that grow 141 without fragmentation, because they do not reach the maturity size m. The ﬁrst two terms in 142 Eq. (3b) −ibixi + (i −1)bi−1xi−1 describe the change in xi due to the group growth. The 143 7 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. 2.4 Population dynamics under a deterministic life cycle 123 165 To ﬁnd the evolutionary optimal life cycle, it is necessary to ﬁnd values of λ for all life cy- 166 cles of interest. The leading eigenvalue λ is given by the largest solution of the characteristic 167 equation 168 det (A −λI) = 0. (7) det (A −λI) = 0. (7) For a given deterministic life cycle associated to fragmentation at size m according to the 169 partition κ, the characteristic equation (7) reduces to (see Appendix A.2 for a derivation) 170 m For a given deterministic life cycle associated to fragmentation at size m according to the 169 partition κ, the characteristic equation (7) reduces to (see Appendix A.2 for a derivation) 170 Fm+1(λ) + ∆mFm(λ) −b′ m bm m X i=1 πi(κ)Fi(λ) = 0, (8) Fm+1(λ) + ∆mFm(λ) −b′ m bm m X i=1 πi(κ)Fi(λ) = 0, (8) (8) where 171 Fi(λ) = i−1 Y j=1 1 + dj + λ jbj . (9) where 171 Fi(λ) = i−1 Y j=1 1 + dj + λ jbj . Fi(λ) = i−1 Y j=1 1 + dj + λ jbj . (9) (9) The parameter 172 ∆m = m(b′ m −bm) + d′ m −dm mbm (10) ∆m = m(b′ m −bm) + d′ m −dm mbm (10) (10) characterises how costly fragmentation is in terms of risks and delays. In the absence of any 173 costs, we have ∆m = 0. Eq. (8) is a polynomial equation of degree m. In general, we have 174 to solve this equation numerically. 175 2.4 Population dynamics under a deterministic life cycle 123 (4) converges to that of an exponentially growing popula- 157 tion with a stable distribution, i.e., 158 In the long run, the solution of Eq. (4) converges to that of an exponentially growing popula- 157 tion with a stable distribution, i.e., 158 lim t→∞x(t) = eλtw. (6) lim t→∞x(t) = eλtw. (6) lim t→∞x(t) = eλtw. (6) (6) The leading eigenvalue λ gives the total population growth rate, and its associated right eigen- 159 vector w = (w1, . . . , wm) gives the stable distribution of group sizes. In the long term, the 160 fraction of groups of size i in the population is proportional to wi. The leading eigenvalue 161 determines the evolutionary success of a population: In the competition of two populations 162 The leading eigenvalue λ gives the total population growth rate, and its associated right eigen- 159 vector w = (w1, . . . , wm) gives the stable distribution of group sizes. In the long term, the 160 fraction of groups of size i in the population is proportional to wi. The leading eigenvalue 161 determines the evolutionary success of a population: In the competition of two populations 162 8 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint utilizing different life cycles (and hence different λ), the one with larger growth rate will 163 outcompete the other one. Thus, natural selection would promote the life cycle that provides 164 the largest λ. We call this the evolutionary optimal life cycle. 165 utilizing different life cycles (and hence different λ), the one with larger growth rate will 163 outcompete the other one. Thus, natural selection would promote the life cycle that provides 164 the largest λ. We call this the evolutionary optimal life cycle. 2.5 Random ﬁtness landscapes 176 There- 191 fore, one could assume that life cycles that fragment at large group sizes only are strongly 192 disfavoured. 193 2.5 Random ﬁtness landscapes 176 We now numerically investigate the distribution of optimal life cycles on two sets of ﬁtness 177 landscapes: random ﬁtness landscapes and random detrimental ﬁtness landscapes, which 178 strongly disfavour groups. Both sets are explored by 10000 ﬁtness landscapes generated only 179 once and then used to assess all three scenarios: delay, risk, and loss. Within the scope 180 of this study, we are interested in proportion of ﬁtness landscapes promoting each of the 181 classes of life cycles. The amount of collected data provides the relative accuracy about 182 √ 10000/10000 = 0.01, which is enough for our purposes. 183 9 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint In the set of random ﬁtness landscapes, each element of the birth and death rates vector 184 (b and d) was sampled independently from the uniform distribution U(0, 1). 185 In the set of random ﬁtness landscapes, each element of the birth and death rates vector 184 (b and d) was sampled independently from the uniform distribution U(0, 1). 185 In the set of random ﬁtness landscapes, each element of the birth and death rates vector 184 (b and d) was sampled independently from the uniform distribution U(0, 1). 185 In the set of random detrimental ﬁtness landscapes, for each landscape, we initially sam- 186 pled two sequences of n = 19 random numbers, each using the uniform distribution U(0, 1). 187 Then, the ﬁrst sequence has been sorted in descending order to form the vector of the birth 188 rates b and the second sequence has been sorted in ascending order to form the vector of death 189 rates d. Thus, in all detrimental ﬁtness landscape, the values of birth rates monotonically de- 190 creased with the group size, while the values of death rates monotonically increased. 3.1 Some life cycles cannot be evolutionary optimal under any ﬁtness 195 landscape 196 if two partitions τ1 and τ2 exist such as: 212 It can be shown analytically that all three scenarios of the fragmentation cost (delay, risk 209 and loss) lead to the same condition for a life cycle to be forbidden: the life cycle determined 210 by the partition κ is forbidden if two different subsets of offspring with equal combined sizes 211 exist, i.e. if two partitions τ1 and τ2 exist such as: 212 209 τ1 ⊢j, τ2 ⊢j, τ1 ̸= τ2 and τ1 + τ2 ⊂κ, (11) τ1 ⊢j, τ2 ⊢j, τ1 ̸= τ2 and τ1 + τ2 ⊂κ, (11) (11) For any ﬁtness landscape and any fragmentation cost scenario, the life cycle employing such 213 a partition is dominated by one of two life cycles in which one of the subsets occurs twice, 214 while other one is not present, see Appendix A.3 for the proof. 215 The simplest example of the forbidden life cycle is the partition 2+1+1, which has two 216 different offspring subsets: 2 and 1+1, both having the same combined size 2. It is always 217 dominated either by a life cycle with partition 2+2 (subset 2 occurs twice) or by a life cy- 218 cle with partition 1+1+1+1 (subset 1+1 occurs twice), see Fig. 2a for more examples. The 219 proportion of forbidden life cycles rapidly increases with the partition sum (see black bars 220 on Fig. 2b). Individually assessing each of considered 2693 partitions computationally, we 221 found only 687 partitions corresponding to allowed life cycles (this is about a quarter of the 222 total number). 223 The total amount of allowed life cycles is still too large to track each of them individually. 224 Therefore, a classiﬁcation is necessary. We focus on three signiﬁcant subsets: binary frag- 225 mentation, equal split and seeding, see also Fig. 2a. Binary fragmentation partitions have the 226 form κ = a + b. Examples of binary partitions are 2+2 and 7+1. Binary fragmentation cover 227 all scenarios where the parent group divides in two parts. Among the non-binary fragmenta- 228 tion modes, we distinguish equal split and seeding partitions. Equal split partitions have the 229 form κ = a + · · · + a + b such that a > b ≥0 and have more than two parts. Examples 230 of equal splits are 1+1+1 and 3+3+3+2. 3.1 Some life cycles cannot be evolutionary optimal under any ﬁtness 195 landscape 196 To ﬁnd which life cycles can evolve for costly fragmentation we consider a large population 197 of groups that can grow without constraint (see Section 2.4). The growth of any group is 198 limited by the maximal group size n = 19. This leads to 2693 possible life cycles, one for 199 each non-trivial partition of all integers not exceeding 20. The growth rate of a population 200 with any given life cycle can be computed by solving Eq. (8). For each combination of the 201 ﬁtness landscape (Section 2.1) and the fragmentation cost (Section 2.3), one of the 2693 life 202 cycles provides the largest growth rate and, thus, is evolutionary optimal. 203 For any ﬁtness landscape, it is possible to ﬁnd a life cycle which is evolutionary optimal 204 under this ﬁtness landscape. However, the opposite is not true: for some life cycles, it is 205 impossible to ﬁnd any ﬁtness landscape under which it is evolutionary optimal. We label 206 these life cycles “forbidden life cycles”. Consequently, we call a life cycle that is evolutionary 207 optimal under some ﬁtness landscape “allowed life cycle”. 208 10 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint It can be shown analytically that all three scenarios of the fragmentation cost (delay, risk 209 and loss) lead to the same condition for a life cycle to be forbidden: the life cycle determined 210 by the partition κ is forbidden if two different subsets of offspring with equal combined sizes 211 exist, i.e. 3.1 Some life cycles cannot be evolutionary optimal under any ﬁtness 195 landscape 196 For instance, the 238 allowed partitions 4+2+1 and 5+4+4 do not belong to any of these classes. The proportion 239 of binary, equal split and seeding partitions among all allowed partitions decreases with the 240 partition sum, see Fig. 2b). For a system where groups may grow up to n = 19, there are 241 100 binary partitions, 90 equal split partitions, 110 seeding partitions and 387 other allowed 242 partitions, which do not belong to either of these three classes. 243 All binary, equal split and seeding partitions are associated to allowed life cycles. How- 237 ever, not every allowed partition belongs to either of these three subsets. For instance, the 238 allowed partitions 4+2+1 and 5+4+4 do not belong to any of these classes. The proportion 239 of binary, equal split and seeding partitions among all allowed partitions decreases with the 240 partition sum, see Fig. 2b). For a system where groups may grow up to n = 19, there are 241 100 binary partitions, 90 equal split partitions, 110 seeding partitions and 387 other allowed 242 partitions, which do not belong to either of these three classes. 243 3+3+1 2+2+2+1 1 +1 +1 +1 +1 +1 +1 Equal split 6+1 5+2 4+3 Binary 3+1 +1 +1 +1 2+2+1 +1 +1 2+1 +1 +1 +1 +1 Forbidden partitions 3+2+1 +1 Allowed partitions Other 5+1 +1 4+2+1 3+2+2 4+1 +1 +1 a) b) Forbidden partitions Allowed partitions Equal split Seeding Binary Other Seeding Figure 2: Forbidden and allowed partitions. (a) Allowed and forbidden partitions of 7. Allowed partitions are further broken into binary, equal, seeding, and other classes, according to the deﬁnitions in the main text. For each of forbidden partitions, a couple of different subsets of parts with the same sum are underlined (see Eq. (11)). (b) Proportion of forbidden and allowed partitions as a function of the partition sum. For partition sums 2 and 3 all partitions are allowed, starting from 4 some partitions are forbidden (for partition sum 4, it is 2+1+1). The proportion of forbidden partition grows rapidly with the partition sum. Among allowed partitions, the proportions of binary, equal split and seeding classes rapidly declines, consequently the other partitions constitute the majority of allowed fragmentation modes at large partition sums. 3.1 Some life cycles cannot be evolutionary optimal under any ﬁtness 195 landscape 196 Equal splits represent scenarios, where cells are 231 evenly distributed among multiple offspring groups (plus a single smaller remainder group, 232 if needed). Seeding partitions have the form κ = a+b+· · ·+b such that a > b+· · ·+b. Ex- 233 amples of seeding are 3+1+1 and 7+2+2+2. Distinguishing the seeding fragmentation modes 234 is inspired by seeding dispersal exhibited by bioﬁlms, where a small portion of cells leaves 235 The total amount of allowed life cycles is still too large to track each of them individually. 224 Therefore, a classiﬁcation is necessary. We focus on three signiﬁcant subsets: binary frag- 225 mentation, equal split and seeding, see also Fig. 2a. Binary fragmentation partitions have the 226 form κ = a + b. Examples of binary partitions are 2+2 and 7+1. Binary fragmentation cover 227 all scenarios where the parent group divides in two parts. Among the non-binary fragmenta- 228 tion modes, we distinguish equal split and seeding partitions. Equal split partitions have the 229 form κ = a + · · · + a + b such that a > b ≥0 and have more than two parts. Examples 230 of equal splits are 1+1+1 and 3+3+3+2. Equal splits represent scenarios, where cells are 231 evenly distributed among multiple offspring groups (plus a single smaller remainder group, 232 if needed). Seeding partitions have the form κ = a+b+· · ·+b such that a > b+· · ·+b. Ex- 233 amples of seeding are 3+1+1 and 7+2+2+2. Distinguishing the seeding fragmentation modes 234 is inspired by seeding dispersal exhibited by bioﬁlms, where a small portion of cells leaves 235 11 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint All binary, equal split and seeding partitions are associated to allowed life cycles. How- 237 ever, not every allowed partition belongs to either of these three subsets. 3.2 Evolutionary optimal life cycles under random ﬁtness landscapes 244 The previous section introduced the range of potentially optimal life cycles, but it did not 245 give any insight about interconnection between life cycles and ﬁtness landscapes. Some life 246 cycles may be evolutionary optimal under a larger set of ﬁtness landscapes than others. To 247 study the distribution of optimal life cycles for costly fragmentation, we generated a large 248 set of 10000 random ﬁtness landscapes (see section 2.5). For each ﬁtness landscape from 249 this set, we numerically computed the optimal life cycle independently for each of three 250 scenarios of the fragmentation cost (delay, risk, or loss) under a range of cost values (T, R, 251 or L, respectively). 252 3.1 Some life cycles cannot be evolutionary optimal under any ﬁtness 195 landscape 196 It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint 3.1 Some life cycles cannot be evolutionary optimal under any ﬁtness 195 landscape 196 3+3+1 2+2+2+1 1 +1 +1 +1 +1 +1 +1 Equal split 6+1 5+2 4+3 Binary 3+1 +1 +1 +1 2+2+1 +1 +1 2+1 +1 +1 +1 +1 Forbidden partitions 3+2+1 +1 Allowed partitions Other 5+1 +1 4+2+1 3+2+2 4+1 +1 +1 a) Seeding b) Forbidden partitions Allowed partitions Equal split Seeding Binary Other b) Figure 2: Forbidden and allowed partitions. (a) Allowed and forbidden partitions of 7. Allowed partitions are further broken into binary, equal, seeding, and other classes, according to the deﬁnitions in the main text. For each of forbidden partitions, a couple of different subsets of parts with the same sum are underlined (see Eq. (11)). (b) Proportion of forbidden and allowed partitions as a function of the partition sum. For partition sums 2 and 3 all partitions are allowed, starting from 4 some partitions are forbidden (for partition sum 4, it is 2+1+1). The proportion of forbidden partition grows rapidly with the partition sum. Among allowed partitions, the proportions of binary, equal split and seeding classes rapidly declines, consequently the other partitions constitute the majority of allowed fragmentation modes at large partition sums. Figure 2: Forbidden and allowed partitions. (a) Allowed and forbidden partitions of 7. Allowed partitions are further broken into binary, equal, seeding, and other classes, according to the deﬁnitions in the main text. For each of forbidden partitions, a couple of different subsets of parts with the same sum are underlined (see Eq. (11)). (b) Proportion of forbidden and allowed partitions as a function of the partition sum. For partition sums 2 and 3 all partitions are allowed, starting from 4 some partitions are forbidden (for partition sum 4, it is 2+1+1). The proportion of forbidden partition grows rapidly with the partition sum. Among allowed partitions, the proportions of binary, equal split and seeding classes rapidly declines, consequently the other partitions constitute the majority of allowed fragmentation modes at large partition sums. 12 . CC-BY 4.0 International license a ified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: oRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. the increase in fragmentation cost 254 The average maturity size m at which fragmentation occurs and the average size of offspring 255 groups are presented in Fig. 3 a-c. For all three scenarios of the costly fragmentation, the ma- 256 turity size increases with the cost (T, R, or L). For our choice of n = 19, the average maturity 257 size approaches n+1 2 = 10 with an increase in fragmentation delay (T) and the variation ap- 258 proaches q n2−1 12 = √ 30 (see Fig. 3a), because the distribution of maturity sizes approaches 259 a uniform distribution, see Appendix A.4. For fragmentation with risk, the average maturity 260 size steadily grows with risk (R), while the variation of maturity sizes slowly decreases (see 261 Fig. 3b). For fragmentation with losses, the average maturity size steadily increases with 262 cell loss (L) and the variance decreases. At L = n −1 = 18 the maturity size is aways 263 m = n = 19, see Fig. 3c. 264 Also, the number of offspring increases with the cost. For costless fragmentation, the 265 optimal life cycle always produces exactly two offspring groups. With increasing costs, life 266 cycles with fragmentation into multiple parts become optimal, and consequently, the number 267 of produced offspring increases. For fragmentation with delay, the average size of offspring 268 does not change signiﬁcantly with delay (T), see Fig. 3a. For fragmentation with risk, the 269 average size of offspring decreases with risk (R), see Appendix A.5. Combined with the 270 13 b) a) Maturity size Offspring groups Binary partitions Equal split partitions Other allowed partitions c) Seeding partitions Figure 3: Optimal life cycles for costly fragmentation. The top panels present maturity and aver- age offspring sizes in optimal life cycles as a function of the fragmentation cost for a) fragmentation with delay, b) fragmentation with risk and c) fragmentation with cell loss, respectively. Points depict the average value, error bars represent one standard deviation. The bottom panels show the frac- tions of each of binary fragmentation, equal split, seeding, and other allowed partitions as functions of fragmentation cost for the same scenarios of fragmentation cost. While the binary and equal split transitions constitute relatively small portion of available partitions, the corresponding life cycles have high probability to be evolutionary optimal. The increase in fragmentation loss reduces the amount of available life cycles, especially at large L. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint the increase in fragmentation cost 254 CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint increase in the maturity size, this leads to an increase in the number of offspring produced 271 at the fragmentation event. For fragmentation with loss, the size of offspring monotoni- 272 cally decreases with loss (L) and therefore, the offspring number initially increases with loss. 273 However, the number of offspring declines at large L, because this number cannot exceed 274 the number of surviving cells, which is limited by n −L + 1. In our model the number of 275 produced offspring returns to 2 at L = 18. 276 the increase in fragmentation cost 254 Thus, the fraction of life cycles classes at the panel c) does not change smoothly with the fragmentation loss. b) a) Maturity size Offspring groups Binary partitions Equal split partitions Other allowed partitions c) Seeding partitions b) c) b) c) a) Figure 3: Optimal life cycles for costly fragmentation. The top panels present maturity and aver- age offspring sizes in optimal life cycles as a function of the fragmentation cost for a) fragmentation with delay, b) fragmentation with risk and c) fragmentation with cell loss, respectively. Points depict the average value, error bars represent one standard deviation. The bottom panels show the frac- tions of each of binary fragmentation, equal split, seeding, and other allowed partitions as functions of fragmentation cost for the same scenarios of fragmentation cost. While the binary and equal split transitions constitute relatively small portion of available partitions, the corresponding life cycles have high probability to be evolutionary optimal. The increase in fragmentation loss reduces the amount of available life cycles, especially at large L. Thus, the fraction of life cycles classes at the panel c) does not change smoothly with the fragmentation loss. Figure 3: Optimal life cycles for costly fragmentation. The top panels present maturity and aver- age offspring sizes in optimal life cycles as a function of the fragmentation cost for a) fragmentation with delay, b) fragmentation with risk and c) fragmentation with cell loss, respectively. Points depict the average value, error bars represent one standard deviation. The bottom panels show the frac- tions of each of binary fragmentation, equal split, seeding, and other allowed partitions as functions of fragmentation cost for the same scenarios of fragmentation cost. While the binary and equal split transitions constitute relatively small portion of available partitions, the corresponding life cycles have high probability to be evolutionary optimal. The increase in fragmentation loss reduces the amount of available life cycles, especially at large L. Thus, the fraction of life cycles classes at the panel c) does not change smoothly with the fragmentation loss. 14 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . 3.3 Fragmentation cost can drive the formation of multicellular groups 303 Multicellular groups evolve when the existence of cells in a group provides some beneﬁt, 304 expressed for example in a form of better resource acquisition or protection from external 305 threats. However, for costly group fragmentation, even when existence in groups is detri- 306 mental to cells comprising them, formation of multicellular groups may be evolutionary ben- 307 eﬁcial: We have constructed a set of 10000 random detrimental ﬁtness landscapes (see Sec- 308 tion 2.5). For each of them, the death rate increases monotonically with the size of group, 309 while the birth rate monotonically decreases with the group size. For costless fragmentation, 310 the optimal life cycle for all detrimental ﬁtness landscapes is unicellular, i.e. uses the par- 311 tition 1 + 1. With the increase in the value of the fragmentation cost (T, L or R), other – 312 multicellular – life cycles become optimal (see Fig. 4). For all detrimental ﬁtness landscapes 313 and all scenarios of the fragmentation cost, all observed optimal life cycles are equal splits in 314 the form 1 + 1 + · · · + 1 (see Fig. 4a-c). The intuition behind this behaviour is that a soli- 315 tary cell is the most effective state available to the population under the detrimental ﬁtness 316 landscapes, since solitary cells have the largest growth and the lowest death rate among all 317 possible groups sizes. 318 3.2.2 Equal split and binary fragmentation life cycles are overrepresented for random 277 ﬁtness landscapes 278 CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint these classes are evolutionary optimal at roughly the same proportions of ﬁtness landscapes 298 (∼5%). Given that there is a much smaller number of seedings than other partitions, see 299 Fig. 2b, seeding partitions are less suppressed by fragmentation with delay and loss than 300 other partitions. For reproduction with risk, seeding partitions are much less abundant than 301 other partitions. 302 these classes are evolutionary optimal at roughly the same proportions of ﬁtness landscapes 298 (∼5%). Given that there is a much smaller number of seedings than other partitions, see 299 Fig. 2b, seeding partitions are less suppressed by fragmentation with delay and loss than 300 other partitions. For reproduction with risk, seeding partitions are much less abundant than 301 other partitions. 302 3.2.2 Equal split and binary fragmentation life cycles are overrepresented for random 277 ﬁtness landscapes 278 The proportions of different classes of partitions among optimal life cycles change with the 279 fragmentation cost (T, R, or L), see Fig. 3 a-c. 280 If reproduction is costless, only binary partitions can be evolutionary optimal [Pichugin 281 et al., 2017]. At low reproduction costs, binary partitions remain the most abundant class 282 under any scenario of cost implementation. With an increase in costs, the fraction of ﬁtness 283 landscapes promoting binary fragmentation declines. For reproduction with delay, this frac- 284 tion stabilizes at about 60% (see Fig. 3a), while for reproduction with risk, it falls below 5% 285 (see Fig. 3b). For fragmentation with loss, the binary fragmentation increase in abundance 286 up to L ≈15 on, see Fig. 3c. This is connected to the decrease in the number of available 287 partitions once the fragmentation loss become compatible with the maximal available group 288 size L ∼n (such as at L = 18, the only possible partition is 1+1, which is a binary one). 289 Equal split fragmentations constitute another major class of observed reproduction modes. 290 Equal split fragmentations constitute another major class of observed reproduction modes. 290 For reproduction with risk and with (moderate) losses, equal splits are evolutionary optimal 291 for the vast majority of ﬁtness landscapes. For reproduction with delay, equal splits are the 292 second most abundant class of optimal life cycles. Equal splits are promoted by natural se- 293 lection, because they maximize the number of offspring groups per act of fragmentation and 294 thus share the cost among the largest number of offspring groups. 295 Seeding and other fragmentation modes contribute only a small portion of optimal life 296 cycles in all three scenarios of reproduction cost. For reproduction with delay and loss, both 297 Seeding and other fragmentation modes contribute only a small portion of optimal life 296 cycles in all three scenarios of reproduction cost. For reproduction with delay and loss, both 297 15 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . 4 Discussion 319 A key factor considered in the present study is the cost of reproduction – an act of making 320 offspring results in less net biomass than the growth without reproduction. How much is it the 321 case for the natural populations? A number of evidences from observations and experimental 322 16 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; /325670 1 +...+1 1 +1 Maturity size Offspring groups c) a) b) Figure 4: Fragmentation cost can drive the evolution of multicellular groups in detrimental ﬁtness landscapes. The top panels show the average size of the parental and offspring groups in optimal life cycles as a function of fragmentation cost for a) fragmentation with delay, b) fragmentation with risk and c) fragmentation with cell loss, respectively. Points show the average value, error bars represent one standard deviation. The set of ﬁtness landscapes is given by monotonic random sequences (see main text). The size of offspring groups is strictly one, which means that all observed equal split fragmentations had all offspring being independent cells. The bottom panels shows the fractions of unicellular (1+1) and multicellular modes of fragmentation. At no cost, all detrimental ﬁtness landscapes promote the unicellular life cycles. For fragmentation with delay, the fraction of unicellular life cycles rapidly decreases and approaches zero at T = 0.3. However, starting from T ≈5.9 unicellular life cycles become sometimes optimal again. For fragmentation with risk, multicellular life cycles are not observed below R ≈3.9. Nevertheless, by R ≈18.9 under all ﬁtness landscapes, the optimal life cycles become multicellular. 4 Discussion 319 Points show the average value, error bars represent one standard deviation. The set of ﬁtness landscapes is given by monotonic random sequences (see main text). The size of offspring groups is strictly one, which means that all observed equal split fragmentations had all offspring being independent cells. The bottom panels shows the fractions of unicellular (1+1) and multicellular modes of fragmentation. At no cost, all detrimental ﬁtness landscapes promote the unicellular life cycles. For fragmentation with delay, the fraction of unicellular life cycles rapidly decreases and approaches zero at T = 0.3. However, starting from T ≈5.9 unicellular life cycles become sometimes optimal again. For fragmentation with risk, multicellular life cycles are not observed below R ≈3.9. Nevertheless, by R ≈18.9 under all ﬁtness landscapes, the optimal life cycles become multicellular. For fragmentation with loss, the partition 1+1 corresponds to the fragmentation at the minimal possible size. The proportion of ﬁtness landscapes promoting the partition 1+1 decreases rapidly. However, the number of available fragmentation partitions decreases with L such as at L = 18 the only possible partition is 1+1. Figure 4: Fragmentation cost can drive the evolution of multicellular groups in detrimental ﬁtness landscapes. The top panels show the average size of the parental and offspring groups in optimal life cycles as a function of fragmentation cost for a) fragmentation with delay, b) fragmentation with risk and c) fragmentation with cell loss, respectively. Points show the average value, error bars represent one standard deviation. The set of ﬁtness landscapes is given by monotonic random sequences (see main text). The size of offspring groups is strictly one, which means that all observed equal split fragmentations had all offspring being independent cells. The bottom panels shows the fractions of unicellular (1+1) and multicellular modes of fragmentation. At no cost, all detrimental ﬁtness landscapes promote the unicellular life cycles. For fragmentation with delay, the fraction of unicellular life cycles rapidly decreases and approaches zero at T = 0.3. However, starting from T ≈5.9 unicellular life cycles become sometimes optimal again. For fragmentation with risk, multicellular life cycles are not observed below R ≈3.9. Nevertheless, by R ≈18.9 under all ﬁtness landscapes, the optimal life cycles become multicellular. For fragmentation with loss, the partition 1+1 corresponds to the fragmentation at the minimal possible size. The proportion of ﬁtness landscapes promoting the partition 1+1 decreases rapidly. 4 Discussion 319 For fragmentation with loss, the partition 1+1 corresponds to the fragmentation at the minimal possible size. The proportion of ﬁtness landscapes promoting the partition 1+1 decreases rapidly. However, the number of available fragmentation partitions decreases with L such as at L = 18 the only possible 1 +...+1 1 +1 Maturity size Offspring groups c) a) b) b) b) c) a) Figure 4: Fragmentation cost can drive the evolution of multicellular groups in detrimental ﬁtness landscapes The top panels show the average size of the parental and offspring groups in optimal life cycles as Figure 4: Fragmentation cost can drive the evolution of multicellular groups in detrimental ﬁtness Figure 4: Fragmentation cost can drive the evolution of multicellular groups in detrimental ﬁtness landscapes. The top panels show the average size of the parental and offspring groups in optimal life cycles as a function of fragmentation cost for a) fragmentation with delay, b) fragmentation with risk and c) fragmentation with cell loss, respectively. Points show the average value, error bars represent one standard deviation. The set of ﬁtness landscapes is given by monotonic random sequences (see main text). The size of offspring groups is strictly one, which means that all observed equal split fragmentations had all offspring being independent cells. The bottom panels shows the fractions of unicellular (1+1) and multicellular modes of fragmentation. At no cost, all detrimental ﬁtness landscapes promote the unicellular life cycles. For fragmentation with delay, the fraction of unicellular life cycles rapidly decreases and approaches zero at T = 0.3. However, starting from T ≈5.9 unicellular life cycles become sometimes optimal again. For fragmentation with risk, multicellular life cycles are not observed below R ≈3.9. Nevertheless, by R ≈18.9 under all ﬁtness landscapes, the optimal life cycles become multicellular. For fragmentation with loss, the partition 1+1 corresponds to the fragmentation at the minimal possible size. The proportion of ﬁtness landscapes promoting the partition 1+1 decreases rapidly. However, the number of available fragmentation partitions decreases with L such as at L = 18 the only possible partition is 1+1. Figure 4: Fragmentation cost can drive the evolution of multicellular groups in detrimental ﬁtness landscapes. The top panels show the average size of the parental and offspring groups in optimal life cycles as a function of fragmentation cost for a) fragmentation with delay, b) fragmentation with risk and c) fragmentation with cell loss, respectively. 4 Discussion 319 However, the number of available fragmentation partitions decreases with L such as at L = 18 the only possible partition is 1+1. landscapes. The top panels show the average size of the parental and offspring groups in optimal life cycles as a function of fragmentation cost for a) fragmentation with delay, b) fragmentation with risk and c) fragmentation with cell loss, respectively. Points show the average value, error bars represent one standard deviation. The set of ﬁtness landscapes is given by monotonic random sequences (see main text). The size of offspring groups is strictly one, which means that all observed equal split fragmentations had all offspring being independent cells. landscapes. The top panels show the average size of the parental and offspring groups in optimal life cycles as a function of fragmentation cost for a) fragmentation with delay, b) fragmentation with risk and c) fragmentation with cell loss, respectively. Points show the average value, error bars represent one standard deviation. The set of ﬁtness landscapes is given by monotonic random sequences (see main text). The size of offspring groups is strictly one, which means that all observed equal split fragmentations had all offspring being independent cells. The bottom panels shows the fractions of unicellular (1+1) and multicellular modes of fragmentation At no 17 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint studies shows that reproduction can be indeed costly. Such a costs come in different forms. 323 For instance, consider streptococcus bacteria, which naturally forms cell chains held together 324 by cell walls. To fragment, these cell walls must be broken and the process of unchaining 325 requires the expression of autolysin [Lominski et al., 1958, Shaikh and Stewart-Tull, 1975, 326 Mou et al., 1976]. Autolytic-defective mutants unable to fragment and form long chains 327 [Soper and Winter, 1973, Shungu et al., 1979]. 4 Discussion 319 It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint force of evolution in natural populations is an open experimental question. Nevertheless, we 351 can consider known cases of fragmentation into multiple parts and assess whether a group 352 reproduction is associated with any costs. 353 The ﬁrst example is the bacterium Metabacterium polyspora, inhabiting the gastrointesti- 354 nal tract of guinea pig. The life cycle of this bacterium involves repeatable passages through 355 the tract of multiple hosts. In order to survive such a process, multiple endospores are pro- 356 duced within a single cell [Angert and Losick, 1998], see Fig. 5a). Up to nine endospores 357 can be formed in a single bacterial cell, which make this life cycle a clear example of a frag- 358 mentation into multiple parts. The most apparent cost of reproduction in M. polyspora is that 359 the maternal cell is discarded after the release of endospores. Moreover, the formation of 360 endospores in bacteria is signiﬁcantly different from the normal binary cell division, since 361 the resulting object must survive through much higher stress than the parent cell [Nicholson 362 et al., 2000]. Thus, in addition to the normal machinery involved in DNA replication and cell 363 division, a number of additional processes are involved in production and maturation of the 364 endospore (reviewed in [Angert, 2005]). These processes contribute additional costs of the 365 reproductive event. 366 Another example is a group of segmented ﬁlamentous bacteria [Davis and Savage, 1974], 367 where colonies release two independent cells that grow into new colonies. This reproduction 368 mode can be described by the partition x + 1 + 1, i.e. it corresponds to the seeding class. The 369 colony of segmented ﬁlamentous bacteria originates as a single holdfast-bearing cell, which 370 is capable to attach to the host epithelium. Once this cell settles down, it begins to grow and 371 divide, forming the colony. Since the epithelium is repeatedly renewed tissue, colonies have 372 to give rise to new colonies. This requires production of new holdfast-bearing cells. These 373 cells emerge in a process somewhat similar to the production of endospores - asymmetric 374 division with consequent engulfment of a smaller daughter cell by the larger one. 4 Discussion 319 The necessary investment of resources into 328 autolysin production constitutes the cost of group fragmentation in this case (represented by 329 the scenario of fragmentation with delay in our model). 330 Another example is seeding dispersal in bacterial bioﬁlms. Here, the bioﬁlm composed 331 of mostly sessile cells develop cavities ﬁlled with motile cells, who are then released into 332 the environment [Webb et al., 2003a, McDougald et al., 2012, Claessen et al., 2014]. To 333 develop cavities and motile cells, the bioﬁlm changes its structure [Purevdorj-Gage et al., 334 2005], which inevitable bears an investment costs. Moreover, to free up the space for motile 335 cells and provide nutrients for the differentiation, cells in the cavity die [Tolker-Nielsen et al., 336 2000, Webb et al., 2003b]. Therefore seeding in bioﬁlms is related not to one but to two 337 scenarios of reproduction cost considered in our model. 338 A unique mechanism of group fragmentation has been developed by S. cerevisiae colonies 339 in experimental evolution studies [Ratcliff et al., 2013, 2014]. There an initially unicellular 340 budding yeast was subjected to the selection regime favouring formation of cell clusters. 341 Evolved clusters have a tree-like structure. To facilitate a fragmentation, a single cell in the 342 centre of the tree dies, thus, the integrity of the tree cannot be maintained and eventually 343 the colony breaks into several smaller parts. The death of cell is fragmentation cost in this 344 example. While not being a natural world example, this organism shows that in the need 345 of developing an efﬁcient group fragmentation mode, evolution readily accepts the incurring 346 reproduction costs. 347 Comparing our results with the case of costless fragmentation considered in Pichugin 348 et al. [2017] suggests that the evolution of life cycles involving fragmentation into multi- 349 ple parts may be linked with costly group reproduction. Whether this is an actual driving 350 18 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 4 Discussion 319 Notably, 375 once the new holdfast-bearing cells have matured, the cell containing them undergoes lysis in 376 order to release them into the gastrointestinal tract, see Fig. 5b). Thus, these organisms pay a 377 similar cost of reproduction. 378 19 19 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint b) segmented ﬁlamentous bacteria Two cells are released by lysis of the maternal cell two holdfast-bearing cells grow inside a maternal cell c) Many cells grow inside an extracellular matrix Extracellular matrix is discarded and cells are released Cyanobacterium Stanieria a) several cells grow inside a maternal cell Several cells are released by lysis of the maternal cell Metabacterium polyspora Several cells are released by lysis of the maternal cell a) Metabacterium polyspora several cells grow inside a maternal cell a) Several cells are released by lysis of the maternal cell several cells grow inside a maternal cell Two cells are released by lysis of the maternal cell two holdfast-bearing cells grow inside a maternal cell b) segmented ﬁlamentous bacteria b) segmented ﬁlamentous bacteria two holdfast-bearing cells grow inside a maternal cell Two cells are released by lysis of the maternal cell c) Cyanobacterium Stanieria Extracellular matrix is discarded and cells are released Many cells grow inside an extracellular matrix Many cells grow inside an extracellular matrix Extracellular matrix is discarded and cells are released Cyanobacterium Stanieria . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint Figure 5: Examples of multiple fragmentation in nature and their interpretation by means of Figure 5: Examples of multiple fragmentation in nature and their interpretation by means of our model. a) M. polyspora grows multiple endopsora, released after the maternal cell lysis (picture adopted from [Angert and Losick, 1998]). From the viewpoint of our approach, a group of size x + 1 loses one cell and fragments into x groups of one cell each. b) segmented ﬁlamentous bacteria grows two holdfast-bearing cells inside a maternal cell. These cells are released in the result of the maternal cell lysis (picture adopted from [Davis and Savage, 1974]). From the viewpoint of our approach, a group of size x + 2 loses one cell and fragments according to the partition (x −1) + 1 + 1. c) genus Stanieria grows multiple cells within a single body of extracellular matrix. These cells are released simultaneously upon the break of the matrix (Picture adopted from [Waterbury and Stanier, 1978]). From the viewpoint of our approach, a group of size x fragments into x groups of one cell each, loosing the extracellular matrix, which production required a prior investment of resources. 20 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint An example for an organism with fragmentation cost in a form other than cell loss is 379 the Stanieria genus of cyanobacteria. These organisms are born as independent cells. In the 380 course of their life, these cyanobacteria continuously produce an extracellular matrix, which 381 helps the organism to attach to solid surfaces. Shortly before the reproductive event, the cells 382 undergo a rapid succession of ﬁssions, producing between 4 and 1000 cells. Then, the ex- 383 tracellular matrix gets broken, releasing multiple offspring at once [Waterbury and Stanier, 384 1978], see Fig. 5c. Figure 5: Examples of multiple fragmentation in nature and their interpretation by means of CC-BY 4.0 International license a rtified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: ioRxiv preprint the cost of the fragmentation. Both organisms, support the hypothesis as well, but only on a 407 conceptual level. 408 Figure 5: Examples of multiple fragmentation in nature and their interpretation by means of In this case, the fragmentation cost comes in the form of the lost extra- 385 cellular matrix, which protected and sustained the parent organism, but is not transferred to 386 the offspring cells. The production of the extracellular matrix is distributed across the whole 387 lifespan of the organism, therefore, this scenario lays outside of the scope of the current 388 model, where the cost is assumed to be paid at the last step of the organism’s life. Never- 389 theless, the combination of multiple fragmentation in Stanieria and the apparent costs of the 390 reproduction qualitatively support our hypothesis that fragmentation costs can drive life cycle 391 evolution. 392 379 Other notable examples of multiple fragmentation, which are even further away from our 393 model are algae Gonium pectorale and slime molds. G. pectorale also undergoes sexual 394 reproduction, which violates the assumption of asexual reproduction in our model. Slime 395 molds colonies are formed by aggregation of cells and not by the growth of previous member 396 of the colony. Still, both organisms exhibit fragmentation into multiple parts and signiﬁcant 397 fragmentation costs. G. pectorale spends the majority of its life cycle in a form of 16-cell 398 colony. At the fragmentation, the colony dissolves into 16 independent cells, which originate 399 new colonies [Stein, 1958]. Since the maturity size for G. pectorale is 16 cells, but the frag- 400 mentation does not immediately follow the moment of the reaching this size, this organism 401 has an explicit delay of fragmentation. Slime molds, which are popular model organisms 402 in studies on the evolution of cooperation, form a slime composed of multiple cells. The 403 slime further differentiates into fruiting body containing multiple spores and stalk needed to 404 provide some height to the fruiting body, so spores can be distributed across larger territory 405 [Bonner, 1959]. Cells in the stalk die without contributing to the spores, thus the stalk is 406 21 21 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . the cost of the fragmentation. Both organisms, support the hypothesis as well, but only on a 407 conceptual level. 408 CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint have shown that such ongoing beneﬁts of being in a group are not a necessary condition for 435 the evolution of groups. Another, previously overlooked factor capable to drive the evolution 436 of groups is the cost regularly paid at each reproduction event. The impact of the reproduction 437 cost is strong enough that it may promote formation of multicelluar groups even if the group 438 living put cells in disadvantage comparing with solitary existence. Two factors contribute 439 to this effect. First, the growth to larger size takes more time and thus makes reproduction 440 less frequent, so the cost per time unit is smaller. Second, larger group size at fragmentation 441 makes it possible to share the burden of reproduction cost among more units. This reduction 442 of the impact of the reproduction cost is previously overlooked factor, which promotes the 443 formation of multicellular groups. 444 Given the fascinating diversity of biological life cycles observed even in simple organ- 445 isms, it seems daunting to use theoretical models to understand their features. However, our 446 approach shows that even simple models can capture key aspects of this process and pro- 447 duce results for a whole variety of life cycles. At the same time, these models point towards 448 fragmentation costs as potential drivers of this diversity. 449 5 Acknowledgements 450 We are grateful to David Rogers and Philippe Remigi for fruitful discussions and biological 451 insights. 452 the cost of the fragmentation. Both organisms, support the hypothesis as well, but only on a 407 conceptual level. 408 Another aspect of our study is that all three considered scenarios of the fragmentation 409 cost share the same set of potentially optimal life cycles. For fragmentation with delay, risk 410 and cell loss, only these life cycles, which partitions do not contain two different partitions 411 with the same sum, can be evolutionary optimal. Given the difference between the ways how 412 the considered costs affect the life of a single organism in a population, this result is striking. 413 For costless fragmentation and fragmentation with proportional costs, only binary frag- 414 mentation can be evolutionary optimal [Pichugin et al., 2017], which vastly reduces the num- 415 ber of possible life cycles. For instance, if the group size limited by n = 19, there are only 99 416 binary fragmentations which can evolve for costless fragmentation. The introduction of the 417 ﬁxed fragmentation cost expands the space of optimal life cycles. For costly fragmentation, 418 the number of potentially optimal life cycles is almost 7 times larger: 687. 419 Among all potentially optimal life cycles, we discriminate two special classes: binary 420 fragmentation and equal split. They constitute only a small fraction of all allowed life cycles, 421 see Fig. 2b. However, these two narrow classes of fragmentation modes are evolutionary 422 optimal under majority of random ﬁtness landscapes for all three scenarios of the fragmenta- 423 tion cost, see Fig. 3. Among the natural bacterial populations and simple eukaryotic species, 424 binary ﬁssion is the dominant mode of reproduction (see [Angert, 2005]). The majority of 425 species, which utilize the fragmentation into more than two parts, do it by ﬁssion in multiple 426 unicellular propagules, as discussed above. A notable exception is the reproduction mode 427 of segmented ﬁlamentous bacteria [Davis and Savage, 1974] (see above). Thus, binary frag- 428 mentation and equal split are not only promoted by our model, but also relatively widespread 429 in nature. 430 The evolution of groups from unicellular ancestors is often considered to be driven by 431 some ongoing beneﬁts provided by the group membership such as better protection [Stanley, 432 1973], access to novel resources[Rainey and Travisano, 1998] and the opportunity to coop- 433 erate (reviewed in [Kaiser, 2001] and in [Grosberg and Strassmann, 2007]). In our work we 434 22 . mal under any ﬁtness landscape 455 Following Pichugin et al. [2017], the state of the population can be described by the vector x, 456 where xi denotes the abundance of groups of size i. All processes changing the state vector 457 x – birth, death and fragmentation – occur with a constant rate. Thus, the dynamics of the 458 23 . 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It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint population state can be described by a set of linear differential equations or, equivalently, by 459 a matrix differential equation 460 population state can be described by a set of linear differential equations or, equivalently, by 459 a matrix differential equation 460 ˙x = Ax, (12) ˙x = Ax, (12) (12) where A is a projection matrix deﬁned by demographics of the population [Caswell, 2001]. 461 An element ai,j of the projection matrix describes the rate of change of the number of groups 462 of size i caused by processes occurring with groups of size j. 463 To construct the projection matrix elements, consider groups of a certain size j. We 464 denote by qj,κ the probability that upon the growth from size j to j + 1, the group will 465 fragment by a partition κ ⊢j′ ≤j + 1 (where the “≤” indicates that cells can be lost 466 upon fragmentation). Among these partitions we distinguish the trivial partition of j + 1 that 467 corresponds to the growth without fragmentation; we denote this by qj,(j+1). The combined 468 probability of all outcomes is equal to one: 469 X κ qj,κ = 1. (13) (13) For deterministic life cycles, only one partition occurs in all groups in a population. Thus, 470 for group sizes j up to maturity size m, the trivial partition occurs with probability one 471 (qj,(j+1) = 1), while all other partitions have zero probability. mal under any ﬁtness landscape 455 Once the group grows from 472 the maturity size, a certain non-trivial partition of j′ ≤m + 1 occurs with probability one. In 473 a stochastic life cycle, more than one partition has non-zero probability at least at one group 474 size. Therefore, the projection matrix is different from Eq. (5). 475 To show that stochastic life cycles are dominated by deterministic ones, we construct the 476 projection matrix for an arbitrary stochastic life cycle. Groups grow by one cell at a time, 477 thus no process can increase the size of group by more than one unit at once, so ai,j = 0 for 478 all i > j + 1. Thus, the projection matrix may contain non-zero elements only in the upper 479 right triangle (emergence of smaller groups during fragmentation), on the main diagonal 480 (fragmentation, growth and death of clusters), and on the ﬁrst lower subdiagonal (growth of 481 clusters to sizes larger by one cell). 482 The ﬁrst lower subdiagonal describes the rate of emergence of new larger groups in a 483 result of group growth without fragmentation. These rates are equal to the product of the 484 24 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a rtified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: ioRxiv preprint basic growth rate and the probability of the group to grow: 485 aj+1,j = jbjqj,(j+1). (14) aj+1,j = jbjqj,(j+1). (14) The upper right triangle of the matrix describes the emergence of new groups in a result of 486 fragmentation of larger groups. For a given partition κ and given size of the newborn group 487 i, the rate of production of new groups is equal to the product of the fragmentation rate (jb′ j), 488 the probability to fragment according to the given partition (qj,κ), and the number of groups 489 of given size produced in the act of fragmentation with this partition (πi(κ)). mal under any ﬁtness landscape 455 The value of an 490 element ai,j in the upper left triangle is equal to the sum of rates provided by all partitions 491 available to groups of size j: 492 The upper right triangle of the matrix describes the emergence of new groups in a result of 486 fragmentation of larger groups. For a given partition κ and given size of the newborn group 487 i, the rate of production of new groups is equal to the product of the fragmentation rate (jb′ j), 488 the probability to fragment according to the given partition (qj,κ), and the number of groups 489 of given size produced in the act of fragmentation with this partition (πi(κ)). The value of an 490 element ai,j in the upper left triangle is equal to the sum of rates provided by all partitions 491 available to groups of size j: 492 ai,j = jb′ j X κ qj,κπi(κ). (15) ai,j = jb′ j X κ qj,κπi(κ). (15) (15) κ The main diagonal ai,i describes the changes in groups numbers due to growth and frag- 493 mentations as well as the death of groups. The ﬁrst component of ai,i is given by the fact that 494 once a group of size j grows or fragments, the number of groups of that size decreases. The 495 rates of decrease are equal to jbjqj,(j+1) due to the growth and jb′ j P κ qj,κ due to the fragmen- 496 tations. The second component is provided by the fragmentation with partition κ = j + 1, 497 which produce groups of size equal to the size of parent. This leads to an increase in the num- 498 ber of groups of size j at rate jb′ jqj,j+1πj(j + 1), where π1 (1 + 1) = 2 and πj (j + 1) = 1 if 499 j > 1. The last component of ai,i comes from the death of groups, which leads to a decrease 500 in their number at rate djqj,(j+1) + d′ j P κ qj,κ, where the ﬁrst term describes the death rate in 501 the absence of the fragmentation and the second term describes the death rate of fragmenting 502 groups. Combined, the diagonal elements of projection matrix are 503 aj,j = −jbjqj,(j+1) −jb′ j X κ qj,κ + jb′ jqj,j+1πj(j + 1) −djqj,(j+1) −d′ j X κ qj,κ. mal under any ﬁtness landscape 455 (16) aj,j = −jbjqj,(j+1) −jb′ j X κ qj,κ + jb′ jqj,j+1πj(j + 1) −djqj,(j+1) −d′ j X κ qj,κ. (16) (16) All elements of the projection matrix given by Eqs. (14)-(16) are linear with respect to 504 any probability qj,κ. As shown in Pichugin et al. [2017], in this case the optimal life cycle is 505 always deterministic, independent of the parameter values, such as the ﬁtness landscape and 506 the scenario of the fragmentation cost. 507 25 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a fied by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: oRxiv preprint A.2 Characteristic equation of a deterministic fragmentation mode 508 CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint Substituting Eq. (18) and Eq. (19) into Eq. (17) and simplifying, we obtain 521 A.2 Characteristic equation of a deterministic fragmentation mode 508 Consider a deterministic fragmentation mode in which groups grow up to the maturity size 509 m and once the next cell is born, fragment according to a partition κ ⊢j′ ≤m + 1. The 510 corresponding projection matrix is an m × m matrix of the form 511 A =          −b1 −d1 0 0 0 · · · mb′ mπ1(κ) b1 −2b2 −d2 0 0 · · · mb′ mπ2(κ) 0 2b2 −3b3 −d3 0 · · · mb′ mπ3(κ) 0 0 ... ... ... ... 0 0 0 · · · (m −1)bm−1 mb′ mπm(κ) −mb′ m −d′ m          . The population growth rate is given by the leading eigenvalue λ1 of A, i.e., the largest 512 solution of the characteristic equation 513 The population growth rate is given by the leading eigenvalue λ1 of A, i.e., the largest 512 solution of the characteristic equation 513 The population growth rate is given by the leading eigenvalue λ1 of A, i.e., the largest 512 solution of the characteristic equation 513 p p g g y g g 1 , , g solution of the characteristic equation 513 det (A −λI) = 0. (17) det (A −λI) = 0. (17) (17) By using a Laplace expansion along the last column of A −λI, we can rewrite the left hand 514 side of the above expression (i.e., the characteristic polynomial of A) as 515 det (A −λI) = m−1 X i=1 (−1)i+mmb′ mπi(κ)Mi,m+ (−1)2m (mb′ mπm(κ) −mb′ m −d′ m −λ) Mm,m = m X i=1 (−1)i+mmb′ mπi(κ)Mi,m −(mb′ m + d′ m + λ) Mm,m (18) (18) where Mi,m is the (i, m) minor of A −λI. For all i = 1, . . . , m, the minor Mi,m is the 516 determinant of a block diagonal matrix, and hence equal to the product of the determinants of 517 the diagonal blocks. Moreover, each diagonal block is either a lower triangular or an upper 518 triangular matrix, whose determinant is given by the product of the elements in their main 519 diagonals. We can then write 520 Mi,m = i−1 Y j=1 (−jbj −dj −λ) m−1 Y j=i jbj. (19) (19) 26 . Substituting Eq. (18) and Eq. (19) into Eq. (17) and simplifying, we obtain 521 (−1)m−1 m X i=1 mb′ mπi(κ) i−1 Y j=1 (jbj + dj + λ) m−1 Y j=i jbj (−1)m−1 m X i=1 mb′ mπi(κ) i−1 Y j=1 (jbj + dj + λ) m−1 Y j=i jbj −(−1)m−1 (mb′ m + d′ m + λ) m−1 Y j=1 (jbj + dj + λ) = 0. −(−1)m−1 (mb′ m + d′ m + λ) m−1 Y j=1 (jbj + dj + λ) = 0. Dividing both sides by 522 (−1)m m Y j=1 jbj, we get 523 (−1)m m Y j=1 jbj, we get 523 mb′ m + d′ m + λ mbm m−1 Y j=1 1 + dj + λ jbj − m X i=1 b′ m bm πi(κ) i−1 Y j=1 1 + dj + λ jbj = 0. mb′ m + d′ m + λ mbm m−1 Y j=1 1 + dj + λ jbj − m X i=1 b′ m bm πi(κ) i−1 Y j=1 1 + dj + λ jbj = 0. To move the ﬁrst multiplier with λ into the product, we rewrite it as 524 To move the ﬁrst multiplier with λ into the product, we rewrite it as 524 mb′ m + d′ m + λ mbm = 1 + dm + λ mbm + m(b′ m −bm) + d′ m −dm mbm . Thus, 525 m Y j=1 1 + dj + λ jbj + m(b′ m −bm) + d′ m −dm mbm m−1 Y j=1 1 + dj + λ jbj − m X i=1 b′ m bm πi(κ) i−1 Y j=1 1 + dj + λ jbj = 0. Simplifying this, we ﬁnally obtain that the characteristic equation (17) can be written as 526 Fm+1(λ) + ∆mFm(λ) −b′ m bm m X i=1 πi(κ)Fi(λ) = 0, (20) (20) where 527 where 527 Fi(λ) = i−1 Y j=1 1 + dj + λ jbj . (21) and 528 ∆i = i(b′ i −bi) + d′ i −di ibi . (22) where 527 Fi(λ) = i−1 Y j=1 1 + dj + λ jbj . and 528 where 527 Fi(λ) = i−1 Y j=1 1 + dj + λ jbj . Substituting Eq. (18) and Eq. (19) into Eq. (17) and simplifying, we obtain 521 (21) and 528 i(b′ b ) + d′ d where 527 Fi(λ) = i−1 Y j=1 1 + dj + λ jbj . (21) (21) and 528 ∆i = i(b′ i −bi) + d′ i −di ibi . (22) ∆i = i(b′ i −bi) + d′ i −di ibi . (22) (22) 27 27 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint Note that two transformations preserve Eq. (20): 529 d →d −r, λ1 →λ1 + r, d′ →d′ −r, r ≤min(d), (23) and 530 d →sd, b →sb, b′ →sb′, d′ →sd′, λ1 →sλ1, s > 0. d →d −r, λ1 →λ1 + r, d′ →d′ −r, r ≤min(d), (23) (23) and 530 and 530 d →sd, b →sb, b′ →sb′, d′ →sd′, λ1 →sλ1, s > 0. These transformations allow us to set b1 = 1 and min(d) = 0 without loss of generality. 531 These transformations allow us to set b1 = 1 and min(d) = 0 without loss of generality. 531 A.3 Forbidden fragmentation modes 532 Hence, it must be that only one of the following three scenarios is satisﬁed: (i) p2(λ(κ1)) < 557 0 < p3(λ(κ1)), (ii) p2(λ(κ1)) = p3(λ(κ1)) = 0, or (iii) p2(λ(κ1)) > 0 > p3(λ(κ1)). If 558 p2(λ(κ1)) < 0 < p3(λ(κ1)), and by virtue of Eq. (25) and Bolzano’s theorem (if a continuous 559 function has values of opposite sign inside an interval, then it has a root in that interval), p2(λ) 560 has a root between λ(κ1) and ∞. Therefore, λ(κ1) ≤λ(κ2) holds. Likewise, if p2(λ(κ1)) > 561 0 > p3(λ(κ1)), then λ(κ1) ≤λ(κ3) holds. Finally, if p2(λ(κ1)) = p3(λ(κ1)) = 0, then 562 both λ(κ1) ≤λ(κ2) and λ(κ1) ≤λ(κ3) hold. We conclude that either λ(κ1) ≤λ(κ2) or 563 λ(κ1) ≤λ(κ3) must hold. 564 A.3 Forbidden fragmentation modes 532 It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint To prove the statement above, let us deﬁne the polynomial pi(λ) as the left hand side of 550 Eq. (20) with κ = κi, so that λ(κi) is the largest root of pi(λ). We obtain 551 To prove the statement above, let us deﬁne the polynomial pi(λ) as the left hand side of 550 Eq. (20) with κ = κi, so that λ(κi) is the largest root of pi(λ). We obtain 551 Eq. (20) with κ = κi, so that λ(κi) is the largest root of pi(λ). We obtain 551 p1(λ) = Fm+1(λ) + ∆mFm(λ) −b′ m bm m X i=1 πi(τ1)Fi(λ) + m X i=1 πi(τ2)Fi(λ) + m X i=1 πi(φ)Fi(λ) ! (24a) p2(λ) = Fm+1(λ) + ∆mFm(λ) −b′ m bm 2 m X i=1 πi(τ1)Fi(λ) + m X i=1 πi(φ)Fi(λ) ! (24b) p3(λ) = Fm+1(λ) + ∆mFm(λ) −b′ m bm 2 m X i=1 πi(τ2)Fi(λ) + m X i=1 πi(φ)Fi(λ) ! . (24c) (24a) (24b) These polynomials satisfy the following two properties. First, 552 These polynomials satisfy the following two properties. First, 552 lim λ→∞pi(λ) = ∞, (25) lim λ→∞pi(λ) = ∞, (25) as the leading coefﬁcient of the left hand side of Eq. (20) is given by (b1 · b2 · . . . · bmm!)−1, 553 which is always positive. Second, 554 as the leading coefﬁcient of the left hand side of Eq. (20) is given by (b1 · b2 · . . . · bmm!)−1, 553 which is always positive. Second, 554 as the leading coefﬁcient of the left hand side of Eq. (20) is given by (b1 · b2 · . . . · bmm!)−1, 553 which is always positive. Second, 554 p1(λ) = p2(λ) + p3(λ) 2 . (26) p1(λ) = p2(λ) + p3(λ) 2 . (26) (26) Evaluating Eq. (26) at λ(κ1), and since λ(κ1) is a root of p1(λ), p1(λ(κ1)) = 0, it then follows 555 that 556 Evaluating Eq. (26) at λ(κ1), and since λ(κ1) is a root of p1(λ), p1(λ(κ1)) = 0, it then follows 555 that 556 p2(λ(κ1)) = −p3(λ(κ1)). p2(λ(κ1)) = −p3(λ(κ1)). p2(λ(κ1)) = −p3(λ(κ1)). A.3 Forbidden fragmentation modes 532 For any ﬁtness landscape, for any combination of the fragmentation delay, risk and ﬁxed loss, 533 the fragmentation mode having two different subsets of offspring with the same combined 534 size is dominated. To prove this, we use approach similar to one used in Appendix E in 535 [Pichugin et al., 2017]. Consider positive integers m, j, k such that m + 1 ≥2j + k, two 536 partitions τ1 ⊢j and τ2 ⊢j such that τ1 ̸= τ2, and an arbitrary partition φ ⊢k, and the 537 following three deterministic fragmentation modes: 538 1. κ1 = τ1 + τ2 + φ ⊢2j + k ≤m + 1, whereby a complex fragments upon growth from 539 size m into a number of offspring given by partitions τ1, τ2, and φ. 540 1. κ1 = τ1 + τ2 + φ ⊢2j + k ≤m + 1, whereby a complex fragments upon growth from 539 size m into a number of offspring given by partitions τ1, τ2, and φ. 540 2. κ2 = τ1 + τ1 + φ ⊢2j + k ≤m + 1, whereby a complex fragments upon growth from 541 size m into a number of offspring given by two partitions τ1 and one partition φ. 542 3. κ3 = τ2 + τ2 + φ ⊢2j + k ≤m + 1, whereby a complex fragments upon growth from 543 size m into a number of offspring given by two partitions τ2 and one partition φ. 544 Denoting by λ(κi) the leading eigenvalue of the projection matrix induced by fragmenta- 545 tion mode κi, we can show that, for any ﬁtness landscape, either λ(κ1) ≤λ(κ2) or λ(κ1) ≤ 546 λ(κ3) holds. This means that a fragmentation mode with two different subsets of offspring 547 with the same combined size is dominated by a mode where one of these subsets repeats 548 twice, while another one is not present. 549 28 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a rtified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. A.4 Optimal life cycles under large delay of fragmentation 565 Consider the deterministic life cycle that follows partition κ. Its proliferation rate is given 566 by Eq. (8). Under fragmentation with delay, the birth rate at the fragmentation size changes 567 29 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint according to 568 according to 568 1 mb′ m = 1 mbm + T. (27) 1 mb′ m = 1 mbm + T. (27) (27) At large delay T ≫ 1 mbm, b′ m can be approximated as 569 b′ m ≈ 1 mT ≪1 (28) At large delay T ≫ 1 mbm, b′ m can be approximated as 569 At large delay T ≫ 1 mbm, b′ m can be approximated as 569 b′ m ≈ 1 mT ≪1 (28) b′ m ≈ 1 mT ≪1 (28) (28) Thus, ∆m given by Eq. (22) can be approximated by: 570 Thus, ∆m given by Eq. (22) can be approximated by: 570 Thus, ∆m given by Eq. (22) can be approximated by: 570 ∆m = m(b′ m −bm) + d′ m −dm mbm ≈ 1 bmmT −1. (29) ∆m = m(b′ m −bm) + d′ m −dm mbm ≈ 1 bmmT −1. (29) ∆m = m(b′ m −bm) + d′ m −dm mbm ≈ 1 bmmT −1. (29) Therefore, Eq. (20) becomes: 571 1 1 m (29) mbm bmmT Therefore, Eq. (20) becomes: 571 Fm+1(λ) + 1 b mT −1 Fm(λ) − 1 b mT m X πi(κ)Fi(λ) = 0, (30) Therefore, Eq. (20) becomes: 571 Fm+1(λ) + 1 bmmT −1 Fm(λ) − 1 bmmT m X i=1 πi(κ)Fi(λ) = 0, (30) (30) The delay value T contribute to this equation only in a form of factor 1 T ≪1. To analyse the 572 solutions of obtained equation, we ﬁrst discard all terms containing 1 T in Eq. A.4 Optimal life cycles under large delay of fragmentation 565 (30) and get 573 The delay value T contribute to this equation only in a form of factor 1 T ≪1. To analyse the 572 solutions of obtained equation, we ﬁrst discard all terms containing 1 T in Eq. (30) and get 573 Fm+1(λ) −Fm(λ) = 0, Fm+1(λ) −Fm(λ) = 0, Substituting the expression of Fi(λ) from Eq. (21) we get 574 Substituting the expression of Fi(λ) from Eq. (21) we get 574 dm + λ mbm m−1 Y j=1 1 + dj + λ jbj = 0. (31) (31) We denote the solutions of this equation as λ0. There are m solutions of this equation: 575 one solution λ0 m,m = −dm and m −1 solutions in a form λ0 j,m = −(jbj + dj), where 576 j ∈{1, 2, · · · , m −1}. For any solution in a form λ0 j,m = −(jbj + dj), we can ﬁnd another 577 life cycle fragmenting already at size j < m for which the solution λ0 j,j = −dj > λ0 j,m exists. 578 Thus, the proliferation rate of the optimal life cycle must have the form λ0 = −dm + O 1 T . 579 As a consequence, for high fragmentation delay, under the optimal life cycle, group fragments 580 after reaching the most protected state with the minimal di. 581 To ﬁnd which of many fragmentation modes available to the group reproducing at the 582 most protected state is evolutionary optimal, we consider the ﬁrst order approximation of the 583 growth rate given by 584 λ ≈λ0 + 1 T λ1 = −dm + 1 T λ1. (32) (32) 30 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint To ﬁnd λ1 we substitute Eq. (32) into Eq. (30), 585 Fm+1 −dm + 1 T λ1 −Fm −dm + 1 T λ1 + 1 bmmT " Fm −dm + 1 T λ1 − m X i=1 πi(κ)Fi −dm + 1 T λ1 # = 0, Then we use expressions of Fi(λ) from Eq. (21) and discard all terms smaller than 586 Then we use expressions of Fi(λ) from Eq. (21) and discard all terms smaller than 1 T 586 λ1 mbmT Fm(−dm) + 1 bmmT " Fm(−dm) − m X i=1 πi(κ)Fi(−dm) # = 0. Thus, 587 Thus, 587 λ1 = Pm i=1 πi(κ)Fi(−dm) Fm(−dm) −1. λ1 = Pm i=1 πi(κ)Fi(−dm) Fm(−dm) −1. In the optimal life cycle under high delay of fragmentation, groups fragment according to the 588 partition that provides the highest value of λ1. 589 In the optimal life cycle under high delay of fragmentation, groups fragment according to the 588 partition that provides the highest value of λ1. 589 For the special case of the constant death rate, the optimal life cycle can be found explic- 590 itly. In this case, the death rate can be set to d = 0 (see Eq. (23)), so 591 For the special case of the constant death rate, the optimal life cycle can be found explic- 590 itly. In this case, the death rate can be set to d = 0 (see Eq. (23)), so 591 λ1 = Pm i=1 πi(κ)Fi(0) Fm(0) −1. λ1 = Pm i=1 πi(κ)Fi(0) Fm(0) −1. λ1 = Pm i=1 πi(κ)Fi(0) Fm(0) −1. At d = 0, Fi(0) = 1, so: 592 At d = 0, Fi(0) = 1, so: 592 λ1 = m X i=1 πi(κ) −1, λ1 = m X i=1 πi(κ) −1, λ1 = m X i=1 πi(κ) −1, the right hand side of this expression is the number of produced offspring groups minus one. 593 This expression is maximized by the life cycle producing the maximal number of offspring 594 groups, i.e. by the equal split life cycle producing only unicellular propagules. 595 For the set of random ﬁtness landscapes used in section 3.2, the minimum of di is evenly 596 distributed across all considered sizes {1, 2, · · · , 19}. To ﬁnd λ1 we substitute Eq. (32) into Eq. (30), Thus, at large delay, the size of frag- 597 mentation should be evenly distributed as well, which corresponds to average fragmentation 598 size equal to 10 and standard variation of sizes equal to √ 30. The mean and standard varia- 599 tion of the observed distribution of fragmentation sizes quickly approach these values in our 600 numerical simulations, cf. Fig. 3. 601 the right hand side of this expression is the number of produced offspring groups minus one. 593 This expression is maximized by the life cycle producing the maximal number of offspring 594 groups, i.e. by the equal split life cycle producing only unicellular propagules. 595 For the set of random ﬁtness landscapes used in section 3.2, the minimum of di is evenly 596 distributed across all considered sizes {1, 2, · · · , 19}. Thus, at large delay, the size of frag- 597 mentation should be evenly distributed as well, which corresponds to average fragmentation 598 size equal to 10 and standard variation of sizes equal to √ 30. The mean and standard varia- 599 tion of the observed distribution of fragmentation sizes quickly approach these values in our 600 numerical simulations, cf. Fig. 3. 601 31 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint For the set of ﬁtness landscapes detrimental to larger groups used in section 3.3, the 602 minimum of di is achieved always at d1. Therefore, the maturity size for large delay is 1, 603 which corresponds to the unique fragmentation pattern 1 + 1. In our simulations the initial 604 increase in T resulted in the gradual decrease of the fraction of ﬁtness landscapes promoting 605 1 + 1 to zero. To ﬁnd λ1 we substitute Eq. (32) into Eq. (30), However, further increase of T make some ﬁtness landscapes promote 1 + 1 606 again, and above some intermediary value of T, the fraction of ﬁtness landscapes promoting 607 1 + 1 begin to increase, see Fig. 4a. 608 602 A.5 Optimal life cycles under high risk of fragmentation 609 Consider the deterministic life cycle that follows partition κ. It’s proliferation rate is given 610 by Eq. (8). For fragmentation with risk, the death rate at the fragmentation size changes 611 according to 612 d′ i = di + R (33) d′ i = di + R (33) Thus, ∆m given by Eq. (10) becomes 613 Thus, ∆m given by Eq. (10) becomes 613 ∆m = m(b′ m −bm) + d′ m −dm mbm = R mbm . (34) (34) Therefore, Eq. (8) becomes 614 Therefore, Eq. (8) becomes 614 Fm+1(λ) + R mbm Fm(λ) − m X i=1 πi(κ)Fi(λ) = 0, (35) Or, after dividing by R, 15 Fm+1(λ) + R mbm Fm(λ) − m X i=1 πi(κ)Fi(λ) = 0, (35) (35) m i=1 Or, after dividing by R, 615 1 mbm Fm(λ) + 1 R Fm+1(λ) − m X i=1 πi(κ)Fi(λ) ! = 0, (36) Or, after dividing by R, 615 , g y , 1 mbm Fm(λ) + 1 R Fm+1(λ) − m X i=1 πi(κ)Fi(λ) ! = 0, (36) 1 mbm Fm(λ) + 1 R Fm+1(λ) − m X i=1 πi(κ)Fi(λ) ! = 0, (36) (36) To analyse the solutions of obtained equation, we ﬁrst discard all terms containing 1 R and get 616 To analyse the solutions of obtained equation, we ﬁrst discard all terms containin 6 1 mbm m−1 Y j=1 1 + dj + λ0 jbj = 0, (37) (37) For m > 1, this equation has m −1 solutions in a form λ0 = −jbj −dj. Thus, the ﬁrst 617 approximation of the proliferation rate, given by the maximal root of this equation, is equal 618 to 619 λ0 = −min 0<i<m(ibi + di). λ0 = −min 0<i<m(ibi + di). 32 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. A.5 Optimal life cycles under high risk of fragmentation 609 ; https://doi.org/10.1101/325670 doi: bioRxiv preprint For m = 1, this equation has no solution, instead the proliferation rate of the population 620 undergoing 1+1 life cycles (the only life cycle with m = 1) is given by 621 For m = 1, this equation has no solution, instead the proliferation rate of the population 620 undergoing 1+1 life cycles (the only life cycle with m = 1) is given by 621 1 b1 F1(λ) + 1 R (F2(λ) −2F1(λ)) = 1 b1 + 1 R 1 + λ + d1 b1 −2 = 0, Thus, for κ = 1 + 1, the proliferation rate is given by 622 λ1+1 = −R −d1 + b1 ≪−1 λ1+1 = −R −d1 + b1 ≪−1 Thus, under high risk of fragmentation, the life cycle with κ = 1 + 1 is dominated by any 623 other life cycle. Accordingly, in our simulations, the proportion of unicellular life cycles 624 monotonically decreases with the increase in R, see Figs. 3e) and 4. Therefore, according to 625 the approximation, natural selection promotes life cycles with maturity size m greater than 626 the critical value i∗minimizing expression ibi + di. 627 Thus, under high risk of fragmentation, the life cycle with κ = 1 + 1 is dominated by any 623 other life cycle. Accordingly, in our simulations, the proportion of unicellular life cycles 624 monotonically decreases with the increase in R, see Figs. 3e) and 4. Therefore, according to 625 the approximation, natural selection promotes life cycles with maturity size m greater than 626 the critical value i∗minimizing expression ibi + di. 627 To distinguish between such life cycles, we consider the ﬁrst order approximation of the 628 growth rate given by 629 λ ≈λ0 + 1 Rλ1 = −min 0<i<m(ibi + di) + 1 Rλ1. (38) (38) We substitute λ in the form of Eq. (38) into Eq. (36) and discard all terms smaller than 1 R 630 We substitute λ in the form of Eq. (38) into Eq. (36) and discard all term 630 We substitute λ in the form of Eq. (38) into Eq. A.5 Optimal life cycles under high risk of fragmentation 609 640 For the special case where d = 0 and bi does not decrease faster than i−1, the sequence 638 ibi +di monotonically increases. Hence, i∗= 1, so the optimal life cycle is the fragmentation 639 into unicellular propagules. 640 For the set of random ﬁtness landscapes used in section 3.2, the expression ibi + di tend 641 to grow with i, so its minimum i∗is more likely to be achieved at small values of i. Since, i∗ 642 establishes an upper limit on the size of offspring groups, our analysis suggests that this size 643 should decrease with R. 644 For the set of random ﬁtness landscapes used in section 3.2, the expression ibi + di tend 641 to grow with i, so its minimum i∗is more likely to be achieved at small values of i. Since, i∗ 642 establishes an upper limit on the size of offspring groups, our analysis suggests that this size 643 should decrease with R. 644 A.5 Optimal life cycles under high risk of fragmentation 609 (36) and discard all terms smaller than 1 R 630 1 mbm λ1 Ri∗bi∗ Y i∈(1,··· ,m−1)\i∗ 1 + λ0 + di ibi −1 R i∗ X i=1 πi(κ)Fi(λ0) = 0 (39) (39) Note, that offspring groups of size larger than i∗do not contribute to sum at the end of the 631 expression at the left hand side, because Fi>i∗(λ0) = 0. The term linear with respect to 1 R is 632 equal to 633 i∗ Note, that offspring groups of size larger than i∗do not contribute to sum at the end of the 631 expression at the left hand side, because Fi>i∗(λ0) = 0. The term linear with respect to 1 R is 632 equal to 633 i∗ λ1 = mbmi∗bi∗ Pi∗ i=1 πi(κ)Fi(λ0) Q i∈(1,··· ,m−1)\i∗ 1 + λ0+di ibi (40) (40) The optimal life cycle maximizes this expression. 634 optimal life cycle maximizes this expression. The optimal life cycle maximizes this expression. 634 The optimal life cycle maximizes this expression. 634 For any given maturity size m, the life cycle producing more offspring groups with size 635 not exceeding than i∗has higher λ1. Thus, under the optimal life cycle, the size of offspring 636 cannot be larger than i∗. 637 For any given maturity size m, the life cycle producing more offspring groups with size 635 not exceeding than i∗has higher λ1. Thus, under the optimal life cycle, the size of offspring 636 cannot be larger than i∗. 637 33 33 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted May 18, 2018. ; https://doi.org/10.1101/325670 doi: bioRxiv preprint For the special case where d = 0 and bi does not decrease faster than i−1, the sequence 638 ibi +di monotonically increases. Hence, i∗= 1, so the optimal life cycle is the fragmentation 639 into unicellular propagules. References 645 George E. Andrews. The Theory of Partitions. Cambridge University Press, Cambridge, UK, 646 1998. 647 E.R. Angert. Alternatives to binary ﬁssion in bacteria. Nature Reviews Microbiology, 3(3): 648 214–224, 2005. 649 E.R. Angert and R. M. Losick. Propagation by sporulation in the guinea pig symbiont 650 metabacterium polyspora. Proceedings of the National Academy of Sciences, 95(17):10218 651 – 10233, 1998. 652 C.B. Basbaum and W. Zena. 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https://openalex.org/W2767835635	https://zenodo.org/records/1121203/files/10.21307_stattrans-2016-070.pdf	English	null	A Multidimensional and Dynamised Classification of Polish Provinces Based on Selected Features of Higher Education in 2002–2013	Statistics in Transition	2,017	cc-by	6,662	STATISTICS IN TRANSITION new series, June 2017 Vol. 18, No. 2, pp. 271–290, DOI: 10.21307/stattrans-2016-070 STATISTICS IN TRANSITION new series, June 2017 Vol. 18, No. 2, pp. 271–290, DOI: 10.21307/stattrans-2016-070 1 Faculty of Management, President Wojciechowski Higher Vocational State School, Kalisz, Poland. E-mail: w.lukaszonek@g.pl. 2 Rankings by Perspektywy and Rzeczpospolita/Gazeta Prawna (2002–2015); Shanghai Ranking (2010–2015). A MULTIDIMENSIONAL AND DYNAMISED CLASSIFICATION OF POLISH PROVINCES BASED ON SELECTED FEATURES OF HIGHER EDUCATION IN 2002–2013 Wojciech Łukaszonek1 ABSTRACT For close to two decades after the fall of communism in 1989, Polish higher education enjoyed an unprecedented period of development. Favourable political, economic, social and demographic changes led to a fivefold increase in the number of students and the number of higher educational institutions. The dynamic changes and their effects did not occur uniformly, in either space or time. An attempt is made here to identify and analyse the regional differentiation between Polish provinces in terms of features relating to higher education. To investigate the changes in higher education in the period of economic and social transformation, observations were made of fundamental characteristics of higher education in the years 2002–2013. The applied procedure uses new statistical methods applicable to a space of doubly multivariate data. The covariance matrix used to construct principal components is given the structure of a Kronecker product. The results led to the identification of six groups of provinces, including two consisting of a single province – Mazowieckie and Małopolskie provinces – which contain the largest and the highest-ranked2 higher educational institutions in Poland: the University of Warsaw and Jagiellonian University. Key words: higher education, doubly multivariate data, cluster analysis, dendrite method, covariance matrix, Kronecker product. 1. Introduction The systemic changes that took place in Poland in the late 1980s and early 1990s led to transformation of the country in many different areas, including economic, social and cultural ones (Golinowska, ed. 2005). The process of changes in the 1990s also affected education, and its effects made a strong impression on the system of higher education. The economic reforms forced changes in the labour market, which led to increased needs for highly qualified staff. One consequence was a rapid growth in the number of people entering higher education. A degree seemed to be a guarantee of well-paid work, the possibility of further development, economic independence and improved social status (Sikorska 1998, Mach 2003). It had previously been an elite attribute, as reflected in the number of graduates in the population. In the centrally planned Polish economy of the 1970s and 1980s, the higher education system was closely controlled by the authorities, and student numbers were centrally regulated (Wnuk-Lipińska 1996, Antonowicz 2012, Kwiek 2014). In 1990 the percentage of the Polish population holding degrees was approximately 6%, this being a result of the policy applied in previous years. The adoption of democratic principles, giving more freedom to citizens, had a strong impact on social behaviours. There was an increase in Poles’ educational aspirations, linked to the economic changes that were reflected in the dynamic expansion of the private sector (Ziółkowski 2000, Kwiek 2014). The increased demand for employment was accompanied by demographic changes, manifested in an increase in the population aged 19–24, the time at which higher education is undertaken. In effect, the number of students increased extremely rapidly. Over the years 1990–2005 the total number increased almost fivefold, from approximately 400,000 to almost 2 million (Fig. 1). Figure 1. Numbers of students and of higher educational institutions in 1990–2013 Source: based on Higher Education 2014, GUS 2015. Figure 1. Numbers of students and of higher educational institutions in 1990–2013 Source: based on Higher Education 2014, GUS 2015. The universities and colleges then existing in Poland were not prepared (particularly in terms of infrastructure) for such a rapid rise in the number of people interested in studying. In 1990 there were 112 higher educational institutions, all of them – apart from the Catholic University of Lublin – being state-run (public) institutions. The free market principles adopted at that time, and the Act on higher education (Dz.U. 1990 no. 3 On the GUS website, the net index of student numbers is defined as the ratio of the number of persons (in a given age group) in a given level of education at the start of the school year to the total population (at 31 December) in the age group corresponding to that level of education. For example, the net index for the primary school level is calculated by dividing the number of primary school pupils aged 7–12 (the age assigned to that level) at the start of a given school year by the total population aged 7–12 at 31 December of the same year. The result is given as a percentage. 1. Introduction 65 item 385), enabled the foundation of Poland’s first private higher educational institutions. The requirements for such an establishment were very liberal and relatively easy to fulfil, which gave an impetus to an unprecedented, and in effect uncontrolled, process of privatisation of higher education (Kwiek 2014). In the 15 years following the fall of communism, a total of 315 non-public (private) higher educational institutions were established in Poland, filling the gap in the market that had arisen due to the increasing public desire to study and the inability of state institutions to meet that need (Misztal 2000, Wasielewski 2013). From 1990 to 2010 the number of higher educational institutions in Poland increased almost fivefold (similarly to the growth in the number of students; Fig. 1). The educational boom of that period naturally led to an increase in numbers of graduates. The net index of student numbers3 (among persons aged 19–24) increased from 9.8% in 1990 to 40.8% in 2010, which is in agreement with the growth in the number of students (Fig. 2). Since 2005 the index has remained above 38%, one of the highest values among the countries of the European Union (GUS 2009, 2012, 2015). Figure 2. Number of students, number of persons aged 19–24, net index of student numbers, 2002–2013 Source: based on Higher Education 2014, GUS 2015. Figure 2. Number of students, number of persons aged 19–24, net index of student numbers, 2002–2013 Source: based on Higher Education 2014, GUS 2015. The 1990s, which introduced democratic norms into social and political life, and free market values into the economy, brought to light social inequalities (not previously noticed in the post-war period) and the phenomena causing them (including unemployment, educational levels inadequate to the needs of the labour market, physical disability, and other factors). The period saw a growth in the importance of statutory measures aimed at counteracting social exclusion. One such decision was the Act on vocational colleges (Dz.U. 1997 no. 96 item 590), whose aims included enabling persons in difficult economic and life situations to undertake higher education. The establishment of national vocational colleges in smaller cities, bringing higher education closer to places distant from large academic centres, enabled the powerful development of local communities. These colleges were intended as a response to the needs of local labour markets, providing teaching oriented towards the professional and practical dimensions of academic subjects. The decision to find such institutions was in line with the international trend towards making higher education more widely available to the general population (Trow 1973). The speed of changes in the Polish higher education system, whose scale was unprecedented anywhere in Europe, and the consequences of those changes for economic and social development in Poland, provided the motivation for the present work. The aim of the research is to identify and investigate the regional differentiation between Polish provinces in terms of features relating to higher education in the period of economic and social transformation. Observations were made in doubly multivariate way presenting: (i) fundamental characteristics of higher education and (ii) their changes in the years 2002–2013. The next section of the article will present the features relating to higher education that were selected as diagnostic variables. The third section will describe the research procedure and statistical methods used. Since the study was based on doubly multivariate data, a method of principal component analysis for data of that type was applied. Delimitation of provinces was performed using the dendrite method (Florek et al. 1951, Kruskal 1956, Prim 1957) applied to the obtained principal components. The results of the classification of Polish provinces obtained by the described algorithm are set out in the fourth section. The final conclusions are preceded by an analysis of the identified clusters. 4 For example: Szkolnictwo Wyższe w Polsce 2013, Ministry of Science and Higher Education; Szkoły wyższe i ich finanse 2013, GUS. 5 The reports are denoted in the GUS databases as follows: F-01/s: Report on revenue, costs and financial results of higher educational institutions; S-10: Report on higher education; S-11: Report on material and social assistance to students and doctoral students; S-12: Report on academic scholarships, post-graduate and doctoral studies and employment in higher educational institutions and scientific and research institutes. 2. Diagnostic variables The study was carried out using a set of diagnostic variables that are cited in many reports assessing the state of higher education and in analyses of educational systems4. The first variable is the number of higher educational institutions per 10,000 population (X1). During the first decade following the fall of communism, approximately 200 non-public higher educational institutions were established in Poland. These had an impact on the availability and diversity of study courses offered within the provinces in which the institutions were located. The second variable considered is the number of students per 1000 population (X2). The aforementioned rise in student numbers has led to variation between provinces in terms of features relating to higher education over the past decades. Changes in numbers of students have been closely linked to changes in the number of higher educational institutions (Fig. 1). One consequence of the rising number of students is an increase in the number of graduates per 1000 population, which is taken as variable X3 in the model. The structure and size of teaching and academic staff affect the prestige enjoyed by higher educational institutions. This is reflected in the academic potential of the provinces in which those institutions are situated. The analysis included two values relating to staffing: the number of academic teachers per 10,000 population (X4) and the number of academic teachers with the title of professor per 10,000 population (X5). Research activity and specialist education are represented by two variables: the number of post-graduate students per 10,000 population (X6) and the number of doctoral students per 10,000 population (X7). The analysis of the spatial variation between provinces was based on the set of seven diagnostic variables presented above. The data used are taken from the Local Data Bank (http://stat.gov.pl/bdl/), the original source being the annual reports of higher educational institutions5. Missing values were acquired from the Statistical Yearbooks of Provinces, published by the Central Statistical Office (GUS). The available absolute figures were divided by the numbers of inhabitants of the relevant provinces. To ensure correctness of the analysis, zero unitarization was applied (Walesiak 2014). 3. Research procedure The algorithm for spatial delimitation of provinces consisted of three stages: data normalisation, construction of principal components, and cluster analysis. Data normalisation was performed using the method of zero unitarization (Walesiak 2014). The fact that all of the observed values are stimulants (having positive impact) meant that a single common normalisation formula could be used. The source values of the observed features were transformed according to (1): ௝௣ൌ ௝௣െ݉݅݊൛௝௣ൟ ݉ܽݔ൛௝௣ൟെ݉݅݊൛௝௣ൟ (1) (1) where zjp is the normalised value of the pth variable for the jth object, and xjp is the value of the pth variable for the jth object. The method gives normal values of the observed features in the interval <0; 1>, reducing the effect of disproportions in these values on the principal component analysis carried out in the second stage. The second stage of the procedure involved principal component analysis of doubly multivariate data for a covariance matrix with Kronecker product structure. Let us assume that we have an n-element sample consisting of objects characterised by p statistical features measured at T different time points. Data of this type are called doubly multivariate. Let Xjk denote the column vector of measurements of p features on the jth object at the kth time point, j=1,2,…,n, k=1,2,…,T. Let Xj=(Xj1, Xj2,…, XjT) be a p × T matrix, and xj=vec(Xj) be a pT- dimensional column vector of measurements of p features for the jth object at successive time points k, j=1,2,…,n. We assume that xj ~ NpT ( μ , ષ ), j=1, 2, …, n, where ષ is a positive definite covariance matrix. Based on the estimator of the covariance matrix ષ we construct the principal components (Hotelling 1933). The estimator of the matrix ષ constructed from an n-element sample is positive definite with probability 1 if and only if n>pT (e.g. Giri 1996). This condition implies a need to have a very large sample, which is not always possible. We, therefore, assume that the matrix ષ has the structure of a Kronecker product (e.g. Gałecki 1994, Naik and Rao 2001, Roy and Khattree 2005, Krzyśko et al. 2011): ષൌ܄6ǡ (2) ષൌ܄6ǡ (2) where ܄ is the positive definite matrix of covariance between time points, with dimension T×T, and 66 is the positive definite matrix of covariance between all statistical features, with dimension p×p. 3. Research procedure When the matrix ષ has this structure, its estimator is positive definite (with probability 1) if and only if n>max(p, T), which significantly weakens the condition on the size of the sample. Bearing in mind that the matrix ܄ represents variability over time, we may consider three models: Model 1. We assume that the observations xj are independent and that xj ~ NpT(μ, ܄6), where ܄ is a T×T positive definite matrix,6 is a p×p positive definite matrix, and n>max(p, T). We do not impose any additional restrictions on ܄. Model 2. Weadopt the same assumptions as in Model 1, but also assume that the matrix ܄ is completely symmetric, that is it has the form: ܄ൌെ ͳ ͳ െɏ ൣሺͳ െɏሻધ்൅ɏ૚்૚் ܂൧ǡ (3) (3) where ɏ is the coefficient of correlation, and ૚் is a T-dimensional column vector of ones. where ɏ is the coefficient of correlation, and ૚் is a T-dimensional column vector of ones. Model 3. We adopt the same assumptions as in Model 1, but also assume that the matrix ܄ has the structure of a first-order autoregression (Krzyśko et al. 2011), that is it has the form: Model 3. We adopt the same assumptions as in Model 1, but also assume that the matrix ܄ has the structure of a first-order autoregression (Krzyśko et al. 2011), that is it has the form: ܄ൌ ͳ ͳ െɏଶ൫ɏȁ୰ିୱȁ൯୰ǡୱୀଵ ் ǡ (4) (4) where ɏ is the coefficient of correlation. 6 The shortest dendrite is the tree for which the sum of the weights on the edges is the smallest. The weights are taken as the distance between the tree nodes representing the studied objects. The shortest dendrite was constructed using Kruskal’s algorithm (1956). where: u,v=1, 2, …, n, i=1, 2, …, p. where: u,v=1, 2, …, n, i=1, 2, …, p. In the shortest dendrite we determine the mean and standard deviation of the weights of all edges (distances between objects). The critical value, providing a criterion for the removal of edges from the dendrite, was taken to be the sum of and . The removal of edges whose weight exceeds the critical value leads to a division of the dendrite, and consequently to the separation of clusters. where ɏ is the coefficient of correlation. where ɏ is the coefficient of correlation. In all three models the unknown parameters are estimated by the maximum likelihood method, solving appropriate systems of simultaneous equations iteratively until the selected “stop” criterion is attained (Srivastava et. al 2008, Krzyśko and Skorzybut 2009). We construct principal components based on the matrix ષ෡ൌ܄෡66෡(Deręgowski and Krzyśko 2009). If n>max( p, T ), then the matrix ܄෡6෡ is positive definite with probability 1, and so all eigenvalues are real and positive. If α1, α2, .. , αT are the eigenvalues of ܄෡ and β1, β2, … , βp are the eigenvalues of 6෡, the eigenvalues of ܄෡6෡ are pT numbers of the form αrβs , where r=1, 2, … ,T, s=1, 2, … ,p. Based on the eigenvalues so defined, we construct the principal components of the matrix ષ෡ൌ܄෡6෡. The principal components constructed in this way were used in the cluster analysis that formed the last stage of the study procedure. A hierarchical algorithm was used, based on the Wrocław taxonomy (Florek et al. 1951), involving the construction of the shortest dendrite6 over a set of n objects, based on a selected measure of dissimilarity (Euclidean distance in this case): ɏሺ௨ǡ ௩ሻൌ൫ሺ௨െ௩ሻᇱሺ௨െ௩ሻ൯ భ మൌቌ෍ሺ௨௜െ௩௜ሻଶ ௣ ௜ୀଵ ቍ భ మ ǡ (5) (5) 4. Classification of provinces Statistical analysis of the higher education data was performed in several stages (steps), with a different number of provinces considered each time. Each of the stages was based on the dendrite method, where the critical value was taken to be the mean length of an edge of the dendrite plus the standard deviation of the lengths. The method produced a division into six groups of provinces in four steps. In the first step, the principal components were constructed for all 16 provinces, taking account of the three models for the structure of the matrix ܄. The goodness criterion was taken to be the index W, being the ratio of the sum of the variances of the first two principal components to the sum of the variances of all principal components, expressed as a percentage (Table 1). Model 2 was found to preserve the largest proportion of the variation of the data (73.55%). Table 1. The goodness criterion for the models in the first step The goodness criterion Model 1 Model 2 Model 3 W index 63.27 73.55 63.4 Table 1. The goodness criterion for the models in the first step Source: own calculations. Source: own calculations. Source: own calculations. Source: own calculations. Source: own calculations. Figure 3 shows a projection of the provinces in the plane of the first two principal components, together with a dendrite over the points representing the provinces. The dotted line marks an edge longer than the critical value of 2.1591 (dendrite connections and edge lengths are given in Table 2). 1 – łódzkie 2 – mazowieckie 3 – małopolskie 4 – śląskie 5 – lubelskie 6 – podkarpackie 7 – podlaskie 8 – świętokrzyskie 9 – lubuskie 10 – wielkopolskie 11 – zachodniopomorskie 12 – dolnośląskie 13 – opolskie 14 – kujawsko-pomorskie 15 – pomorskie 16 – warmińsko-mazurskie 1 – łódzkie 2 – mazowieckie 3 – małopolskie 4 – śląskie 5 – lubelskie 6 – podkarpackie 7 – podlaskie 8 – świętokrzyskie 9 – lubuskie 10 – wielkopolskie 11 – zachodniopomorskie 12 – dolnośląskie 13 – opolskie 14 – kujawsko-pomorskie 15 – pomorskie 16 – warmińsko-mazurskie Figure 3. Dendrite over points representing provinces in the plane of the first two principal components, model 2 in the first step Figure 3. Dendrite over points representing provinces in the plane of the first two principal components, model 2 in the first step Source: own calculations. Table 2. Dendrite connections and edge lengths Table 2. Dendrite connections and edge lengths Pairs of provinces Edge length Pairs of provinces Edge length 1-10 0.8752 7-15 1.4587 2-12 3.7373 10-12 1.4477 3-12 1.7664 10-15 1.2559 4-14 0.8875 11-15 1.3666 5-10 1.1293 13-16 1.2440 6-8 1.7859 14-15 1.5957 6-9 1.0788 14-16 1.3620 6-13 1.1570 engths Pairs of provinces Edge length 7-15 1.4587 10-12 1.4477 10-15 1.2559 11-15 1.3666 13-16 1.2440 14-15 1.5957 14-16 1.3620 g Pairs of provinces Edge length 1-10 0.8752 2-12 3.7373 3-12 1.7664 4-14 0.8875 5-10 1.1293 6-8 1.7859 6-9 1.0788 6-13 1.1570 The result of the analysis in the first step reveals the identification of two clusters, one of which is an isolated (single-element) cluster consisting of Mazowieckie province, further denoted Cluster 1. The remaining provinces, which make up the second cluster, underwent analysis in the second step. The greatest part of the variation, expressed by the index W, is preserved by the principal components in the second model (71.16%; Table 3). Table 3. The goodness criterion for the models in the second step le 3. The goodness criterion for the models in the second step Table 3. Source: own calculations. The goodness criterion for the models in the second step The goodness criterion Model 1 Model 2 Model 3 W index 61.65 71.16 61.98 Source: own calculations. Figure 4 shows a projection of the provinces in the plane of the first two principal components, together with the constructed dendrite. The edges that exceed the critical value (1.5989) are marked by a dotted line. 1 – łódzkie 2 – małopolskie 3 – śląskie 4 – lubelskie 5 – podkarpackie 6 – podlaskie 7 – świętokrzyskie 8 – lubuskie 9 – wielkopolskie 10 – zachodniopomorskie 11 – dolnośląskie 12 – opolskie 13 – kujawsko-pomorskie 14 – pomorskie 15 – warmińsko-mazurskie 1 – łódzkie 2 – małopolskie 3 – śląskie 4 – lubelskie 5 – podkarpackie 6 – podlaskie 7 – świętokrzyskie 8 – lubuskie 9 – wielkopolskie 10 – zachodniopomorskie 11 – dolnośląskie 12 – opolskie 13 – kujawsko-pomorskie 14 – pomorskie 15 – warmińsko-mazurskie Figure 4. Dendrite over points representing provinces in the plane of the first two principal components, model 2 in the second step Figure 4. Dendrite over points representing provinces in the plane of the first two principal components, model 2 in the second step Source: own calculations. Source: own calculations. In the second step three clusters were identified, of which two are isolated clusters: x Cluster 2 consisting of Małopolskie province; x Cluster 6 consisting of Świętokrzyskie province. In the third step, analysis was applied to the third cluster from the previous stage, consisting of 13 provinces: Śląskie, Podkarpackie, Lubuskie, Opolskie, Kujawsko-Pomorskie, Warmińsko-Mazurskie, Łódzkie, Lubelskie, Podlaskie, Wielkopolskie, Zachodniopomorskie, Dolnośląskie and Pomorskie. The values of W for the three considered cases (Table 4) clearly show that Model 2 is again the most adequate to the data, explaining 67.15% of the variation. Table 4. The goodness criterion for the models in the third step Table 4. The goodness criterion for the models in the third step Table 4. The goodness criterion for the models in the third step The goodness criterion Model 1 Model 2 Model 3 W index 61.18 67.15 60.37 Source: own calculations. Figure 5 shows a projection of the 13 provinces in the plane of the first two principal components, together with a dendrite constructed on the points representing them. The edges whose length exceed the critical value (1.462) are marked by dotted lines. Source: own calculations. This leads to a division into two clusters, consisting of the provinces: x Łódzkie, Lubelskie, Podlaskie, Wielkopolskie, Zachodniopomorskie, Dolnośląskie, Pomorskie (denoted as Cluster 3); x Śląskie, Podkarpackie, Lubuskie, Opolskie, Kujawsko-Pomorskie, Warmińsko-Mazurskie (denoted temporarily as Cluster 4). x Śląskie, Podkarpackie, Lubuskie, Opolskie, Kujawsko-Pomorskie, Warmińsko-Mazurskie (denoted temporarily as Cluster 4). 1 – łódzkie 2 – śląskie 3 – lubelskie 4 – podkarpackie 5 – podlaskie 6 – lubuskie 7 – wielkopolskie 8 – zachodniopomorskie 9 – dolnośląskie 10 – opolskie 11 – kujawsko-pomorskie 12 – pomorskie 13 – warmińsko-mazurskie 1 – łódzkie 2 – śląskie 3 – lubelskie 4 – podkarpackie 5 – podlaskie 6 – lubuskie 7 – wielkopolskie 8 – zachodniopomorskie 9 – dolnośląskie 10 – opolskie 11 – kujawsko-pomorskie 12 – pomorskie 13 – warmińsko-mazurskie Figure 5. Dendrite over points representing provinces in the plane of the first two principal components, model 2 in the third step Figure 5. Dendrite over points representing provinces in the plane of the first two principal components, model 2 in the third step Source: own calculations. Source: own calculations. The fourth step, the final stage of the analysis, concerned Śląskie, Podkarpackie, Lubuskie, Opolskie, Kujawsko-Pomorskie and Warmińsko- Mazurskie provinces, contained in the temporary Cluster 4. The greatest part of the variation (67.75%) is preserved by the first two principal components in Model 2 (Table 5). le 5. The goodness criterion for the models in the fourth step Table 5. The goodness criterion for the models in the fourth step Table 5. The goodness criterion for the models in the fourth step The goodness criterion Model 1 Model 2 Model 3 W index 55.76 67.75 57.88 Source: own calculations. Source: own calculations. The critical edge length value (1.3246) in the dendrite was exceeded by the pairing of Kujawsko-Pomorskie and Warmińsko-Mazurskie provinces. This led to a division of the considered provinces into two clusters: x Cluster 4 consisting of Śląskie and Kujawsko-Pomorskie; x Cluster 5 consisting of the remaining provinces: Podkarpackie, Lubuskie, Opolskie and Warmińsko-Mazurskie. x Cluster 5 consisting of the remaining provinces: Podkarpackie, Lubuskie, Opolskie and Warmińsko-Mazurskie. The projection of the six provinces in the plane of the first two principal components, together with the constructed dendrite, is shown in Figure 6. Figure 6. Dendrite over points representing provinces in the plane of the first two principal components, model 2 in the fourth step 1 – śląskie 2 – podkarpackie 3 – lubuskie 4 – opolskie 5 – kujawsko-pomorskie 6 – warmińsko-mazurskie Figure 6. Dendrite over points representing provinces in the plane of the first two i i l t d l 2 i th f th t 1 – śląskie 2 – podkarpackie 3 – lubuskie 4 – opolskie 5 – kujawsko-pomorskie 6 – warmińsko-mazurskie Figure 6. Dendrite over points representing provinces in the plane of the first two principal components, model 2 in the fourth step Figure 6. Dendrite over points representing provinces in the plane of the first two principal components, model 2 in the fourth step Figure 6. Dendrite over points representing provinces in the plane of the first two principal components, model 2 in the fourth step Source: own calculations. The provinces constituting Cluster 3 were also analysed, but no basis was found for any further division of that cluster. As a result of the four-stage classification process described above, the provinces were divided into a total of six groups (Fig. 7). Figure 7. Dendrite for all 16 provinces, with clusters shown Cluster 1 mazowieckie (2) Cluster 2 małopolskie (3) Cluster 3 łódzkie (1), zachodniopomorskie (11), wielkopolskie (10), lubelskie (5), dolnośląskie (12), pomorskie (15), podlaskie (7) Cluster 4 śląskie (4), kujawsko-pomorskie (14) Cluster 5 podkarpackie (6), lubuskie (9), opolskie (13), warmińsko-mazurskie (16) Cluster 6 świętokrzyskie (8) Figure 7. Dendrite for all 16 provinces, with clusters shown Note: circles contain the numbers of the provinces belonging to the identified groups, and squares contain the numbers used to denote the clusters. In the legend, the numbers assigned to the provinces in the computational procedure are given in brackets. Note: circles contain the numbers of the provinces belonging to the identified groups, and squares contain the numbers used to denote the clusters. In the legend, the numbers assigned to the provinces in the computational procedure are given in brackets. Source: own calculations. 7 Perspektywy ranking of higher educational institutions 2015. 8 The top 40 higher educational institutions in the 2015 Perspektywy ranking included, in order: Adam Mickiewicz University in Poznań, Wrocław University of Technology, the University of Wrocław, Gdańsk Medical University, Łódź University of Technology, Poznań Medical University, Poznań University of Technology, Wrocław Medical University, the University of Łódź, Gdańsk University of Technology, the University of Gdańsk, Łódź Medical University, Poznań University of Economics, Białystok Medical University, Lublin Medical University, Maria Curie-Skłodowska University in Lublin, the Pomeranian Medical University in Szczecin, Poznań University of Life Sciences, Wrocław University of Environmental and Life Sciences, the Catholic University of Lublin, Lublin University of Technology, and the University of Białystok. 5. Analysis In the first two steps of the algorithm, two single-element clusters were identified, consisting of Mazowieckie and Małopolskie provinces (Fig. 8). These regions contain the two largest and most renowned academic centres in Poland: Warsaw and Kraków. In national rankings, the University of Warsaw and Jagiellonian University are the two highest ranked higher educational institutions (Perspektywy ranking of higher educational institutions7, Polityka ranking of higher educational institutions). They are also the only Polish institutions to appear on the Shanghai Ranking of the world’s 500 leading universities (2015). It should also be noted that Warsaw and Kraków are the largest cities in Poland (in terms of population). As the national capital, Warsaw is also a financial, political and cultural centre. Mention should also be made of other higher educational institutions in these two provinces, which appear in the top ten of the aforementioned ranking: Warsaw University of Technology, the Warsaw School of Economics (SGH), and AGH University of Science and Technology in Kraków. Among non-public institutions offering master’s degree courses, the leading ten (in the aforementioned ranking) include six institutions in Warsaw and one in Kraków. The concentration of so many leading institutions in those provinces explains their strong position in the higher education market, and is visible on the dendrite (Fig. 7) in the form of the large distance separating those regions from the remainder. Figure 8. Spatial classification of provinces based on features relating to higher education (2002–2013) S l i Figure 8. Spatial classification of provinces based on features relating to higher education (2002–2013) Figure 8. Spatial classification of provinces based on features relating to higher education (2002–2013) Source: own analysis. Source: own analysis. Cluster 3 consists of seven provinces containing higher educational institutions that are well-renowned within Poland and have a long-established tradition8. Most of the capitals of provinces in this cluster are among Poland’s largest cities: Łódź, Wrocław, Poznań, Gdańsk (together with Gdynia and Sopot), Szczecin. The group also includes Lubelskie and Podlaskie provinces. These two eastern regions owe their membership of this cluster to the presence of higher educational institutions with notable values: the Catholic University of Lublin, Maria Curie-Skłodowska University in Lublin, the University of Białystok, Białystok Medical University, and the theological colleges in Białystok, Łomża and Drohiczyn. In the course of the delimitation procedure, Cluster 4 was separated from Cluster 5. The dendrite (Fig. Source: own calculations. 5. Analysis 7) shows the closeness of Śląskie and Kujawsko- Pomorskie provinces both to the group of provinces with the smallest potential (Cluster 5) and to the numerous group (Cluster 3), occupying the central part of the diagram. Cluster 5 contains the Polish provinces with the lowest values of the analysed parameters. The cartogram reveals the peripheral nature of these regions, as well as their relative closeness to regions with higher potential. The last of the identified clusters, consisting of Świętokrzyskie province, deviates from the axial arrangement of clusters seen on the dendrite. The values of some of the analysed higher education parameters were such as to place this province in the central group (a shift to the right on the horizontal axis of the dendrite) while others indicated that it belonged to the group with the smallest academic potential (a shift upwards on the vertical axis). This dual nature of observed values is well illustrated by the dendrite (Fig. 7). The above analysis is complemented by a characterisation of the identified clusters in terms of descriptive statistics (Table 6). Table 6. Descriptive statistics of the diagnostic variables for distinguished (identified) clusters Variable Clusters Overall mean Coefficient of variation between groups 1 2 3 4 5 6 Mean value within group X1 19.60 9.84 11.60 9.21 6.75 11.20 11.37 38.59% X2 63.44 60.12 47.33 40.02 34.76 38.59 47.38 25.31% X3 14.12 12.11 10.84 9.66 8.68 10.80 11.03 17.28% X4 3.15 3.66 2.69 2.04 1.58 1.43 2.43 36.75% X5 0.85 0.75 0.59 0.47 0.38 0.39 0.57 34.01% X6 10.35 4.14 3.37 2.67 2.45 2.86 4.31 70.12% X7 1.71 1.49 0.87 0.58 0.23 0.08 0.82 80.09% Meanings of variables: X1 – the number of higher educational institutions per 10,000 population; X2 – the number of students per 1000 population; X3 – the number of graduates per 1000 population; X4 – the number of academic teachers per 1000 population; X5 – the number of academic teachers with the title of professor per 10,000 population; X6 – the number of post-graduate students per 10,000 population; X7 – the number of doctoral students per 10,000 population. Meanings of variables: X1 – the number of higher educational institutions per 10,000 population; X2 – the number of students per 1000 population; X3 – the number of graduates per 1000 population; X4 – the number of academic teachers per 1000 population; X5 – the number of academic teachers with the title of professor per 10,000 population; X6 – the number of post-graduate students per 10,000 population; X7 – the number of doctoral students per 10,000 population. 5. Analysis Descriptive statistics of the diagnostic variables for distinguished (identified) clusters Meanings of variables: X1 – the number of higher educational institutions per 10,000 population; X2 – the number of students per 1000 population; X3 – the number of graduates per 1000 population; X4 – the number of academic teachers per 1000 population; X5 – the number of academic teachers with the title of professor per 10,000 population; X6 – the number of post-graduate students per 10,000 population; X7 – the number of doctoral students per 10,000 population. Meanings of variables: X1 – the number of higher educational institutions per 10,000 population; X2 – the number of students per 1000 population; X3 – the number of graduates per 1000 population; X4 – the number of academic teachers per 1000 population; X5 – the number of academic teachers with the title of professor per 10,000 population; X6 – the number of post-graduate students per 10,000 population; X7 – the number of doctoral students per 10,000 population. Source: own calculations. The mean values of the analysed features exhibit variation between the identified clusters. Mazowieckie province has the highest values for six out of the seven features. The single-element Cluster 2, consisting of Małopolskie province, has the highest number of academic teachers per 10,000 population, while in the other categories it lies second only to Mazowieckie province (often coming only slightly behind). The other clusters are separated from the leading two by a significant distance. The values recorded for Świętokrzyskie province clearly reveal its dual nature: the number of higher educational institutions per 10,000 population, the number of graduates per 1000 population and the number of post- graduate students per 10,000 population have values close to those for the high- potential clusters, while the values of number of academic teachers per 1000 population, the number of academic teachers with the title of professor per 10,000 population and the number of doctoral students per 10,000 population would place that province in the weakest group. It should be noted that, in terms of the values of the observed features, Cluster 4 differs from Cluster 3 (with higher potential) to a similar degree as from Cluster 5 (with lower potential), from which it was separated out. 5. Analysis The differentiation of the identified groups is greatest in the case of the variables representing numbers of post-graduate and doctoral students, for which the coefficients of variation are 70% and 80% respectively. The number of graduates per 1000 population, on the other hand, is relatively similar for all clusters, with a coefficient of variation not exceeding 20%. The large disproportions in the values of variables between the two isolated clusters (Mazowieckie and Małopolskie provinces) and the other groups, the large group of provinces with moderate academic potential containing renowned centres of learning, and the isolated position of Świętokrzyskie province, deviating from the axial arrangement of the other clusters, create a characteristic picture of the spatial variation between Polish provinces based on the selected parameters relating to higher education. 6. Conclusions The analysis has confirmed the dominance of Mazowieckie and Małopolskie provinces in the Polish higher education market. The higher educational institutions of these regions have been ranked the highest in national rankings for many years, as well as being Poland’s only representatives in important international rankings. The applied delimitation model revealed relations between the provinces in terms of the analysed features. A detailed analysis of the results obtained and consideration of additional parameters relating to economic, demographic and social features would enable a better and more comprehensive presentation of the differences between the regions. A wider-ranging analysis of the Polish regions, covering features relating to human capital and the quality of life, would appear to be a natural development of the research reported here, and will form a part of the author’s future work. REFERENCES ANTONOWICZ, D., (2012). External influences and local responses. 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Innowacyjność a konserwatyzm: uczelnie polskie w procesie przemian społecznych. Wydawnictwo Instytutu Studiów Politycznych PAN. REFERENCES Models with a Kronecker product covariance structure: estimation and testing. Mathematical Methods of Statistics, 17 (4), pp. 357–370. TROW, M., (1973). Problems in the transition from elite to mass higher education. Carnegie Commission on Higher Education, Berkeley. WALESIAK, M., (2014). Przegląd formuł normalizacji wartości zmiennych oraz ich własności w statystycznej analizie wielowymiarowej. Przegląd Statystyczny, 61 (4), pp. 363–372. WASIELEWSKI, K., (2013). Caught in the trap of mass education – transformations in the Polish higher education after 1989, in: SZAFRANIEC K. and KONSTANTINOVSKIY D. (eds.). Polish and Russian Youth: Education and Work in Changing Society. IS RAS, Moskwa. WNUK-LIPIŃSKA, E., (1996). Innowacyjność a konserwatyzm: uczelnie polskie w procesie przemian społecznych. Wydawnictwo Instytutu Studiów Politycznych PAN. ZIÓŁKOWSKI, M., (2000). Przemiany interesów i wartości społeczeństwa polskiego. Teorie, tendencje, interpretacje. Wydawnictwo Fundacji Humaniora, Poznań. ZIÓŁKOWSKI, M., (2000). Przemiany interesów i wartości społeczeństwa polskiego. Teorie, tendencje, interpretacje. Wydawnictwo Fundacji Humaniora, Poznań.
https://openalex.org/W4288594854	https://zenodo.org/record/2556784/files/polifi-airtime-policy.pdf	English	null	PoliFi: Airtime Policy Enforcement for WiFi	arXiv (Cornell University)	2,019	cc-by-sa	5,795	PoliFi: Airtime Policy Enforcement for WiFi Per Hurtig Dept. of Computer Science Karlstad University, Sweden per.hurtig@kau.se Toke Høiland-Jørgensen Dept. of Computer Science Karlstad University, Sweden toke.hoiland-jorgensen@kau.se Anna Brunstrom Dept. of Computer Science Karlstad University, Sweden anna.brunstrom@kau.se between stations in a WiFi network by making appropriate scheduling decisions at the AP [2]. Given such a mechanism to enforce fairness, a natural question is whether it can be extended to express different capacity sharing policies. In this work we answer this question in the afﬁrmative, in the form of a workable solution to airtime policy enforcement in WiFi, which we have named PoliFi. between stations in a WiFi network by making appropriate scheduling decisions at the AP [2]. Given such a mechanism to enforce fairness, a natural question is whether it can be extended to express different capacity sharing policies. In this work we answer this question in the afﬁrmative, in the form of a workable solution to airtime policy enforcement in WiFi, which we have named PoliFi. Abstract—As WiFi grows ever more popular, airtime con- tention becomes an increasing problem. One way to alleviate this is through network policy enforcement. Unfortunately, WiFi lacks protocol support for conﬁguring policies for its usage, and since network-wide coordination cannot generally be ensured, enforcing policy is challenging. However, as we have shown in previous work, an access point can inﬂuence the behaviour of connected devices by changing its scheduling of transmission opportunities, which can be used to achieve airtime fairness. In this work, we show that this mechanism can be extended to successfully enforce airtime usage policies in WiFi networks. We implement this as an extension our previous airtime fairness work, and present PoliFi, the resulting policy enforcement system. The number of possible policies one might want to express is all but inﬁnite. Therefore, to focus our discussion, we deﬁne the following three representative policy use cases: 1) Prioritising devices. It should be possible to conﬁgure one or more devices to receive a higher share of network resources than other devices on the network. Our evaluation shows that PoliFi makes it possible to express a range of useful policies. These include prioritisation of speciﬁc devices; balancing groups of devices for sharing between different logical networks or network slices; and limiting groups of devices to implement guest networks or other low-priority services. We also show how these can be used to improve the performance of a real-world DASH video streaming application. I. INTRODUCTION WiFi is increasingly becoming the ubiquitous connectivity technology in homes as well as in enterprises. The ability for anyone to set up an access point and connect any device to it is one of the driving factors behind this increase of pop- ularity. However, increased popularity also means increased contention for resources as more devices are deployed. PoliFi makes it possible for the user to express all of these policies. Our design builds on our previous airtime scheduler for the Linux kernel, but extends it by (a) generalising the implementation from a speciﬁc driver to the common kernel WiFi stack, (b) extending the kernel scheduler to support weighted scheduling of stations, and (c) adding a userspace policy daemon that transforms the higher-level policy deci- sions into conﬁguration of the kernel scheduling mechanism. Since no two devices can transmit at the same time on a given frequency, the sparse resource that determines perfor- mance in WiFi networks is the time spent transmitting, also known as airtime usage. The 802.11 Media Access Control (MAC) protocol used in WiFi networks does not, in itself, guarantee a fair usage of this sparse resource. In fact it is well known that devices transmitting at lower rates can use more than their fair share of the airtime [1]. The rest of this paper presents PoliFi in detail, and is structured as follows: Section II summarises related work. Section III describes our design, with a performance analysis presented in Section IV. Finally, Section V concludes. PoliFi: Airtime Policy Enforcement for WiFi 2) Balancing device groups. In this use case, the network should be conﬁgured to share the available resources between groups of devices in a given way. For instance, this could be used to implement the “network slicing” concept often seen in 5G architectures [3]. 3) Limiting groups of devices to a maximum capacity share, in order to implement a lower-priority service, such as a guest network. Set station weights The concept of airtime policy enforcement appears in the concept of network slicing, which is an important part of the upcoming 5G mobile network architecture [3]. Network slicing involves splitting up a network into several virtual parts that are conceptually isolated from one another, which is a form of policy enforcement. A description of how to achieve network slicing in WiFi networks is given in [6], which corresponds roughly to our second use-case. The authors implement a prototype in simulation. Our mechanism builds on the same basic concept of computing per-device weights from group weights, but we solve a number of issues that prevent it from being implemented on real hardware. In addition, [6] only covers the second of our three policy use-cases. Another approach to splitting a wireless network into multi- ple parts is presented in [7], which describes a scheme where a separate software router is installed in the access point. This software router queues packets and enforces capacity sharing. However, the capacity sharing is implemented at the bandwidth level which, as mentioned above, is not the sparse resource in a WiFi network. Fig. 1: The high-level design of PoliFi. The kernel maintains data structures for every station, containing its current airtime deﬁcits and conﬁgured weight. The scheduler uses this to decide which station to transmit to next. The hardware reports airtime usage on TX completion. The userspace daemon tracks the associated stations and their queue state, and updates the weights in the kernel based on user policy preferences. A description of a scheme for network slicing in a home network is described in [8]. The authors describe a design that uses Software Deﬁned Networking (SDN) to split a home network into different parts, but do not discuss any mechanism for how the sharing is achieved. Finally, some enterprise APs offer features related to airtime fairness and policy conﬁguration, e.g., [9]. Unfortunately, no technical description of how these policies are enforced is generally available, which prevents us from comparing them to our solution. which makes it a natural place to implement airtime policy as well. The module we have added to hostapd can be conﬁgured in three modes, corresponding to the three use cases described in the introduction: static mode, dynamic mode and limit mode. II. RELATED WORK One way to improve performance of a network under contention is to apply different policies to different devices on the network, which works best if applied directly to the sparse resource instead of a proxy such as byte-level throughput. However, WiFi is decentralised at the protocol level, and thus lacks protocol support for enforcing policies on airtime usage. Fortunately, it turns out that in the common infrastructure deployment scenario, the access point can exert quite a bit of inﬂuence on the transmission behaviour of clients, or stations, as they are commonly called. In previous work, we have shown that this makes it possible to achieve airtime fairness Network policies are, in general, nothing new. For instance, standardisation of different trafﬁc classes has occurred in the form of the DiffServ framework [4]. In the WiFi world, the 802.11e standard deﬁnes different priority levels, which can be mapped to DiffServ code points [5]. However, this is all related to applying policies to different types of trafﬁc, whereas PoliFi deals with realising different capacity sharing policies between devices on the same network at the airtime usage level. As such, PoliFi is orthogonal so DiffServ, 802.11e and other trafﬁc class policy mechanisms. Kernel mac80211 subsystem Weighted DRR scheduler WiFi hardware Device driver Userspace Policy daemon Assoc / disassoc notifications Station queue state Set station weights Airtime usage Station Weight Deficits Station Weight Deficits Station Weight Deficits User configuration Station state tracker Fig. 1: The high-level design of PoliFi. The kernel maintains data structures for every station, containing its current airtime deﬁcits and conﬁgured weight. The scheduler uses this to decide which station to transmit to next. The hardware reports airtime usage on TX completion. The userspace daemon tracks the associated stations and their queue state, and updates the weights in the kernel based on user policy preferences. Kernel mac80211 subsystem Weighted DRR scheduler WiFi hardware Device driver Userspace Policy daemon Assoc / disassoc notifications Station queue state S t t ti i ht Airtime usage Station Weight Deficits Station Weight Deficits Station Weight Deficits User configuration Station state tracker As mentioned above, PoliFi is an extension of our previous work implementing an airtime fairness enforcement mecha- nism in Linux [2]. Set station weights The user can conﬁgure each of these modes per physical WiFi domain, and assign parameters for individual stations (based on their MAC addresses), or for entire Basic Service Sets (BSSes). The latter is a natural grouping mechanism, since this corresponds to logical networks conﬁgured on the same device (e.g., a primary and a guest network). However, extending the design to any other logical grouping mechanism is straight forward. III. THE POLIFI DESIGN We have designed PoliFi as a two-part solution, where a user-space daemon is conﬁgured by the user, and in turn conﬁgures a scheduling mechanism in the kernel. In this section, we describe our design in detail. A diagram of the design is shown in Figure 1. We begin by describing the user space daemon that conﬁgures the policies. Following this, we describe how the weighted Deﬁcit Round-Robin (DRR) scheduling mechanism is used to achieve the desired policies, and ﬁnally we describe how the mechanism is integrated into the Linux kernel WiFi stack. In static mode, the daemon will simply assign static weights to stations when they associate to the access point. Weights can be conﬁgured for individual stations, while a default weight can be set for each BSS, which will be applied to all stations that do not have an explicit value conﬁgured. This implements the basic use case of assigning higher priorities to speciﬁc devices, but does not guarantee any speciﬁc total share. II. RELATED WORK Compared to this previous work, PoliFi adds the policy enforcement component, and also generalises the mechanism by moving it out of the device drivers and into the common WiFi subsystem in Linux, thus making it applicable to more device drivers. Station state tracker User configuration Station queue state B. Weighted Airtime DRR First, the approach outlined above assumes that we have knowledge of which stations are active at any given time. This might look trivial at ﬁrst glance, since an access point needs to maintain some amount of state for all currently associated clients in any case. However, clients can be associated to an access point without sending or receiving any data, and thus without consuming any airtime. This means that association state in itself is not sufﬁcient to ascertain the set of currently active clients. Fortunately, we have another piece of data: The queue backlog for each device. Monitoring the backlog gives us a straight-forward indicator for activity without hav- ing to monitor actual packet ﬂows; we can simply consider any device that has had a non-zero queue backlog within a suitably short time span as active, and use that number in our calculations. The fairness mechanism that we are starting from (described in detail in [2]) is a Deﬁcit Round-Robin scheduler, which operates by accounting airtime usage as reported by the WiFi hardware after a transmission has completed, and scheduling transmissions to ensure that the aggregate usage over time is the same for all active stations. Using the airtime information provided after transmission completes means that retrans- missions can be accounted for, which improves accuracy especially for stations with low signal quality. Furthermore, as we have shown in our previous work, for TCP trafﬁc we can provide fairness even for transmissions transfers coming from each station. This is achieved by accounting the airtime of received packets, which causes the scheduler to throttle the rate of TCP ACKs going back to the station. The second difﬁculty lies in the fact that we need to transform the total weights between groups of stations into weights for each individual station. As shown in [6], this is conceptually just a simple division. However, when imple- menting this in an operating system kernel, we are limited to integer arithmetic, which means that to get accurate weights, we need to ensure that the division works when conﬁned to the integers. To achieve this, we ﬁrst limit our conﬁguration language to be expressed as integer weights between groups. A. Userspace Policy Daemon We implement the userspace policy daemon as part of the hostapd access point management daemon. This is the daemon responsible for conﬁguring wireless devices in access point mode in Linux. This means it already implements policies for other aspects of client behaviour (such as authentication), The dynamic and limit modes work by assigning weights use case described in [6]: each group is assigned a weight signifying its share relative to the other groups; from these group weights, each device in that group is assigned a weight computed by dividing the group weight with the number of active devices in that group. to each BSS, which are interpreted as their relative shares of the total airtime, regardless of how many stations they each have associated. Additionally, in limit mode, one or more BSSes can be marked as limited. BBSes that are marked as limited are not allowed to exceed their conﬁgured share, whereas no limitations are imposed on unmarked BSSes. Thus, dynamic mode implements the second use case, while limit mode implements the third. The ﬁnal use case requires limiting one or more groups of stations to a ﬁxed share of the available capacity. This can be illustrated with the guest network use case, where an example policy could be that a guest network is not allowed to exceed 50% of the available capacity. If this policy is implemented as a ﬁxed share between groups, however, a single station on the guest network would be able to get the same capacity as, say, ﬁve users of the regular network, which is not what we want. Thus, a different policy is needed: a group can be limited, and should have its weight adjusted only if it would get more than the conﬁgured share, not if it gets less. Thus, this becomes a two-step procedure that ﬁrst assigns unit weights to all devices (which is the default when no policy is applied), and calculates whether or not this results in the limited group using more than its conﬁgured share of the airtime. If it does, a policy is computed in the same way as for the dynamic use case, which results in the limited group being assigned exactly its conﬁgured airtime share. A. Userspace Policy Daemon For both dynamic mode and limit mode, the daemon period- ically polls the kernel to discover which stations are currently active, using the queue backlog as a measure of activity, as discussed below. After each polling event, per-station weights are computed based on the number of active stations in each BSS, and these weights are conﬁgured in the kernel. The details of the weight computation, and how this is used to achieve the desired policy, is discussed in the next section. Selecting the polling frequency is a tradeoff between reaction time and system load overhead. The polling interval defaults to 100 ms, which we have found to offer good reaction times (see Section IV-B), while having a negligible overhead on our test system. While our implementation is focused on the single access point case, where the access point enforces a single conﬁgured policy, the userspace daemon could just as well pull its policy conﬁguration from a central cloud-based management service, while retaining the same policy enforcement mechanism. 2) Computing the Weights: Having established that our desired policies can be expressed in terms of weights, we turn to the practical difﬁculties of applying this to a real WiFi system. B. Weighted Airtime DRR Then, to ensure that we can divide these weights with the number of active stations, we multiply them by a suitable constant, chosen as follows: 1) Adding Weighted Scheduling: Given this effective air- time fairness scheduler, we can realise arbitrary division of the available capacity between different stations, by simply assigning them different scheduling weights. For the DRR scheduling algorithm employed by our scheduler, this is achieved by using different quantums per station. Thus, to apply this to airtime policy enforcement, we need to express the desired policy as a number of different service weights for each of the active devices. The ﬁrst use case is trivially expressed in terms of weights: simply assign the prioritised device a higher weight; for instance, to double its priority, assign it a weight of 2. The second use case, where capacity should be split between groups of devices has been covered in the network slicing We are given the set of groups I, where each group i has a conﬁgured group weight Wi and Ni active stations. We then deﬁne the multiplication constant C = Q i∈I Ni. Multiplying all group weights by this same constant maintains their relative ratio, and the choice of constant ensures that each group’s weight can be divided by the number of active stations in that group. This gives us the following expression for the per- station weight for group i: Algorithm 1 Airtime fairness scheduler. The schedule function is part of the device driver and is called on packet arrival and on transmission completion. The account_airtime function is called by the driver when it receives airtime usage information on TX completion or packet reception. 1: function SCHEDULE(qoslvl) 2: tid ←GET_NEXT_TID(qoslvl) 3: BUILD_AGGREGATE(tid) 4: RETURN_TID(tid) 5: function GET_NEXT_TID(qoslvl) 6: tid ←FIND_FIRST(active_tids, qoslvl) 7: stn ←tid.station 8: deﬁcit ←stn.deﬁcit[qoslvl] 9: if deﬁcit ≤0 then 10: stn.deﬁcit[qoslvl] ←deﬁcit + stn.weight 11: LIST_MOVE_TAIL(tid, active_tids) 12: restart 13: LIST_REMOVE(tid, active_tids) 14: return tid 15: function RETURN_TID(tid) 16: if tid.queue is not empty then 17: LIST_ADD_HEAD(tid, active_tids) 18: 19: function ACCOUNT_AIRTIME(tid, airtime) 20: stn ←tid.station 21: qoslvl ←tid.qoslvl 22: stn.deﬁcit[qoslvl] ←stn.deﬁcit[qoslvl] −airtime W s i = WiC Ni (1) (1) The third issue we need to deal with is converting the weights to the per-station time quantums that are used in the scheduler, and which are expressed in microseconds. B. Weighted Airtime DRR These should be kept at a reasonable absolute size, because larger weights result in coarser time granularity of the scheduler, making each scheduler round take longer and impacting la- tency of all devices in the network. We convert the calculated weights into ﬁnal quantums by normalising them so they fall within a range of 100−1000 µs, but preferring smaller values if the ratio between the smallest and largest weight is more than 10×. IV. EVALUATION In this section we evaluate how effectively PoliFi is able to implement the desired policies. We examine steady state behaviour as well as the reaction time of the dynamic and limit modes with a changing number of active stations. To show how airtime policies can provide beneﬁts for speciﬁc applications, we also include an DASH video streaming use case in our evaluations. We perform the experiments on our testbed with four WiFi devices. The details of our setup are omitted here due to space constraints, but are available in an online appendix [11]. In order to move the scheduling decision out of the drivers, we deﬁne a new driver API, shown in Algorithm 1. The device driver runs the schedule() function, and asks mac80211 for the next TID queue to schedule, using the get_next_tid() API function. The driver then services this queue until no more packets can be scheduled (typically because the hardware queue is full, or the TID queue runs empty). After this, the driver uses the return_tid() API function to return the TID queue to the scheduler. A third API function, account_airtime(), allows the driver to register airtime usage for each station, which is typically done asynchronously as packets are completed or received. C. Kernel Airtime Scheduler We implement the kernel part of PoliFi in the WiFi protocol subsystem of the Linux kernel (called mac80211). Our imple- mentation builds on our previous airtime fairness scheduler, described in [2], which implemented a queueing system in this layer. In this queueing system, packets are assigned a Trafﬁc ID (TID) before enqueue, and a separate queueing structure is created for each TID, of which there are 16 per station. These per-TID queues then form the basis of the scheduling of different stations. The queueing structure itself is based on the FQ-CoDel queue management scheme [10] and ensures ﬂow isolation and low queueing latency. deﬁcit scheduler approach described above. As for the previous airtime fairness scheduler in the driver, airtime deﬁcits are kept separately for each of the four hardware QoS levels, to match the split of the hardware transmission queue scheduling. The algorithm is implemented as part of the get_next_tid() function as shown in Algorithm 1. The PoliFi scheduler has been accepted into the upstream Linux kernel and will appear in Linux 5.1 with support for the ath9k and ath10k drivers for Qualcomm Atheros 802.11n and 802.11ac hardware. While our previous implementation implemented queueing in the general WiFi layer, scheduling and tracking of each active station’s airtime usage was still the responsibility of the driver. In PoliFi, we move the scheduling decisions into mac80211, where it can be leveraged by all device drivers. In addition, we modify the scheduler to support the weight- based policy enforcement capability described above. The weights can be set by userspace through the standard nl80211 conﬁguration API. A. Steady state measurements 2: Aggregate airtime usage share of four stations, over a 30-second bulk transfer. Graph columns correspond to the different policy modes. In static mode stations 2 and 3 are assigned weights of 3 and 4, respectively. In dynamic and limit mode, stations 1-3 are on one BSS while station 4 is on another; both BSSes have the same weight, and the second BSS is set to limited. The plots are box plots of 30 test runs, but look like lines due to the low variation between runs. (a) Dynamic mode 0 10 20 30 40 Time (s) 0.00 0.25 0.50 0.75 1.00 Airtime share (b) Limit mode (a) Dynamic mode Fig. 4: Airtime usage over time with changing number of active stations, in dynamic and limit mode. UDP ﬂows to each station start 5 seconds apart. The purple station (starting ﬁrst) is on one BSS, while the remaining three stations are on the other BSS. a single station in it is set to limited, which in this case means that its natural airtime share is less than the conﬁgured share, and thus that no limiting is necessary to enforce the conﬁgured policy. We test this to ensure that the algorithm correctly allows the group that is not marked as limited to exceed its conﬁgured airtime share. Fig. 2: Aggregate airtime usage share of four stations, over a 30-second bulk transfer. Graph columns correspond to the different policy modes. In static mode stations 2 and 3 are assigned weights of 3 and 4, respectively. In dynamic and limit mode, stations 1-3 are on one BSS while station 4 is on another; both BSSes have the same weight, and the second BSS is set to limited. The plots are box plots of 30 test runs, but look like lines due to the low variation between runs. Fig. 2: Aggregate airtime usage share of four stations, over a 30-second bulk transfer. Graph columns correspond to the different policy modes. In static mode stations 2 and 3 are assigned weights of 3 and 4, respectively. In dynamic and limit mode, stations 1-3 are on one BSS while station 4 is on another; both BSSes have the same weight, and the second BSS is set to limited. The plots are box plots of 30 test runs, but look like lines due to the low variation between runs. A. Steady state measurements The aggregate airtime usage of the stations and BSSes is seen in ﬁgures 2 and 3, respectively. With no policy conﬁgured, the scheduler simply enforces fairness between the active stations. In the static policy mode, relative weights of 3 and 4 are assigned to stations 2 and 3, respectively. These weights are clearly reﬂected in the airtime shares achieved by each station in the second column of the graphs in Figure 2, showing that static policy assignment works as designed. BSS 1 BSS 2 BSS 1 BSS 2 0.0 0.2 0.4 0.6 0.8 1.0 Airtime share Dynamic Limit (a) UDP trafﬁc BSS 1 BSS 2 BSS 1 BSS 2 0.0 0.2 0.4 0.6 0.8 1.0 Airtime share Dynamic Limit (b) TCP trafﬁc Fig. 3: Aggregate airtime usage of the two BSSes, for the same test as that shown in Figure 2. BSS 1 BSS 2 BSS 1 BSS 2 0.0 0.2 0.4 0.6 0.8 1.0 Airtime share Dynamic Limit (b) TCP trafﬁc BSS 1 BSS 2 BSS 1 BSS 2 0.0 0.2 0.4 0.6 0.8 1.0 Airtime share Dynamic Limit (a) UDP trafﬁc g p y g g Turning to the group modes, Figure 3 shows the aggregate airtime for each of the two conﬁgured BSSes. In dynamic mode, the scheduler enforces equal sharing between the two BSSes, which translates to the single station in BSS 2 getting three times as much airtime as the other three, as is seen in the third column of Figure 2. In limit mode, BSS 2 is limited to at most half of the airtime, but because there is only one station connected to it, its fair share is already less than the limit, and so this corresponds to the case where no policy is enforced. Thus, the tests show that the scheduler successfully enforces the conﬁgured policies for all three use cases. A. Steady state measurements The steady state tests consist of running a bulk ﬂow (either UDP or TCP) to each of four stations associated to the access point running PoliFi. Three of the stations are associated to one BSS on the access point, while the fourth is on a separate BSS. These two BSSes are the groups the algorithm balances in dynamic and limit mode. Both groups are given equal weights, meaning that they should receive the same total airtime share. When testing the limit mode use case, the BSS with only Using this API, mac80211 has enough information to implement airtime policy enforcement using the weighted 0 10 20 30 40 Time (s) 0.00 0.25 0.50 0.75 1.00 Airtime share (a) Dynamic mode 0 10 20 30 40 Time (s) 0.00 0.25 0.50 0.75 1.00 Airtime share (b) Limit mode Fig. 4: Airtime usage over time with changing number of active stations, in dynamic and limit mode. UDP ﬂows to each station start 5 seconds apart. The purple station (starting ﬁrst) is on one BSS, while the remaining three stations are on the other BSS. Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 0.0 0.1 0.2 0.3 0.4 0.5 Airtime share No policy Static Dynamic Limit (a) UDP trafﬁc Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 0.0 0.1 0.2 0.3 0.4 0.5 Airtime share No policy Static Dynamic Limit (b) TCP trafﬁc Fig 2: Aggregate airtime usage share of four stations over 0 10 20 30 40 Time (s) 0.00 0.25 0.50 0.75 1.00 Airtime share Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 0.0 0.1 0.2 0.3 0.4 0.5 Airtime share No policy Static Dynamic Limit (a) UDP trafﬁc Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 Station 1 Station 2 Station 3 Station 4 0.0 0.1 0.2 0.3 0.4 0.5 Airtime share No policy Static Dynamic Limit (b) TCP trafﬁc Fig. C. DASH Trafﬁc Test To showcase an example real-world use case that can be improved by airtime policy enforcement, we examine a DASH video streaming application. In this scenario, we add a station with poor signal quality to the network, representing a streaming device that is connected to the wireless network at a location where signal quality is poor. Moving the device is not an option, so other measures are necessary to improve the video quality. We stream the Big Buck Bunny [12] video using the dash.js [13] player running in the Chromium browser on the slow station. We determine that the maximum video bitrate the device can reliably achieve in this scenario (with no competing trafﬁc) is 2 Mbps. However, when the other devices are active, the video bitrate drops to 1 Mbps because of contention. [2] T. Høiland-Jørgensen, M. Kazior, D. Täht, P. Hurtig, and A. Brunstrom, “Ending the anomaly: Achieving low latency and airtime fairness in wiﬁ,” in 2017 USENIX Annual Technical Conference (USENIX ATC 17), 2017. [3] X. Foukas, G. Patounas, A. Elmokashﬁ, and M. K. Marina, “Network slicing in 5g: Survey and challenges,” IEEE Communications Magazine, vol. 55, no. 5, pp. 94–100, 2017. [4] J. Babiarz, K. Chan, and F. Baker, “Conﬁguration Guidelines for DiffServ Service Classes,” RFC 4594 (Informational), RFC Editor, Aug. 2006, updated by RFC 5865. https://www.rfc-editor.org/rfc/rfc4594.txt p y p g [5] T. Szigeti, J. Henry, and F. Baker, “Mapping Diffserv to IEEE 802.11,” RFC 8325 (Proposed Standard), RFC Editor, Feb. 2018. https://www. rfc-editor.org/rfc/rfc8325.txt g [6] M. Richart, J. Baliosian, J. Serrat, J.-L. Gorricho, R. Agüero, and N. Agoulmine, “Resource allocation for network slicing in WiFi access points,” in 13th International Conference on Network and Service Management, CNSM, 2017, 2017. [7] K. Katsalis, K. Choumas, T. Korakis, and L. Tassiulas, “Virtual 802.11 wireless networks with guaranteed throughout sharing,” in 2015 IEEE Symposium on Computers and Communication (ISCC), Jul 2015. Figure 5 shows the achieved video bitrate along with the data goodput of the video ﬂow, while three other stations are simultaneously receiving bulk data. With no policy set, the video bitrate drops to 1 Mbps, as described above. However, when we prioritise the station (to half the available airtime in this case), the achieved bitrate stays at 2 Mbps throughout the 10-minute video. This shows how PoliFi can improve the performance of a speciﬁc real-world application. [8] Y. Yiakoumis, K.-K. Yap, S. Katti, G. Parulkar, and N. C. DASH Trafﬁc Test McKeown, “Slicing home networks,” in Proceedings of the 2Nd ACM SIGCOMM Workshop on Home Networks, ser. HomeNets ’11. ACM, 2011. [9] “Air Time Fairness (ATF) Phase1 and Phase 2 Deployment Guide,” Cisco systems, 2015. https://www.cisco.com/c/en/us/td/docs/wireless/ technology/mesh/8-2/b_Air_Time_Fairness_Phase1_and_Phase2_ Deployment_Guide.html [10] T. Hoeiland-Joergensen, P. McKenney, D. Taht, J. Gettys, and E. Du- mazet, “The Flow Queue CoDel Packet Scheduler and Active Queue Management Algorithm,” RFC 8290 (Experimental), RFC Editor, Jan. 2018. https://www.rfc-editor.org/rfc/rfc8290.txt REFERENCES [1] M. Heusse, F. Rousseau, G. Berger-Sabbatel, and A. Duda, “Perfor- mance anomaly of 802.11 b,” in IEEE INFOCOM 2003. Twenty-Second Annual Joint Conference of the IEEE Computer and Communications Societies, vol. 2. IEEE, 2003, pp. 836–843. B. Dynamic measurements Fig. 3: Aggregate airtime usage of the two BSSes, for the same test as that shown in Figure 2. To evaluate the reaction time of the scheduler as station activity varies, we perform another set of UDP tests where 0 100 200 300 400 500 600 Time (s) 0 2 4 Mbits/s Fig. 5: DASH video throughput with prioritisation (solid lines) and without (dashed lines). The straight lines (orange) show the video bitrate picked by the player, while the others show the actual data stream goodput. 0 100 200 300 400 500 600 Time (s) 0 2 4 Mbits/s we start the ﬂows to each of the stations ﬁve seconds apart. We perform this test for the dynamic and limit modes, as these are the cases where the scheduler needs to react to changes in station activity. The results of this dynamic test is shown in Figure 4 as time series graphs of airtime share in each 200 ms measurement interval. The station that starts ﬁrst is Station 4 from the previous graphs, i.e., the station that is on BSS 2. In dynamic mode, as seen in Figure 4a, the ﬁrst station is limited to half the available airtime as soon as the second station starts transmitting. And because the two groups are set to share the airtime evenly, as more stations are added, the ﬁrst station keeps using half the available airtime, while the others share the remaining half. Fig. 5: DASH video throughput with prioritisation (solid lines) and without (dashed lines). The straight lines (orange) show the video bitrate picked by the player, while the others show the actual data stream goodput. In limit mode, as we saw before, the airtime shares of each of the four stations correspond to their fair share. This is also seen in Figure 4b, where all stations share the airtime equally as new stations are added. PoliFi can improve performance of WiFi networks with high airtime contention, and enables novel network usages such as network slicing. For this reason we believe it to be an important addition to modern WiFi networks, which is made widely available through its inclusion in the upstream Linux WiFi stack. These dynamic results show that PoliFi has a short reaction time, and can continuously enforce airtime usage policies as station activity changes. This is important for deployment in a real network with varying activity levels. [12] Big Buck Bunny, Blender Foundation, 2018. https://peach.blender.org/ [13] “dash.js reference DASH player,” Dash Industry Forum, 2018. https: //github.com/Dash-Industry-Forum/dash.js/wiki V. CONCLUSION [11] T. Høiland-Jørgensen, P. Hurtig, and A. Brunstrom, “PoliFi: Airtime Pol- icy Enforcement for WiFi,” Feb. 2019. https://doi.org/10.5281/zenodo. 2556784 We have presented PoliFi, a solution for enforcing airtime usage policies in WiFi networks. Our evaluation shows that PoliFi makes it possible to express a range of useful policies, including prioritisation of speciﬁc devices, and balancing or limiting of groups of devices. We have also shown how the policy enforcement can improve the performance of a real- world DASH video streaming application. [12] “Big Buck Bunny,” Blender Foundation, 2018. https://peach.blender.org/ [13] “dash.js reference DASH player,” Dash Industry Forum, 2018. https: //github.com/Dash-Industry-Forum/dash.js/wiki
https://openalex.org/W2106364637	https://ccsenet.org/journal/index.php/jedp/article/download/12571/11065	English	null	Motivational Beliefs and Self-regulated Learning in Low Vocational Training Track Students	Journal of educational and developmental psychology	2,012	cc-by	8,372	Vol. 2, No. 1; May 2012 Vol. 2, No. 1; May 2012 Journal of Educational and Developmental Psychology www.ccsenet.org/jedp Abstract The present study investigates students’ self-regulation of cognition and motivation and how they relate to motivational beliefs across three topics. Three types of topic-specific motivational beliefs (self-efficacy beliefs, achievement goals, and perceived instrumentality) and cognitive (cognitive and metacognitive strategy use in reading comprehension, writing, and mathematics) and motivational self-regulation were assessed by self-report in a sample of 243 vocational students in low vocational training tracks. The results indicate that perceived instrumentality is the prominent predictor of cognitive self-regulation across the three topics whereas mastery goals and self-efficacy play a significant but minor role in that prediction.Moreover, a significant self-efficacy by perceived instrumentality interaction effect emerged regarding cognitive self-regulation in mathematics. Motivational self-regulation is predicted consistently by self-efficacy, mastery goals, and work-avoidance goals. The differential effects of motivational beliefs on the facets of self-regulation are discussed. Keywords: Self-regulated learning, Vocational education and training, Motivation, Motivation Strategy use Published by Canadian Center of Science and Education 1. Introduction The ability to self-regulate one’s learning is recognized as an important determinant of school success and more generally as being crucial to lifelong learning (Boekaerts, 1997). Pintrich (2000) has defined self-regulated learning (SRL) as “an active, constructive process whereby learners set goals for their learning and then attempt to monitor, regulate, and control their cognition, motivation, and behavior, guided and constrained by their goals and the contextual features in the environment” (p. 453). An extended literature shows that SRL consistently relates to numerous motivational beliefs (Schunk & Zimmerman, 2008; Zimmerman, 2011). Accordingly, SRL is described as a motivationally driven process: Due to the need for concentration, effort, and attentive self-reflection in applying self-regulation strategies, only the most valued skills or outcomes are typically self-regulated (Lens & Vansteenkiste, 2008; Zimmerman, 2011). However, research has mainly focused on the regulation of cognition, i.e., the use of cognitive and metacognitive learning strategies, and less attention has been given to how the regulation of motivation relates to students’ motivational beliefs (De Corte, Depaepe, & Verschaffel, 2011; Wolters, 2011). Furthermore, most knowledge of SRL is based on students in primary to university programs, disregarding some groups of students, such as vocational students and students in the lower tracks of education, for whom improvements in SRL abilities may be more important as they tend to self-regulate to a lesser extent or with less efficiency than conventional students (Zimmerman, 2000). Bearing in mind the lack of studies considering several domains (or areas) of regulation such as the cognitive and motivational ones, and the limited evidence regarding vocational students with learning difficulties, the current study investigated the differential effect of multiple motivational beliefs (self-efficacy, goal orientation, and instrumentality) on the regulation of cognition and motivation in vocational students across several topics. 1.1 Self-regulated Learning as Cognitive and Motivational Regulation Motivational Beliefs and Self-regulated Learning in Low Vocational Training Track Students Jean-Louis Berger Swiss Federal Institute for Vocational Education and Training Avenue de Longemalle 1, Case Postale 192, 1000 Lausanne Malley 16, Switzerland Tel: 41-21-621-8231 E-mail: Jean-Louis.Berger@iffp-suisse.ch Received: December 20, 2011 Accepted: January 6, 2012 Published: May 1, 2012 doi:10.5539/jedp.v2n1p37 URL: http://dx.doi.org/10.5539/jedp.v2n1p37 Received: December 20, 2011 Accepted: January 6, 2012 Published: May 1, 2012 doi:10.5539/jedp.v2n1p37 URL: http://dx.doi.org/10.5539/jedp.v2n1p37 1.1 Self-regulated Learning as Cognitive and Motivational Regulation 1.1 Self-regulated Learning as Cognitive and Motivational Regulation While learning, students use strategies in order to acquire, understand, memorize, and transfer knowledge (Weinstein, Acee, & Jung, 2011). In other words, they regulate their cognition for using adaptive strategies to learn the material. Cognitive regulation refers to selecting appropriate strategies such as rehearsal or organization 37 Published by Canadian Center of Science and Education Journal of Educational and Developmental Psychology Vol. 2, No. 1; May 2012 www.ccsenet.org/jedp (i.e., cognitive strategies), and overseeing learning using metacognitive strategies such as planning or monitoring (Brown, 1987). There is strong empirical evidence supporting the positive relationship between the degree of cognitive regulation and students' achievement (Zimmerman & Martinez-Pons, 1988). In studies of SRL in vocational education, students are generally characterized by their teachers as cognitively passive when confronted with learning tasks (Elke et al., 2010): They do not spontaneously use strategies either because they do not know how to proceed or because they are not motivated to do so. While our own research has shown that they can learn self-regulation strategies successfully (Berger, Kipfer, & Büchel, 2008), less is known about how their motivation influences their cognitive regulation. In addition to students’ ability to regulate their cognition, their capacity to regulate their motivation is known to have an important impact on the quality of their learning and their level of achievement (Wolters, 2003, 2011). In other words, students must use resource management strategies, modulate their effort (increase their persistence or time on task), and manage their affective and motivation processes to achieve their goals (Boekaerts, 1997; Corno, 2008; Kuhl, 1987). Some studies have even revealed that “self-discipline” is more important than intelligence and prior grades in school success explaining more variance than either of these two variables (Duckworth & Seligman, 2005). Regulation of motivation could be represented by two perspectives: Students' use of strategies for the regulation of their motivation (Wolters, 2003), paralleling the use of learning strategies for cognitive regulation, or students' ability to regulate their motivation effectively (volitional perspective; Kuhl, 1984). The second perspective is adopted here, focusing on the extent to which a student can self-regulate rather than on the tools he or she can develop and use for self-regulation. Among the concepts developed to theorize this regulation, we have chosen “action control”, introduced by Kuhl (1984), to represent regulation of motivation. 1.1 Self-regulated Learning as Cognitive and Motivational Regulation Kuhl (1987) observed that although the expectancy-value theory can explain what individuals intend to do, it would best be extended to explicate to which extent they actually perform actions to realize these intentions; namely the ability to control the enactment of an intention. In this way, Kuhl enriched the expectancy-value theory through the addition of “action control”, which may help explain the performance deficit observed by the learned helplessness phenomena. A basic distinction is made regarding the focus of attention in the time between the intention to reach a goal and performance of the necessary activities: Attention can focus on action-related information, thus facilitating performance, or on state-related information, thus hindering performance. State-orientation is potentially related to the processing of more information than what is necessary, whereas action-orientation may be related to a more parsimonious processing of information for attainment of a goal. Hence, action-oriented students have limited difficulties in enacting their intention even if they are faced with obstacles. For example, an action-oriented student faced with solving a challenging mathematical problem will initiate the solving process and persevere even if she cannot find a solution quickly. Contrarily, a state-oriented student in the same situation will tend to ruminate on the difficulty, become anxious, switch to another task or stop studying in favor of a more enjoyable activity such as watching TV (Kuhl, 1984). Motivational Beliefs, Regulation of Cognition, and Regulation of Motivation 1.2.2 Achievement Goals and SRL 1.2.2 Achievement Goals and SRL The achievement goals framework postulates reasons students engage in learning, including the standard they use for judging their competencies (Pintrich, 2000). Several types of goals have been proposed to describe motivation to learn, such as mastery goals and work-avoidance goals. Mastery goals are adopted by students wanting to understand, master, and learn the material, whereas students aiming to be successful with the least effort embrace work-avoidance goals (Nicholls, 1989). Over the past 25 years, research on achievement goals and learning strategies has established strong connections between goals and the use of cognitive regulation strategies at the topic level. Most studies found that the use of learning strategies is positively correlated with mastery and negatively associated with work-avoidance goals (e.g., Dowson & McInerney, 2001; Dupeyrat & Mariné, 2005; Vrugt & Oort, 2008). Furthermore, in a study with 263 vocational students learning professional mathematics, it was found that mastery goals were positively correlated with rehearsal, elaboration, and metacognitive strategies whereas work-avoidance goals were significantly correlated only with rehearsal strategies (Berger, 2012). This last type of strategies does not favor long term and flexible learning (Weinstein et al., 2011), which is indicative of the indirect effect achievement goals may have on learning. Linnenbrink and Pintrich (2000) assume that the diverse types of achievement goals are linked differentially to behavioral processes and therefore to motivational regulation. Students adopting mastery goals would have a tendency to be highly persistent and to exert more effort in comparison to students adopting other types of goals. Contrarily, those adopting work-avoidance goals would tend to be less persistent and to regulate their motivation to a lesser degree. In one of the rare empirical studies on achievement goals and motivational regulation, Wolters and Rosenthal (2000) found that mastery goals were a consistent and strong predictor of the use of several types of strategies to regulate one’s motivation. 1.2.3 Perceived Instrumentality and SRL Husman and Lens (1999) have defined perceived instrumentality as “an individual’s understanding of the instrumental value of a present behavior for the attainment of future goals” (p. 116). Research evidence indicates that instrumentality has a positive effect on cognitive and motivational regulations, contrary to other forms of extrinsic motivation based on rewards or punishments, which have been found to hinder students’ SRL (Lens, Simons, & Dewitte, 2002). 1.3 Aim of the Study 1.3 Aim of the Study In sum, research evidence indicates that all motivational beliefs, be they self-efficacy, achievement goals or instrumentality, can sustain and trigger cognitive and motivational regulations. However, few studies have combined multiple motivational beliefs to investigate how they additively, and perhaps interactively, predict SRL. Moreover, studies with vocational students are rare, meaning that we do not know whether the patterns of relations between motivational beliefs and SRL in these students differ from what was observed with other types of students. Therefore, our aim in the present study is to investigate the motivational predictors of cognitive and motivational self-regulation in students in low vocational training tracks. 1.2.2 Achievement Goals and SRL In a study with 184 first-year nursing students, Simons, Dewitte and Lens (2004) investigated the effect of course perceived instrumentality on cognitive (deep vs. surface level of processing) and motivational (persistence) regulation. They found that the more students believed a course was useful for their future occupation, the more they persevered when studying for this same course. Although no direct effect on cognitive regulation was found, further path analysis of the data showed that perceived instrumentality indirectly predicted cognitive regulation through the mediation of task-orientation (akin to mastery goals). Similarly, in two empirical studies with 297 and 269 (respectively) high school students learning mathematics, Miller, Greene, Montalvo, Ravindran, and Nichols (1996) consistently found that perceptions of instrumentality of course performance to the attainment of future goals was predictive of the cognitive regulation, over and above the effect of achievement goals and perceived math ability (akin to self-efficacy). Accordingly, perceived instrumentality seems to play a key role in adolescents’ SRL. 1.2 Motivational Beliefs, Regulation of Cognition, and Regulation of Motivation 1.2 Motivational Beliefs, Regulation of Cognition, and Regulation of Motivatio Numerous studies have indicated that motivational beliefs are predictive of SRL, which in turn explain school achievement (e.g., Hong & Peng, 2008; Metallidou & Vlachou, 2007; Pintrich & DeGroot, 1990). Therefore, we consider motivational beliefs as predictors of SRL in the present study. Within the multiplicity of motivational beliefs, self-efficacy, achievement goals, and instrumentality were especially targeted for examination in relation to SRL. These associations are briefly reviewed below. 1.2.1 Self-efficacy and SRL Self-efficacy is defined as the perceived capability to execute a task or to succeed in a specific topic (Bandura, 1997). Applied to students, this means the extent to which they feel able to learn a certain topic or successfully solve a task in school. Considerable evidence has documented the relationship between expectancy for success (typically assessed as self-efficacy beliefs) and the adaptive use of cognitive and metacognitive strategies. Student who have more confidence in their abilities to learn are better at monitoring their work time and modifying their learning strategy when necessary (Pajares, 2008). Berger & Karabenick (2011) emphasized that in longitudinal studies, self-efficacy explained additional variance over time in higher-order cognitive and metacognitive strategy use; that is, higher self-efficacy is related to an increased use of deep-processing strategies over time. These results are supported by Borkowski, Chan, and Muthukrishna's (2000) heuristic developmental model of self-regulated learning, which assumes reciprocal effects of motivational beliefs and cognitive and metacognitive strategy use. According to this model, self-efficacy beliefs lead to the efficient use of learning strategies and improved performance, including the increased use of metacognitive strategies. According to Schunk and Pajares (2005), self-efficacy beliefs are positively predictive of the quantity of effort to Schunk and Pajares (2005), self-efficacy beliefs are positively predictive of the quantity of effor According to Schunk and Pajares (2005), self-efficacy beliefs are positively predictive of the ISSN 1927-0526 E-ISSN 1927-0534 ISSN 1927-0526 E-ISSN 1927-0534 38 Journal of Educational and Developmental Psychology Vol. 2, No. 1; May 2012 www.ccsenet.org/jedp students will exert on a task, how long they will persevere when facing difficulties, and how resilient they will be when confronted with adverse situations. This suggests a positive association between self-efficacy and ability to regulate one’s motivation. Accordingly, Pajares’ recent literature review (2008) concluded that the higher students' self-efficacy beliefs, the more persistence when confronted with challenges, regardless of prior achievement or ability. 2. Method 2.1 Participants Published by Canadian Center of Science and Education 2.1 Participants nts in this study were 243 vocational students in low-performing tracks. In accordance with school The participants in this study were 243 vocational students in low-performing tracks. In accordance with school 39 Published by Canadian Center of Science and Education Journal of Educational and Developmental Psychology Vol. 2, No. 1; May 2012 www.ccsenet.org/jedp principals, they were recruited in three vocational schools in the area of Geneva, Switzerland. The criterion for inclusion in the sample was the type of vocational training (2-years of training leading to a Federal Certificate of Vocational education and training, an intellectually less demanding training than the typical 3-4 years of training leading to a Federal Diploma of Vocational education and training; Stalder & Nägele, 2011). The mean age was 18 years and 8 months (SD = 2 yrs. 2 months), 61% were male and 39.9% were Swiss. They were apprentices in various professional fields, students from several fields being combined in the classes. 2.2.2 Regulation of Motivation Regulation of motivation was assessed by seven items for the language topic and seven items for the math topic in parallel. All items were from the QaL and measured the extent to which the student perceives herself or himself as able to regulate her or his motivation (e.g., “Even if I have to do difficult math tasks, I begin them right away.”). In the QaL validation study, this scale showed adequate internal consistency (respectively α = .87 - .81). 2.2 Instruments In addition to demographic information related to sex, nationality, and age, participants completed several scales. The motivational beliefs (excluding instrumentality) and regulation of motivation in math and language scales were taken from the Questions about Learning (QaL; Büchel, Berger, & Kipfer, 2011), a self-report instrument designed to assess motivation, social influences, and learning strategies in vocational students. Details about the construct validity of the QaL scales are available in Büchel et al. (2011). The regulation of cognition scales were designed for the present study. All items were rated on a 4-point Likert scale (1 = “not at all true of me” to 4 = “very true of me” for the motivation items; 1 = “nearly never” to 4 “nearly always” for the cognitive and motivational regulation items). 2.2.1 Motivational Beliefs Perceived instrumentality was assessed with a total of twelve items, four items for each of three subjects: Reading comprehension, writing (e.g., “Knowing how to correctly write a text will help me find a job after apprenticeship.”), and mathematics. All of the items refer to the value the participants placed on their future (distal goal) rather than on passing the exam. The items assessing perceived instrumentality of mathematics were also used by Berger (2008) with vocational students; they were found to strongly load on a single factor and yield an internally consistent score (α = .77). Self-efficacy beliefs were measured using two scales from the QaL (Büchel et al., 2011): Three items for language (e.g., “I feel able to succeed in reading, grammar and orthography.”) and three items for mathematics (e.g., “I feel able to succeed in math problem solving.”). In the QaL validation study (Büchel et al., 2011), this scale showed adequate internal consistency (respectively α = .69 - .60). Mastery goals were assessed by six items from the QaL, three for math and three for language. These items measured the extent to which the student is oriented toward learning, understanding, and mastering the material (e.g., “I want to learn as much as possible in math.”). In the QaL validation study, this scale showed adequate internal consistency (respectively α = .82 - .68). Work-avoidance goals were assessed by six items from the QaL, three for math and three for language, and measured the extent to which the student is oriented toward doing the least effort necessary and avoiding the difficult or challenging tasks (e.g., “Do you wish to avoid doing math tasks that require to think a lot?”). In the QaL validation study, this scale showed adequate internal consistency (respectively α = .58 - .59). 2.2.3 Regulation of Cognition 2.2.3 Regulation of Cognition Learning strategy scales were developed for three topics all students learn in vocational schools. Reading comprehension strategies were assessed with 11 items: Seven for cognitive strategies (e.g., “When I read a difficult text, I take notes.”) and four for metacognitive strategies (e.g., “When I read the title and first lines, I ask myself what the text is about.”). Writing strategies were assessed with 13 items: Six for cognitive strategies (e.g., “When I write a text, I ask help if I have some doubts.”) and seven for metacognitive strategies (e.g., “Before beginning to write a text, I take a while to think about what I am going to write.”). Mathematics strategies were assessed with 16 items: Nine for cognitive strategies (e.g., “When I learn mathematics, I try to establish relationships between what I learn in other topics.”) and seven for metacognitive strategies (e.g., “When I am finished with a math task, I check my calculations.”). ISSN 1927-0526 E-ISSN 1927-0534 40 Journal of Educational and Developmental Psychology Vol. 2, No. 1; May 2012 www.ccsenet.org/jedp 3. Results Exploratory factor analyses revealed that, within each topic, all cognitive and metacognitive strategy items loaded on a single factor rather than on two distinct factors. Therefore, students do not appear to distinguish in their answers the use of the two types of strategies; rather, they tend to use both strategies in concert. A confirmatory factor analysis was then performed specifying three factors, each representing regulation of cognition in the three topics. One item pertaining to reading comprehension strategies was removed for non-significant loading. Furthermore, four covariances between residuals were added to the CFA model, all justified by common wording in the items. The model fit was judged adequate given the large number of items: χ2 (683) = 1073.99, χ2/df = 1.57, RMSEA = .05, CFI = .83. In comparison, a two factor model (one factor for reading comprehension and writing strategies and another for math) has a worse fit to the data (χ2 (701) = 1228.08, χ2/df = 1.75, RMSEA = .06, CFI = .79). Descriptive statistics, internal consistency, and correlations are shown in Table 1. Reliability ranges from α = .71 to .88. Most scales taken from the QaL have higher internal consistency in the present study than in the validation study (Büchel et al., 2011). 2.3 Procedure The author and a graduate assistant collected the data during whole-class sessions usually devoted to general knowledge courses. Students were first presented with the purpose of the study and told that their responses would be anonymous and participation was voluntary. Students completed the instruments in random order. Teachers were asked to leave the room during data collection so that the students felt comfortable responding freely. Insert Table 1 Here As preliminary analyses, mean differences between topics were tested using paired-sample t-tests. The results show that students reported both more work-avoidance (t(250) = 3.409, p = .001, Cohen’s d = 0.26) and instrumentality (t(250) = 4.276, p < .001, d = 0.25) for learning math than language. No significant differences were found for self-efficacy and mastery-goals. Regarding SRL, students reported using more learning strategies (i.e., more cognitive regulation) for math than for reading comprehension (t(250) = 3.244, p = .001, d = .0.21) or redaction (t(250) = 2.987, p = .003, d = 0.17). No significant difference was found between reading comprehension and redaction (p = .47). Finally, the level of motivational regulation was found to be higher for language than for math (t(250) = -2.388, p = .018 d = 0.16). Path analysis was then performed to investigate the relationships between motivational beliefs (predictors) and the two forms of SRL (outcomes). A single model was built, specifying paths from the four variables representing math motivational beliefs to the two variables representing SRL in math, and paths from the four motivational beliefs in language to the three SRL variables related to language (cognitive regulation in reading comprehension, in writing, and motivational regulation). The scores were then corrected for measurement error. After removing the non-significant paths, the model was found to fit the data well: χ2 (23) = 38.72, p = 0.19, RMSEA = .03, CFI = .99. Further, tests of interaction effects revealed that self-efficacy and perceived instrumentality interacted to significantly predict learning strategy use in mathematics. No such interaction effect was found in the prediction of learning strategies in reading comprehension or in writing/redaction. A summary of the path analysis results is shown in Tables 2 and 3 respectively for the topics of math and language. Insert Tables 2 and 3 Here 4. Discussion The present study examined the predictive role of motivational beliefs on SRL in a sample of low vocational training tracks students. Two domains of regulation (cognitive and motivational) were considered across three topics (reading comprehension, writing, and math). Three types of motivational beliefs (self-efficacy, goal orientation, and instrumentality) were included as predictors in addition to individual characteristics. 4.1 The One-dimensionality of Cognitive Self-regulation within a Topic 4.1 The One-dimensionality of Cognitive Self-regulation within a Topic Published by Canadian Center of Science and Education 4.3 The Prediction of Cognitive Self-regulation by Motivational Beliefs We established that perception of instrumentality is the main predictor or cognitive regulation across the three topics, explaining nearly one-third of the variance in learning strategy use. Moreover, we discovered an interaction effect self-efficacy x instrumentality that is predictive of cognitive self-regulation in math. This means that expectancy and value work together to stimulate SRL: When students perceived both the topic to be useful and their ability to learn it to be high, they would then make the most use of learning strategies. Our results furthermore suggest that it may be beneficial to consider the additive and potentially interactive effects of several motivational beliefs when investigating the effects of motivation on SRL in order to obtain a more precise representation, mirroring the results of Miller et al. (1996). The main and interaction effects of instrumentality indicate that this is a key motivational belief in vocational students that might distinguish these students from students in mandatory school or academic curricula. As students engaged in a vocational education program will likely anticipate that it would provide them with useful knowledge for their career, they may be disappointed if they do not perceive instrumentality in the content. This implies that to foster cognitive self-regulation, vocational teachers should focus on their students' perceptions of utility about the tasks and homework they are assigned. One way to accomplish this during instruction is to systematically provide a rationale when asking students to learn new material or simply to work on a task (Jang, 2008; Kember, Ho, & Hong, 2008) or to ask students to think about task relevance (Hulleman, Godes, Hendricks, & Harackiewicz, 2010), two strategies which have been found to be effective in increasing perceived instrumentality for low-performing students. In contrast, mastery goals and self-efficacy were found to have a smaller effect on cognitive self-regulation, contrary to the major role they have played in other studies. This could be attributed to sample specificity in the present study that contrasts with college or high school students on which most knowledge in the field of SRL is based. Therefore, the dynamic of SRL is sensitive to the type of students and contextual differences must be considered in the analysis of this dynamic. Our results call for more research on vocational students’ SRL, notably to study how their regulations differ qualitatively and quantitatively from other students. 4.2 Mean Differences across Topics Several significant, though moderate, differences in motivational beliefs and SRL were found across topics. Vocational students consider math more useful than language. This mirrors results of several studies indicating that this group of students gives more importance to math considering it directly useful for the occupation they are learning (Berger, in press; Cretten, Lens, & Simons, 2001; Prenzel, Kramer, & Dreschel, 2002). Puzzlingly, work-avoidance goals were rated higher for math than for language, suggesting some incoherence in students’ motivational beliefs that might be illustrated by the statement: “I believe math is useful but I am trying to do the least effort to learn it”. A potential explanation is that these students, even while valuing a topic, are not willing to put forth the effort needed to learn due to their past struggles as low-achievers in mandatory schools: They may have found that hard work was not sufficient for success and therefore view effort as a “double-edged sword” (Covington & Omelich, 1979). Furthermore, while students reported to regulate their cognition more intensely in math than in the two language topics, they reported the opposite for motivational regulation. The zero-order correlations however indicate that, across topics, both areas of regulation are positively associated. In sum, motivational beliefs and SRL are, as observed in other students and contexts, to some extent sensitive to the topic (Alexander, Dinsmore, Parkinson, & Winters, 2011). 4.1 The One-dimensionality of Cognitive Self-regulation within a Topic While the cognitive self-regulation scales were developed to assess two sub-forms of regulation (namely the use of cognitive and the use of metacognitive strategies), the results show that students do not discriminate in their answers between these two forms. There is thus a single empirical dimension on a continuum from low to high engagement in cognitive self-regulation. Similar results were found across the three topics and are in accordance with the results from others (e.g., Liu, 2009; Schraw & Dennison, 1994). This one-dimensionality might be seen as a limitation due to the self-reported nature of the data: While students cognitively process such items, they may activate memories that relate not only to the specific self-regulation strategy in question but also to other strategies, leading to answers that may not be as precise as researchers would prefer (Berger & Karabenick, 41 Published by Canadian Center of Science and Education Journal of Educational and Developmental Psychology Vol. 2, No. 1; May 2012 www.ccsenet.org/jedp 2010). Note however that others studying SRL with think aloud or interview methods have also found the diverse strategies to be highly correlated and therefore explained by a single factor (Veenman, Elshout, & Meijer, 1997; Zimmerman & Martinez-Pons, 1988). Consequently, we might argue with Pintrich, Wolters, and Baxter’s (2000) conclusion, from multiple studies revealing large interrelations between strategies, that “students who engage in one component of self-regulated learning also engage in other components. Accordingly, efforts to separate the different components into theoretically smaller subcomponents may not be justified by the empirical data” (p. 81). The present study indicates that whereas sub-forms of cognitive regulation cannot be empirically separated, cognitive and motivational regulations can be distinguished as they correlate only moderately. 4.5 Limitations and Further Research One of the main limitations of this study is the exclusive reliance on self-report. Although the instruments demonstrate adequate psychometric properties, investigation of the validity of students' answers using non-psychometric methodology is warranted to shed light on how students understand, interpret, and formulate answers when questioned about their regulation. Cognitive interviewing or cognitive pretesting may provide such information and therefore, indicate how items could be improved for specific populations (Berger & Karabenick, 2010; Karabenick et al., 2007). Another limitation concerns the cross-sectional research design that does not allow for a causal interpretation of the results. As some longitudinal studies (Berger & Karabenick, 2011; Linnenbrink & Pintrich, 2003; Zusho, Pintrich, & Coppola, 2003) have revealed that motivation influences SRL rather than the opposite, the same reasoning was applied in the present study. It is however critical that longitudinal studies on vocational students' SRL be conducted to provide further information on direction of causality or reciprocal causation between motivational beliefs and SRL. As recent research has shown that regulation is generally considered, even in adolescents’ classrooms, to be the teacher’s responsibility (De Corte et al., 2011; Kistner, Rakoczy, Otto, Dignath-van Ewijk, Büttner, & Klieme, 2010), further study should investigate how to implement methods helping teachers support their students’ use of self-regulation strategies in the classroom. The current study suggests that, as one part of this method, an indirect but efficient way to reach this goal is to change students’ learning environment toward a mastery goal structure and to communicate rationales to students that help them see the instrumental value of what they are taught at vocational schools. These two paths of action will favor both cognitive and motivational regulation, which should increase the chance of school success for students in low tracks of vocational education. 4.4 The Prediction of Motivational Self-regulation by Motivational Beliefs In contrast to the predictors of cognitive self-regulation, achievement goals (mastery positively and work-avoidance negatively) together with self-efficacy, predict largely (between 49% and 67% of explained variance) the regulation of motivation both in language and math. Instrumentality plays no significant role here, in contrast to its huge importance in cognitive self-regulation, which is in accordance with Simons et al.'s (2004) results: Goal orientations were proximal predictors of motivational regulation whereas instrumentality has only ISSN 1927-0526 E-ISSN 1927-0534 42 Journal of Educational and Developmental Psychology Vol. 2, No. 1; May 2012 www.ccsenet.org/jedp an indirect and weak effect. Accordingly, sustaining motivation does not depend on the value of the task but on reasons why one is studying and one’s beliefs about his or her ability to learn the material. These conclusions are consistent across topics and therefore robust. Implications are that vocational teachers should create classroom goal structure oriented toward mastery and help students foster confidence in their ability to learn. In sum, the results indicate that there is a need to focus on motivational beliefs if we are to help vocational students to self-regulate their learning. This focus is complementary to strategic instruction aiming at teaching knowledge about learning strategies (Berger, Kipfer, & Büchel, 2008; Borkowski et al., 2000). and Borkowski, J. G., Chan, L. K. S., & Muthukrishna, N. (2000). A process-oriented model of metacognition: Links between motivation and executive functioning. In G. Schraw & J. C. Impara (Eds.), Issues in the measurement of metacognition (pp. 1-41). 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Bergman, S. Hupka-Brunner, A. Keller, T. Meyer & B. E. Stadler (Eds.), Youth in transition in Switzerland: Results from the TREE panel study (pp. 18-39). Zürich, Switzerland: Seismo. Veenman, M. V. J., Elshout, J. J., & Meijer, J. (1997). The generality vs domain-specificity of metacognitive skills in novice learning across domains. Learning and Instruction, 7(2), 187-209. http://dx.doi.org/10.1016/S0959-4752(96)00025-4 Vrugt, A., & Oort, F. J. (2008). Metacognition, achievement goals, study strategies and academic achievement: pathways to achievement. Metacognition and Learning, 3(2), 123-146. http://dx.doi.org/10.1007/s11409-008-9022-4 Weinstein, C. E., Acee, T. W., & Jung, J. (2011). Self-regulation and learning strategies. New Directions for Teaching and Learning, 126, 45-53. http://dx.doi.org/10.1002/tl.443 Wolters, C. A. (2003). Regulation of motivation: Evaluating an underemphasized aspect of self-regulated learning. Educational Psychologist, 38(4), 189-205. http://dx.doi.org/10.1207/S15326985EP3804_1 Wolters, C. A. (2011). Regulation of motivation: Contextual and social aspects. Teachers College Records, 113(2), 265-283. Wolters, C. A., & Rosenthal, H. (2000). The relation between students’ motivational beliefs and their use of motivational regulation strategies. International Journal of Educational Research, 33, 801-820. http://dx.doi.org/10.1016/S0883-0355(00)00051-3 Zimmerman, B. J. (2000). Attaining self-regulation. A social cognitive perspective. In M. Boekaerts, P. R. Pintrich & M. Zeidner (Eds.), Handbook of self-regulation (pp. 13-39). San Diego, CA: Academic Press. Zimmerman, B. J. (2011). Motivational sources and outcomes of self-regulated learning and performance. In B. J. Zimmerman & D. H. Schunk (Eds.), Handbook of self-regulation of learning and performance (pp. 49-64). New York: Routledge. Zimmerman, B. J., & Martinez Pons, M. (1988). Construct validation of a strategy model of student self-regulated learning. Journal of Educational Psychology, 80(3), 284-290. http://dx.doi.org/10.1037//0022-0663.80.3.284 Zusho, A., Pintrich, P. R., & Coppola, B. (2003). and Skill and will: the role of motivation and cognition in the learning of college chemistry. International Journal of Science Education, 25(9), 1081-1094. http://dx.doi.org/10.1080/0950069032000052207 ISSN 1927-0526 E-ISSN 1927-0534 Summary of path analysis for motivational beliefs and age predicting cognitive and motivational regulation in language Predictors Regulation of cognition and motivation in language Learning strategy use in reading comprehension Learning strategy use in writing/redaction Action control β SE p β SE p β SE p Age .13 .07 .050 .14 .06 .013 - - - Self-efficacy - - - .17 .05 .002 .28 .08 .001 Mastery goals .15 .07 .027 - - - .43 .11 < .001 Work-avoidance goals - - - - - - -.27 .08 .001 Perceived instrumentality .51 .07 < .001 .53 .06 < .001 - - - R2 .40 .40 .67 Note: Nationality and sex are not included in the table because neither were significant predictors of any dependent variable. All predictors are related to language. Table 1. ISSN 1927-0526 E-ISSN 1927-0534 Descriptive statistics and correlations Variable M SD α 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Demographics 1 Sex (0 = male, 1 = female) 0.36 -- -- -.00 .02 -.20 .07 .08 .19 .03 -.10 -.04 .07 .09 .20 -.11 .08 .10 2 Nationality (0 = Swiss, 1 = Foreigner) 0.61 -- -- -- .03 .03 -.04 .13 .11 .05 .09 .06 .07 .10 .03 .11 .12 .07 3 Age (years) 18.59 1.74 -- -- .13 .04 .10 .03 -.02 .02 .14 .16 .09 .04 .22 .19 .22 Motivational beliefs 4 Self-efficacy beliefs in mathematics 2.95 0.54 .76 -- .20 .41 .17 -.18 -.02 .23 .17 .41 .07 .38 .15 .14 5 Self-efficacy beliefs in language 3.04 0.63 .81 -- .20 .47 .12 -.40 .09 .21 .16 .55 .17 .25 .29 6 Mastery goals in mathematics 3.18 0.58 .81 -- .36 -.23 -.17 .42 .40 .50 .25 .46 .27 .20 7 Mastery goals in language 3.25 0.61 .81 -- -.06 -.41 .26 .42 .36 .62 .27 .37 .36 8 Work-avoidance goals in mathematics 2.63 0.67 .71 -- .27 -.18 -.02 -.29 -.01 -.09 .03 .03 9 Work-avoidance goals in language 2.44 0.78 .72 -- -.10 -.12 -.27 -.53 -.07 -.11 -.10 10 Perceived instrumentality of mathematics 3.20 0.64 .78 -- .57 .32 .13 .56 .25 .23 11 Perceived instrumentality of language 3.05 0.56 .84 -- .23 .27 .50 .49 .49 Regulation of motivation 12 Action control in mathematics 2.93 0.48 .84 -- .42 .39 .28 .29 13 Action control in language 3.02 0.58 .86 -- .15 .30 .33 Regulation of cognition 14 Learning strategy use in mathematics 2.64 0.53 .88 -- .52 .61 15 Learning strategy use in reading comprehension 2.53 0.54 .79 -- .75 16 Learning strategy use in writing/redaction 2.55 0.54 .83 -- Note: correlations ≥.13 are significant at p < .05, r ≥.17 are significant at p < .01, and r ≥.22 are significant at p < 001 Table 1. Descriptive statistics and correlations Table 2. ISSN 1927-0526 E-ISSN 1927-0534 ISSN 1927-0526 E-ISSN 1927-0534 46 Journal of Educational and Developmental Psychology Vol. 2, No. 1; May 2012 Journal of Educational and Developmental Psychology Vol. 2, No. 1; May 2012 www.ccsenet.org/jedp www.ccsenet.org/jedp Journal of Educational and Developmental Psychology Vol. 2, No. 1; May 2012 Table 1. Descriptive statistics and correlations Variable M SD α 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Demographics 1 Sex (0 = male, 1 = female) 0.36 -- -- -.00 .02 -.20 .07 .08 .19 .03 -.10 -.04 .07 .09 .20 -.11 .08 .10 2 Nationality (0 = Swiss, 1 = Foreigner) 0.61 -- -- -- .03 .03 -.04 .13 .11 .05 .09 .06 .07 .10 .03 .11 .12 .07 3 Age (years) 18.59 1.74 -- -- .13 .04 .10 .03 -.02 .02 .14 .16 .09 .04 .22 .19 .22 Motivational beliefs 4 Self-efficacy beliefs in mathematics 2.95 0.54 .76 -- .20 .41 .17 -.18 -.02 .23 .17 .41 .07 .38 .15 .14 5 Self-efficacy beliefs in language 3.04 0.63 .81 -- .20 .47 .12 -.40 .09 .21 .16 .55 .17 .25 .29 6 Mastery goals in mathematics 3.18 0.58 .81 -- .36 -.23 -.17 .42 .40 .50 .25 .46 .27 .20 7 Mastery goals in language 3.25 0.61 .81 -- -.06 -.41 .26 .42 .36 .62 .27 .37 .36 8 Work-avoidance goals in mathematics 2.63 0.67 .71 -- .27 -.18 -.02 -.29 -.01 -.09 .03 .03 9 Work-avoidance goals in language 2.44 0.78 .72 -- -.10 -.12 -.27 -.53 -.07 -.11 -.10 10 Perceived instrumentality of mathematics 3.20 0.64 .78 -- .57 .32 .13 .56 .25 .23 11 Perceived instrumentality of language 3.05 0.56 .84 -- .23 .27 .50 .49 .49 Regulation of motivation 12 Action control in mathematics 2.93 0.48 .84 -- .42 .39 .28 .29 13 Action control in language 3.02 0.58 .86 -- .15 .30 .33 Regulation of cognition 14 Learning strategy use in mathematics 2.64 0.53 .88 -- .52 .61 15 Learning strategy use in reading comprehension 2.53 0.54 .79 -- .75 16 Learning strategy use in writing/redaction 2.55 0.54 .83 -- Note: correlations ≥.13 are significant at p < .05, r ≥.17 are significant at p < .01, and r ≥.22 are significant at p < .001 Table 2. ISSN 1927-0526 E-ISSN 1927-0534 ISSN 1927-0526 E-ISSN 1927-0534 Summary of path analysis for motivational beliefs and age predicting cognitive and motivational regulation in language Predictors Regulation of cognition and motivation in language Learning strategy use in reading comprehension Learning strategy use in writing/redaction Action control β SE p β SE p β SE p Age .13 .07 .050 .14 .06 .013 - - - Self-efficacy - - - .17 .05 .002 .28 .08 .001 Mastery goals .15 .07 .027 - - - .43 .11 < .001 Work-avoidance goals - - - - - - -.27 .08 .001 Perceived instrumentality .51 .07 < .001 .53 .06 < .001 - - - R2 .40 .40 .67 Note: Nationality and sex are not included in the table because neither were significant predictors of any dependent variable. All predictors are related to language. e 2. Summary of path analysis for motivational beliefs and age predicting cognitive and moti lation in language Note: Nationality and sex are not included in the table because neither were significant predictors of any dependent variable. All predictors are related to language. 47 Published by Canadian Center of Science and Education 47 Journal of Educational and Developmental Psychology Vol. 2, No. 1; May 2012 www.ccsenet.org/jedp Table 3. Summary of path analysis for motivational beliefs predicting cognitive and motivational regulation in mathematics Predictors Regulation of cognition and motivation in mathematics Learning strategy use in mathematics Action control β SE p β SE p Self-efficacy .18 .07 .015 .26 .11 .016 Mastery goals .25 .07 < .001 .40 .10 < .001 Work-avoidance goals - - - -.27 .08 < .001 Perceived instrumentality .56 .06 < .001 - - - Self-efficacy x Perceived instrumentality .23 .08 .002 - - - R2 .62 .49 Note: Age, nationality and sex are not included in the table because none were significant predictors of any dependent variable. All predictors are related to mathematics. Note: Age, nationality and sex are not included in the table because none were significant predictors of any dependent variable. All predictors are related to mathematics. ISSN 1927-0526 E-ISSN 1927-0534 ISSN 1927-0526 E-ISSN 1927-0534 48
https://openalex.org/W4213018202	http://komunikacie.uniza.sk/doi/10.26552/com.C.2016.3.22-27.pdf	English	null	Mathematical Model for Charter Flights Planning at Airports with High Air Traffic Volume	Komunikácie	2,016	cc-by	4,904	https://doi.org/10.26552/com.C.2016.3.22-27 https://doi.org/10.26552/com.C.2016.3.22-27 Vojtech Graf - Dusan Teichmann - Michal Dorda* * Vojtech Graf, Dusan Teichmann, Michal Dorda Institute of Transport, Faculty of Mechanical Engineering, VSB - Technical University of Ostrava, Ostrava-Poruba, Czech Republic E-mail: vojtech.graf@vsb.cz MATHEMATICAL MODEL FOR CHARTER FLIGHTS PLANNING AT AIRPORTS WITH HIGH AIR TRAFFIC VOLUME MATHEMATICAL MODEL FOR CHARTER FLIGHTS PLANNING AT AIRPORTS WITH HIGH AIR TRAFFIC VOLUME The article is devoted to a problem of flight timetable planning for charter airlines if air transport is operated at airports with high air traffic volume. It is typical for such airports that air transport is subject to time slots. That means aircraft can take-off or land only in given time intervals. A suitable tool for solving such type of the task is linear programming which has been successfully employed for planning in other transport systems. In the article a mathematical model which is able to plan a flight timetable for a given set of flights is presented. Keywords: Aircraft scheduling, linear programming, timetable planning. Keywords: Aircraft scheduling, linear programming, timetable planning. 1. Introduction Transport processes in air transport that have to be planned by airlines can be divided into several groups of elementary problems: To provide reliable and regular operation of air transport all round the world, it is necessary to plan flight timetables. The flight timetables are planned not only by airlines that provide regular air transport but also airlines providing charter air transport. • Schedule design problems. Planning flight timetables is a complex and time-consuming process. The process of flight timetable preparation is usually started a year before its year of validity. The whole process is influenced by many factors. The most important factors are demands of travel agencies, operating times of airports and time slots for taking-off and landing. By means of the time slots (time intervals) airports dictate to airlines when it is possible to plan taking-off or landing at the airport. The problem of flight timetable planning can be considered to be a task of planning service of a given set of flights under different time constraints. Therefore, mathematical modelling can be successfully employed for solving the task. • Fleet assignment problems. • Aircraft maintenance routing problems. • Crew scheduling problems. • Rostering problems. the whole year; that means each airline needs a free time slot for taking-off or landing at the airport. is employed, the metaheuristic is based on simulated annealing metaheuristic. The crew scheduling problem is discussed in publication [4]. The publication is based, in contrast to other publications employing mathematical programming methods, on an unconventional approach that uses artificial neural networks. Each airport can employ several types of the time slots. We can have the time slots for arrivals, departures, using passenger boarding steps or ramps, refuelling and so on. In the mathematical model presented in the article it is not necessary to consider such dividing the time slots. We can assume that each time slot used in the model corresponds to the intersection of all the above mentioned categories of the time slots. That means during the time slot the plane can land or take-off, the passengers can disembark from the plane or board the plane and the plane can be refuelled. The last publication devoted to the single purpose approaches we would like to mention is work [5]. The publication presents a model of the crew scheduling problem. The publication does not apply the model in air transport but the results can be also used for air transport. To solve the model an approach based on column generation technique is applied. The integrated approaches include models and algorithms for solving different combinations of the above mentioned elementary problems. We can mention, for example, publications [6, 7 and 8]. An approach published in [6] is the simplest approach; the approach is sequential. It is based on sequential optimisation of the individual problems in a pre-defined order. The individual problems are solved in the order as given above in the section. The integrated approaches include models and algorithms for solving different combinations of the above mentioned elementary problems. We can mention, for example, publications [6, 7 and 8]. From the point of view of charter airlines that plan flight timetables for the whole season in advance, it is decisive that some airports (especially during the peak season or in seaside resorts) may be loaded by higher air traffic volumes. Therefore, the airline has to take into account the fact that the airport permits arrivals and departures only in the assigned time slots. Because the numbers of the time slots and their lengths may be different for the individual flights, it is necessary to incorporate the fact into the mathematical model. An approach published in [6] is the simplest approach; the approach is sequential. It is based on sequential optimisation of the individual problems in a pre-defined order. The individual problems are solved in the order as given above in the section. Publication [7] includes an integrated approach to the flight scheduling problem together with maintenance planning and crew scheduling within a period of a week. To solve the task a heuristic named cycle checking strategy is employed. The applied strategy splits flight plans in subsets and the subsets are matched one another. Maintenance limitations are checked when splitting the flight plans. Demands for the time slots are submitted to the airports in advance. The time slots at the airports are assigned to the airlines by an airport slot coordinator. After evaluations of all the demands submitted before the season the airport slot coordinator creates a preliminary flight plan to coordinate the individual time slots one another and assigns the time slots to each applicant (the charter airlines). That means the charter airline already knows its assigned time slots when planning the flight timetable and, therefore, has to use the time slots in order not to be sanctioned [9]. Publication [8] discusses an integrated approach to aircraft routing and crew scheduling. To solve the problem a three-step algorithm based on Benders’ decomposition, column generation technique and dynamic constraint generation procedure is employed. • Rostering problems. Approaches that are used for solving the basic above mentioned problems in air transport can be divided into two basic groups – approaches that are used only for solving one of the above mentioned problems (single purpose approaches) and approaches that solve several basic problems at the same time (integrated approaches). From the broad spectrum of publications devoted to the single purpose approaches we can mention, for example, publications [2, 3, 4 or 5]. The article continues in article [1]; in the article a mathematical model that enables to plan flight timetables if two time slots are available for each flight was presented. The original mathematical model was generalised as regards the number of available time slots for the flights; the improved mathematical model is described in the article. The improvement lies in the fact that for each flight any finite number of time slots can be given. Please note that the number of the available time slots can differ for the individual flights. Source [2] presents a model for daily operational flight planning. The total costs of an airline including the costs of passengers caused by flight delays are used as an optimisation criterion. To solve the model the authors proposed a special four- step heuristic. Publication [3] is devoted to aircraft maintenance planning. The total unused legal flying time of the critical aircraft is an optimisation criterion. The publication presents a mathematical model that minimises the value of the optimisation criterion. To solve the model a metaheuristic named compressed annealing 22 ● C O M M U N I C A T I O N S 3 / 2 0 1 6 23 C O M M U N I C A T I O N S 3 / 2 0 1 6 ● 3. Time slots in air transport Let a set I of flights be given, the set contains all the flights that have to be dispatched. For each flight i I ! a set Ji is given; the set contains all the possible time slots. The time slots define time periods in which the flight i I ! can depart (that means the flight has to be dispatched in one of the pre-defined time slots). Each time slot is defined by two values – a value dij is the lower bound of the time slot j Ji ! assigned to the flight i I ! and a value hij is the upper bound of the time slot j Ji ! of the flight i I ! . The article deals with a modification of the mathematical model which enables to plan flight timetables in situations for which it is typical that several time slots for each flight are given. Firstly, let us discuss the time slots in air transport. In general, time slots are defined as time intervals in which certain tasks connected with flights (taking-off or landing) or some aircraft services have to be carried out. The fact whether the airline has to make a request for the time slot or not depends on the airport where the task should be done. From the point of view of assigning the time slots, all the airports can be divided into uncoordinated, partially coordinated and fully coordinated airports. For the uncoordinated airports it is typical that the time slots are not applied for the tasks. The time slots are used for the partially or fully coordinated airports. For such airports high air traffic volumes are typical. The partially coordinated airports can apply the time slots only for some days or seasonally. The fully coordinated airports assign the time slots to the airlines during For each flight i I ! its flight time Ti is given. It corresponds to the time between taking-off and landing. A so-called preparation time is included in the model. The preparation time is a time that is needed for carrying out all tasks after landing the flight and for preparing a consecutive flight I j ! . In order to simplify it, the preparation time consists of three sub-times. , x 0 1 ij ! " , , i I j I 0 , ! ! " , (8) , z 0 1 ij ! " , , i I j Ji ! ! (9) t 0 i $ i I ! (10) aircraft to a ground power unit (GPU), getting off the passengers and unloading the baggage. The second sub-time depends on services which are demanded by the aircraft’s operator and on the flight time of the consecutive flight. The dependence is expressed by a product T k j $ , where Tj is the flight time of the flight I j ! and k is a relative constant. The relative constant expresses the ratio of the flight time Tj of the consecutive flight and the time that is needed for preparation the flight I j ! . The second sub-time contains cargo unloading, cleaning the aircraft, catering replenishment, cabin service (water replenishment, toilette emptying and so on), refuelling, a technical inspection and cargo loading. The third sub-time 2 x includes loading passengers’ baggage, boarding the passengers, removing passenger boarding steps or ramps, disconnecting the ground power unit and taxiing the aircraft from a terminal to a runway. (10) Formula (1) corresponds to the optimisation criterion of the mathematical model. As written earlier in the text, we try to minimise the number of the aircraft we need to serve all the planned flights. Constraints (2) ensure that each flight I j ! has to be dispatched. Constraints (3) model that only one of two possible tasks is assigned to the aircraft after serving the flight i I ! - the aircraft can be assigned to the consecutive flight I j ! or is idle. Constraints (4) ensure that if a consecutive flight is assigned to the aircraft, the assignment is admissible in terms of time. The term on the left side of constraint (4) equals to the departure time of the consecutive flight I j ! minus the sum of the departure time of the preceding flight i I ! ti ^ h, the flight time of the flight i I ! Ti ^ h and the preparation time before serving the flight I j ! T k j 1 2 $ x x + + ^ h. Constraints (5) and (6) ensure the admissible departure times of the flights i I ! . That means the departure times ti have to lie in the pre- defined time slots , d h ij ij . Constraints (7) assure that only single time slot j Ji ! is chosen for dispatching the flight i I ! . Constraints (8), (9) and (10) define the domains of definition of the variables used in the model. In general, the total number of the variables of the model is equal to m n m m 2 2 2 + + , where m is the number of the planned flights and n is the number of the time slots for the flight with the maximum number of the pre- defined time slots. The number of the constraints in the model is m n m m 2 7 2 2 + + . In order to model required decisions and to create necessary logical links 3 groups of variables ,t x i ij and zij are defined in the model. The variables ti model the departure times of the individual flights i I ! ; the variables ti can take non-negative real values. The variables xij model decisions about links between the flights i I ! and I j ! . If x 0 ij = , then the aircraft is not assigned to the flight I j ! after serving the flight i I ! . If x 1 ij = , then the aircraft is assigned to the flight I j ! after serving the flight i I ! . That means both flights are served by the same aircraft in the order i→j. Please note that if x 1 j 0 = then a new aircraft is assigned to the flight I j ! . The last group of the variables zij gives information which time slot j Ji ! is chosen for dispatching the flight i I ! (that means the departure time of the flight i I ! lies in the interval , d h ij ij ). If z 0 ij = , then the time slot j Ji ! is not used for dispatching the flight i I ! . On the other hand, if z 1 ij = , then the departure time of the flight i I ! lies in the time slot j Ji ! . P is a very large positive number. 5. Experiments Calculation experiments were carried out on model examples. The experiments were aimed at finding a dependence of the calculation time on the number of the planned flights and the length of the pre-defined time slots. In addition, we also tried to find out solvability limits of the mathematical model – to estimate for how many flights the model is able to find an optimal solution within a given time limit. In total 39 optimisation calculations were carried out with the model. All the experiments were run on a student (demo) version of optimisation software Express-IVE [10]. To run the experiments we employed a personal computer with processor AMD-8300 Eight-Core 3.3 GHz and 8GB RAM. 24 ● C O M M U N I C A T I O N S 3 / 2 0 1 6 min x j j I 0 !/ (1) x 1 ij i I 0 = , ! " , / j I ! (2) x 1 ij n j J # !/ i I ! (3) t t T T k P xi 1 j i i j j 1 2 $ $ $ x x - + + + + - ^ ^ h h , i I j I ! ! (4) d z t ij ij i j Ji $ # !/ i I ! (5) h z t ij ij i j Ji $ $ !/ i I ! (6) z 1 ij j Ji = !/ i I ! (7) min x j j I 0 !/ (1) x 1 ij i I 0 = , ! " , / j I ! (2) x 1 ij n j J # !/ i I ! (3) t t T T k P xi 1 j i i j j 1 2 $ $ $ x x - + + + + - ^ ^ h h , i I j I ! ! (4) d z t ij ij i j Ji $ # !/ i I ! (5) h z t ij ij i j Ji $ $ !/ i I ! (6) (1) For each flight i I ! its flight time Ti, the length of the time slots and their bounds , d h ij ij are known. A summary of all the values is provided in Table 1. Our goal is to assign the aircraft to the individual flights so that the number of the assigned aircraft is as minimal as possible. The mathematical model of the problem can be defined in the following form: 3. Time slots in air transport The first sub- time 1 x includes stopping the aircraft, placing wheel chocks, putting passenger boarding steps or ramps, connecting the 2 C O M M U N I C A T I O N S 3 / 2 0 1 6 ● 23 5. Experiments The length of the time slots was not constant during the experiments. For the first group of the experiments the length was 5 minutes (see Table 1), for the 24 ● C O M M U N I C A T I O N S 3 / 2 0 1 6 Input data Table 1 Flight Flight time dij hij 1 2 3 4 5 1 2 3 4 5 1 100 0 600 1000 - - 5 605 1005 - - 2 150 400 1150 - - - 405 1155 - - - 3 180 0 300 700 950 - 5 305 705 955 - 4 120 0 350 800 900 1300 5 355 805 905 1305 5 110 450 1200 - - - 455 1205 - - - 6 200 200 550 650 - - 205 555 655 - - 7 180 600 - - - - 605 - - - - 8 100 150 340 900 - - 155 345 905 0 0 9 130 500 1100 - - - 505 1105 - - - 10 160 1300 - - - - 1305 - - - - 11 90 0 250 350 450 650 5 255 355 455 655 12 195 180 620 850 - - 185 625 855 - - 13 120 860 1050 1200 1350 - 865 1055 1205 1355 - 14 140 1260 1320 1400 - - 1265 1325 1405 - - 15 115 50 230 300 450 520 55 235 305 455 525 16 175 90 470 830 1370 - 95 475 835 1375 - 17 95 490 - - - - 495 - - - - 18 135 360 480 1230 - - 365 485 1235 - - Results of experiments for 5-minute time slots Table 2 Number of flights Order of flights Number of aircraft 6 0-1-6 0-3-5-4-2 2 7 0-1-5-6-3-4 0-7-2 2 8 0-1-5-6-8-2 0-3-7-4 2 9 0-1-6-9-4-5 0-3-7-8-2 2 10 0-3-2-1-4-9-10 0-6-7-8-5 2 11 0-3-11-7-4-9-10 0-8-2-6-1-5 2 12 0-1-12-7-4-2 0-3-11-5-6-8-9-10 2 13 0-1-2-3-5 0-8-4-7-13-9 0-12-11-6-10 3 14 0-3-8-9-12-10 0-4-7-1-5 0-11-6-13-2-14 3 Results of experiments for 5-minute time slots second group the length was 10 minutes and for the third group 15 minutes. Please note that all the values are expressed in minutes with beginning at midnight. That means, for example, 6:30 is expressed as 390 minutes. 5. Experiments For each group of the experiments 13 optimisation calculations were carried out; the calculations differed in the number of the planned flights – each group of the experiments was started with 6 flights and ended with 18 flights. For all the optimisation calculations we observed the calculation time. 25 C O M M U N I C A T I O N S 3 / 2 0 1 6 ● Number of flights Order of flights Number of aircraft 6 0-1-4-6 0-2-3-5 2 7 0-1-4-7-5 0-3-2-6 2 8 0-1-8-7-4-5 0-6-3-2 2 9 0-1-7-8-2 0-3-5-6-9-4 2 10 0-1-6-7-8-5 0-4-2-3-9-10 2 11 0-3-2-6-1-5 0-8-11-7-4-9-10 2 12 0-1-8-4-7-12-2 0-11-6-5-3-9-10 2 13 0-1-8-5-6-4-9-10 0-3-11-7-12-2-13 2 14 0-1-6-4-5-14 0-11-8-2-3-9-10 0-12-7-13 3 15 0-1-12-5-13-9-14 0-11-6-8-10 0-15-3-7-4-2 3 16 0-4-15-7-12-16 0-8-2-1-5-14 0-11-6-9-3-13-10 3 17 0-1-12-7-4-9-10 0-3-11-15-16-2-13 0-6-17-8-5-14 3 Fig. 1 The dependence of the calculation times on the number of the flights and the length of the time slots Table 5 and Fig. 1 summarise results of the experiments as regards the dependence of the calculation time (in seconds) on the number of the flights and the length of the time slots. Please note that the length of the time slots is expressed in minutes in Table 5 and the calculation times in seconds. Due to large range Num Fi Ta regard the nu note th Results of experiments for 5-minute time slots Table 2 15 0-1-12-5-6-3-10 0-8-9-4-2 0-15-11-7-13-14 3 16 0-3-11-7-13-2-14 0-4-6-15-8-9-10 0-16-1-12-5 3 17 0-1-8-15-6-3-5 0-12-17-11-13-9-10 0-16-4-7-2-14 3 Results of experiments for 10-minute time slots Table 3 Number of flights Order of flights Number of aircraft 6 0-1-4-2 0-6-3-5 2 7 0-4-2-6-1-5 0-7-3 2 8 0-1-5-3-2 0-6-7-8-4 2 9 0-1-3-7-8-2 0-6-9-4-5 2 10 0-1-8-7-4-9-10 0-6-5-3-2 2 11 0-1-6-5-3-2 0-8-11-7-4-9-10 2 12 0-1-12-7-4-5 0-3-11-2-6-8-9-10 2 13 0-1-7-8-2 0-4-3-6-9 0-11-12-5-13-10 3 14 0-1-12-5-11-8-10 0-2-6-3-4 0-7-13-9-14 3 15 0-4-6-7-3-10 0-11-12-9-8-5 0-15-2-1-13-14 3 16 0-3-2-1-16-5-14 0-6-7-13-10 0-11-8-15-12-9-4 3 17 0-3-5-6-1-10 0-11-8-15-17-12-2-14 0-16-7-13-9-4 3 Number of flights Order of flights Number of aircraft 6 0-1-4-6 0-2-3-5 2 7 0-1-4-7-5 0-3-2-6 2 8 0-1-8-7-4-5 0-6-3-2 2 9 0-1-7-8-2 0-3-5-6-9-4 2 10 0-1-6-7-8-5 0-4-2-3-9-10 2 11 0-3-2-6-1-5 0-8-11-7-4-9-10 2 12 0-1-8-4-7-12-2 0-11-6-5-3-9-10 2 13 0-1-8-5-6-4-9-10 0-3-11-7-12-2-13 2 14 0-1-6-4-5-14 0-11-8-2-3-9-10 0-12-7-13 3 15 0-1-12-5-13-9-14 0-11-6-8-10 0-15-3-7-4-2 3 16 0-4-15-7-12-16 0-8-2-1-5-14 0-11-6-9-3-13-10 3 17 0-1-12-7-4-9-10 0-3-11-15-16-2-13 0-6-17-8-5-14 3 Fig. 1 The dependence of the calculation times on the number of the flights and the length of the time slots Table 5 and Fig. 1 summarise results of the experiments as regards the dependence of the calculation time (in seconds) on the number of the flights and the length of the time slots. Please note that the length of the time slots is expressed in minutes in Table 5 and the calculation times in seconds. 5.1 Results of the experiments Within the experimental part 39 optimisation calculations were carried out. Table 2 presents results of the experiments for which the length of the time slots was 5 minutes. Table 3 summarises results for 10-minute time slots and Table 4 for 15-minute time slots. Each table consists of three columns. The first column gives information about the number of the flights that have to be served. The second column shows orders in which the planned flights should be served. And finally, the third column presents how many aircraft we need to serve all the planned flights. One can notice that in the tables results for 18 flights are missing. That is because for 18 flights the limitations of the student (demo) version of Xpress-IVE were exceeded; the limitations are given by the maximum number of constants and constraints. 25 C O M M U N I C A T I O N S 3 / 2 0 1 6 ● 25 26 ● C O M M U N I C A T I O N S 3 / 2 0 1 6 Due to large range Number of flights Order of flights Number of aircraft 6 0-1-4-6 0-2-3-5 2 7 0-1-4-7-5 0-3-2-6 2 8 0-1-8-7-4-5 0-6-3-2 2 9 0-1-7-8-2 0-3-5-6-9-4 2 10 0-1-6-7-8-5 0-4-2-3-9-10 2 11 0-3-2-6-1-5 0-8-11-7-4-9-10 2 12 0-1-8-4-7-12-2 0-11-6-5-3-9-10 2 13 0-1-8-5-6-4-9-10 0-3-11-7-12-2-13 2 14 0-1-6-4-5-14 0-11-8-2-3-9-10 0-12-7-13 3 15 0-1-12-5-13-9-14 0-11-6-8-10 0-15-3-7-4-2 3 16 0-4-15-7-12-16 0-8-2-1-5-14 0-11-6-9-3-13-10 3 17 0-1-12-7-4-9-10 0-3-11-15-16-2-13 0-6-17-8-5-14 3 Fig. 1 The dependence of the calculation times on the number of the flights and the length of the time slots Results of experiments for 10-minute time slots Table 3 Number of flights Order of flights Number of aircraft 6 0-1-4-2 0-6-3-5 2 7 0-4-2-6-1-5 0-7-3 2 8 0-1-5-3-2 0-6-7-8-4 2 9 0-1-3-7-8-2 0-6-9-4-5 2 10 0-1-8-7-4-9-10 0-6-5-3-2 2 11 0-1-6-5-3-2 0-8-11-7-4-9-10 2 12 0-1-12-7-4-5 0-3-11-2-6-8-9-10 2 13 0-1-7-8-2 0-4-3-6-9 0-11-12-5-13-10 3 14 0-1-12-5-11-8-10 0-2-6-3-4 0-7-13-9-14 3 15 0-4-6-7-3-10 0-11-12-9-8-5 0-15-2-1-13-14 3 16 0-3-2-1-16-5-14 0-6-7-13-10 0-11-8-15-12-9-4 3 17 0-3-5-6-1-10 0-11-8-15-17-12-2-14 0-16-7-13-9-4 3 Fig. 1 The dependence of the calculation times on the number of the flights and the length of the time slots Fig. 1 The dependence of the calculation times on the number of the flights and the length of the time slots Fig. 1 The dependence of the calculation times on the number of the flights and the length of the time slots Table 5 and Fig. 1 summarise results of the experiments as regards the dependence of the calculation time (in seconds) on the number of the flights and the length of the time slots. Please note that the length of the time slots is expressed in minutes in Table 5 and the calculation times in seconds. Due to large range 26 ● C O M M U N I C A T I O N S 3 / 2 0 1 6 Results of experiments – calculation times (in seconds) Table 5 Time slot length Number of flights 6 7 8 9 10 11 12 13 14 15 16 17 5 0.2 0.2 0.3 0.4 0.4 0.7 1.0 17.3 118.6 105.1 1162.2 37.2 10 0.2 0.2 0.3 0.4 0.4 0.8 1.9 9.0 93.9 290.7 133.0 26.0 15 0.2 0.2 0.3 0.4 0.5 0.7 2.3 7.0 18.8 1972.8 15943.5 851.7 Results of experiments – calculation times (in seconds) of the calculation times the logarithmical scale is used for the vertical axis of Fig. 1. the experiments the number of the planned flights and the length of the time slots were changed. The length of the time slots was assumed equal to 5, 10 and 15 minutes and the number of the flights was equal from 6 up to 17. For 18 flights the limitations of the academic (demo) version of Xpress-IVE were exceeded. of the calculation times the logarithmical scale is used for the vertical axis of Fig. 1. 6. Conclusions In our future research we would like to implement some other constraints following from real operation. We can mention, for example, a more detailed analysis of times needed for doing the tasks connected with aircraft services or planning maintenance of the aircraft. Our next target is to test the model in full version of optimisation software Xpress-IVE in order to find out calculation limits for the number of the planned flights. The presented article is devoted to planning flight timetables for charter airlines using mathematical modelling. In the paper the mathematical model that enables to plan flights in situations where due to capacity limitations at the airport the time slots for serving the flights are applied. The model is universal in terms of the number of the time slots. That means the individual flights can have different numbers of the possible time slots. The calculation experiments carried out with model were focused on the calculation times needed to get an optimal References [1] GRAF, V., TEICHMANN, D., DORDA, M.: Mathematical Model of Charter Flight Scheduling with Predefined Time Slots. Proc. of international conference Aplimat, February 2016, Bratislava, pp. 417-423, ISBN 978-80-4531-4. [2] DOZIC, S., KALIC, M., BABIC, O.: Heuristic Approach to the Airline Schedule Disturbances Problem: Single Fleet Case. Social and Behavioral Science, vol. 54, 2012, pp. 1232-1241. [3] BASDERE, M., BILGE, U.: Operational Aircraft Maintenance Routing Problem with Remaining Time Consideration. European J. of Operational Research, vol. 235, 2014, pp. 315-328. [4] LAGERHOLM, M., PETERSON, C., SODERBERG, B.: Airline Crew Scheduling Using Potts Mean Field Techniques. European J. of Operational Research, vol. 120, 2000, pp. 81-96. CEK, J., MARTON, P., KONIORCZYK, M.: The Column Generation and Train Crew Scheduling. Communications - Science of the University of Zilina, vol. 17, No. 1a, 2016, pp. 21-27. [6] BARNHART, C., BELOBABA, P., ODONI, A. R.: Applications of Operations Research in the Air Transport Industry. Transportation Science, vol. 37, No. 4, 2003, pp. 368-391. [7] DIAZ-RAMIREZ, J., HUERTAS, J. I., TRIGOS, F.: Aircraft Maintenance, Routing and Crew Scheduling Planning for Airlines with a Single Fleet and a Single Maintenance and Crew Base. Computers & Industrial Engineering, vol. 75, 2014, pp. 68-78. [8] MERCIER, A., SOUMIS, F.: An Integrated Aircraft Routing, Crew Scheduling and Flight Retiming Model. Computers & Operations Research, vol. 34, 2007, p. 2251-2265. [9] KERNER, L., KULCAK, L., SYKORA, V.: Operational Aspects of Airports (in Czech), Praha : CVUT, 2003, 270 p., ISBN 80-01- 02841-0. [10] FAIR ISAAC CORPORATIN, [online], ©2014 [11-2-2016]. http://www.fico.com/en/products/fico-xpress-optimization [10] FAIR ISAAC CORPORATIN, [online], ©2014 [11-2-2016]. http://www.fico.com/en/products/fico-xpress-optimization-suite/. 27 C O M M U N I C A T I O N S 3 / 2 0 1 6 ● 27
https://openalex.org/W4247781228	https://www.scienceopen.com/document_file/5f6ec43d-e38a-4cdd-a8b6-b6f26703b9b6/ScienceOpen/305_Duarte.pdf	English	null	A Preliminary Assessment of Physical & Virtual Presence in Exergames	Electronic workshops in computing	2,014	cc-by	4,365	1. INTRODUCTION mostly related with running and jogging. They also sport a variety of features for users to analyse their performance during or after workout sessions as well as comparing their data with their friends’. Ultimately, these features can be categorized as persuasive elements as they effectively motivate the user for the current or future exertion activities. Sedentary lifestyles are known to originate health conditions that could be prevented or even minimized by exercising a few hours a day [Mueller 2009]. The increasingly pervasive nature of work in our lives often inhibits individuals from spending a few minutes a day exercising, attempting to improve their physical and cognitive conditions. Unfortunately, many do not have the time to spend exercising, either because they are working late or because there are other matters that take that little time away. Driven by this increasingly worrying issue, researchers and developers began capitalizing on modern smart-phones. Their feature sets (e.g. GPS tracking, health sensor add-ons or high quality cameras) and ubiquitous nature allowed for the creation of applications which aid users in tracking their exertion activities. These tools are typically referred to as exergames, since they capitalize on videogame features and characteristics to entice users. Examples of such applications are Endomondo Sports Tracker1, Nike+2, Adidas miCoach3 or Runtastic4. Exergames are typically used to support exercise activities, The advent of social networking features has given relevance to a particular set of these features: the ability to compare oneself to another (Chittaro 2012). Exergames often integrate functionalities which allow users to compare themselves to others whether offline or during the race by loading specific workout data. If being compared online, the user is steadily fed with information regarding whether his / her performance outmatches the recordings’. Additionally, some exergames now offer the option to track and monitor live workout sessions, enabling audience members to intervene by cheering for their friends. Despite representing a popular feature set for this type of applications, knowledge regarding how individuals working out feel when prompted with feedback from observers or when being compared to another user’s recording is scarce. Furthermore, the way this information is conveyed can also be a pivotal factor into more successfully reaching a user. 1 http://www.endomondo.com 2 http://nikeplus.nike.com/plus/products/gps_app/ 3 http://www.adidas.com/us/micoach/ 4 http://www.runtastic.com/ Following the definition of a design space for the creation of exergames containing notions of © The Authors. Published by BCS Learning and Development Ltd. A Preliminary Assessment of Physical & Virtual Presence in Exergames ís Duarte Paulo Ribeiro André Rodrigues Tiago Gu Universidade de Lisboa Edifício C6,Campo-Grande, 1749-016 Lisboa, Portugal {lduarte, pribeiro}@lasige.di.fc.ul.pt andrefprodrigues91@gmail.com Luís Carriço Luís Duarte Luís Carriço Tiago Guerreiro Paulo Ribeiro {tjvg, lmc}@di.fc.ul.pt {tjvg, lmc}@di.fc.ul.pt Exercising is an activity in which the presence of others can motivate an individual to surpass his / her own limits. In recent years, technology has changed the way we carry out these activities. User commitment has been shown to be dependent on, among other, peer pressure. The introduction of challengeable virtual opponents broadened the spectrum of possibilities, enabling users to be motivated by either a real or virtual partner. In this paper we present an early assessment of the impact that different types of presence exerts on the users’ perceived motivation and competitiveness. In particular, we delve into how virtual entities compare to their real counterparts. We carried out an experiment in which we sought at obtaining amateur athletes perceptions on their motivation and competitiveness when exercising against real and virtual opponents. Results serve not only the purpose of showing that different types of presence counterbalance each other but they also validate a design space for physical and social partners in exertion applications in which our hypothesis are based upon. Exergames. Persuasion. Presence. 5 Benefits of training partners: http://running.competitor.com/2013/09/training/buddy-up- the-benefits-of-training-partners_58448 2.2.1. Physical 2.2.1. Physical Running alongside one or more individuals is a scenario which could take place before exergames were introduced. Reports show that people strive to find a regular running partner and even though there are specialized websites to find individuals to exercise with, people prefer doing it with their friends or acquaintances5. Thus, having a partner to which one can compare to can be pivotal towards steadily improving one’s performance and motivation levels. 2.2.2. Virtual 2.2.2. Virtual Having a preferred exercising partner can have its share of benefits. However, it also introduces potential issues. Among these, the availability of both parties involved emerges as the most compromising. Videogames introduced the idea of using recordings of an individual’s (or any of his / her friends or acquaintances) previous races. Typically denominated ghost recordings, these allow a user to keep awareness about whether his / her performance is better than that recording he / she is comparing to. Since then, this feature has been adopted in exergames extensively. This approach mitigates two problems raised by the need of physical presence: on the one hand, the mutual availability as far as the time of the exercise is concerned; on the other hand it allows for people who are geographically distributed to take part in comparison exercises via ghost recording sharing. 2.1 Bridging Exercise & Entertainment Exergames provide an excellent set of advantages that range from contributing to weight loss and physical health to facilitating social behavior (Mueller 2009; Mueller 2011; Gerling 2012). For this particular instance, these games allow for new and captivating social experiences either by inducing competitive edge or cooperation between players (Mueller 2009; Lindley 2008). Mueller has researched exergames extensively, exploring geographically distributed exertion activities and how users are able to motivate each other when the distance between them is a barrier for design (Mueller 2010). Fueled by the popularity of social networking services, exergames such as Nike+ or Endomondo broadened their focus to not only address the individuals actively exercising, but also those who may have interest in following / monitoring them in the comfort of their own homes / offices. During exercise activities, spectators can send cheering messages to people exercising. Usually, text-to- speech technology is employed to transform textual messages in audio cues. Alternatives to this approach include the usage of cheering / booing sounds to inform the user that he / she is being monitored remotely and that his / her performance is good / poor, respectively. Several other exergames exhibit different approaches towards persuading their users (Chittaro 2012). They rely on social pressure and on comparative data between the game’s participants. Striving to become the best player or surpass a certain opponent is, in its essence, the same as accepting challenges from other individuals in commercially available exergames. Storytelling is an alternative way to entice players (Chittaro 2013), but one which falls outside the scope of our research. 2. RELATED LITERATURE The diversity of existing exergames testifies to the importance and popularity of this type of software. These applications sport a significant feature set, allowing the users to configure options such as the course they desire to tackle or defining personal challenges. Among these characteristics, the usage of persuasive mechanisms is a common trait – if the application is not able to motivate users into exertion activities, then it failed its purpose. We will first provide a brief overview of exergames and then delve into a specific approach utilized to motivate users based on social dynamics. 1. INTRODUCTION Proceedings of HCI 2014, Southport, UK 305 A Preliminary Assessment of Physical & Virtual Presence in Exergames Duarte, Ribeiro, Rodrigues, Guerreiro, Carriço Traditionally, athletes were able to either engage in exercise alone or with a group of acquaintances. Exergames introduced a couple of more options into the landscape of possible participants during exercise. Among these, we emphasize the usage of virtual partners. We will now address each type of peer presence encountered in exergames. physical and virtual presence (Duarte 2014) we sought to assess how users are influenced by these features in this type of applications. This paper reports our initial experimentation on this topic. Despite the limitations of our experimental settings, our ongoing research is reinforced by positive hints retrieved within the context of these trials. We aimed at a subjective assessment in which the users’ perceived motivation and competitiveness level were the main targets. We addressed how the presence of a real opponent, a virtual opponent and a virtual audience impact on an individual. 6 Calories burned while jogging: http://www.myfitnesspal.com/topics/show/134478- accurate-formula-to-determine-calories-burned-jogging 3.2.1. Virtual Ghost One of the social persuasion variants we employed in ExodUS addresses the usage of a virtual partner, henceforth referred to as a ghost. Ghosts are recordings of exercise activities which may or may not belong to the actual application user. In order for these to be properly used, both the location of the recording and the place where the user is currently training should be the same. To keep awareness regarding how the individual is faring against the ghost we opted to utilize audio feedback during the exercise activity (akin to what other exergames adopt). At a given moment (e.g. each 2 minutes, each 1000m) users are relayed feedback about their performance. This cadence is configurable by the user. We compare his / her energy expenditure and / or average speed with the ghost’s and provide appropriate feedback for each situation (e.g. “You’re gaining on him”, “You’re 3.2. Supporting Presence ExodUS encompasses functionalities to support different types of presence. We narrowed our focus towards virtual presence, comprising the ghosts and audience features. 2.2 Social Presence Exercising with the aid of an exergame opens new scenarios and possibilities for individuals. 306 A Preliminary Assessment of Physical & Virtual Presence in Exergames Duarte, Ribeiro, Rodrigues, Guerreiro, Carriço  Cal_DIS = (WEIGHT * 0.75 * DISTANCE)  Cal_INC = (WEIGHT * 0.75 * INCLINE)  TOTAL = Cal_DIS + Cal_INC  Cal_DIS = (WEIGHT * 0.75 * DISTANCE)  Cal_INC = (WEIGHT * 0.75 * INCLINE)  TOTAL = Cal_DIS + Cal_INC  Cal_DIS = (WEIGHT * 0.75 * DISTANCE)  Cal_INC = (WEIGHT * 0.75 * INCLINE)  TOTAL = Cal_DIS + Cal_INC Each formula takes into account a basic user characteristic (weight) and a performance feature (distance and incline, accordingly). With this, Each formula takes into account a basic user characteristic (weight) and a performance feature (distance and incline, accordingly). With this, ExodUS is able to compare different users’ performances and provide them with valuable information regarding their own progress (as utilized in the reported trials). Each formula takes into account a basic user characteristic (weight) and a performance feature (distance and incline, accordingly). With this, ExodUS is able to compare different users’ performances and provide them with valuable information regarding their own progress (as utilized in the reported trials). 3. EXODUS In order to investigate how these three societal persuasive approaches impact on a user’s perceived motivation and sense of competitiveness we envisioned and developed an exergame denominated ExodUS. We followed a user-centred design process, heavily rooted in the analysis of the most common functionalities shared between existing exergames. While the description of this process falls outside the scope of this contribution, we present the core feature-set which enabled the research hereby presented: how user effort is calculated and how the different types of presence are suported. ExodUS aims at hiking and jogging exercises, covering the same type of activities that Nike+ or Endomondo support and was developed for Android 2.1 (and above) devices. We must emphasize that ExodUS is an experimental prototype merely created to aid us exploring the role of physical and virtual presence, impeding it from being a major contribution of this research. Figure 1 – ExodUS’ ghost functionality interface. Figure 1 – ExodUS’ ghost functionality interface. 2.3 Conveying Live Performance Data takes into account the time, distance covered and altitude during the exercise activity according to the following criteria6: Another aspect in which exergames converge is the way persuasive cues are delivered to users. Natural language is a common element in almost all of the studied exergames. The application typically issues an auditory message using natural language to motivate him / her. Music also has a strong presence in these applications. Personalized playlists appear to be a common denominator across exergames. Others extend this feature to include power-songs. Less popular approaches involve the usage of disruptions in a song to metaphorically signal loss of performance (Keep Running). The delivery of applause sounds is gaining momentum especially when spectators are involved. 3.1. Determining User Effort Measuring one’s performance in an Olympic track is a straightforward task: the person with the best individual time wins. When the location chosen to practice is not a regulated one, the task is not as trivial. One may suggest using the distance covered: however, running uphill produces more strain than going downhill. A location independent metric is, thus, paramount to address this problem. Tracking how much energy we expend during practice without specialized equipment is possible due to the variety of sensors integrated in modern smart-phones. Researchers have envisioned a set of formulas capable of giving this information with more or less accuracy. More accurate formulas rely on information such as the average heart rate during the exercise period. Within the context of ExodUS we opted for a simpler formula which 307 A Preliminary Assessment of Physical & Virtual Presence in Exergames Duarte, Ribeiro, Rodrigues, Guerreiro, Carriço bottles of water to keep themselves fresh in- between each trial. bottles of water to keep themselves fresh in- between each trial. slower than your opponent”, etc.), using a male voice from a text-to-speech synthesizer (Pico TTS). Figure 1 depicts the ghost selection interface. On the left we can observe a list of ghost recordings which, upon selection, enable the player to challenge them, replay the ghost recording or comment, as seen on the right side. 3.2.2. Virtual Audience 3.2.2. Virtual Audience The inclusion of virtual spectators contemplates two aspects which will be briefly explained. The first addresses the usage of appropriate cheering and booing sounds. These sounds were cautiously selected to inspire performing inside a crowded stadium. They are also conveyed to the user when: a) the user manually configures ExodUS to give him / her this type of feedback; b) when an authenticated user sends a cheering / booing message to the user. Users can also send text messages which are then translated via a TTS module to the user. ExodUS offers a social feature in which users may share their own performances with each other. We also implemented a monitoring feature which allows people in an individual’s contact list to track exercising sessions live and send cheering / booing feedback to their friends. This is a web-based portal implemented using HTML5 and a MySQL database.  Task 1 – perform a lap in an Olympic track with a real partner (RP). This partner changed pace throughout the race to induce feelings of uncertainty as of whether the subject was outperforming or underperforming. No subject was acquainted with this partner.  Task 2 – perform a lap in an Olympic track using the ExodUS application pre-loaded with a virtual partner in the same track (VP). The ghost performed the track in 60 seconds. No real partner was used in this task. The ghost did not belong to the subject him / herself nor any acquaintance of him / her.  Task 3 – perform a lap in an Olympic track using the ExodUS application with spectator mode on (virtual audience – VA). The user received cheering / booing cues as well as written messages conveyed via the audio channel periodically sent by a couple of persons who were not at the track. No real partner was present in this task. The subject had no knowledge regarding the audience identity. 4. EXPERIMENT We elaborated an experimental period to perform an early assessment of the impact of different types of presence on user perceived motivation and sense of competitiveness. The goal was to collect feedback from users that could be later used to fuel a proper design revamp of ExodUS to further explore distinct ways to persuade individuals. Feedback received during Task 2 was issued based on the subjects’ calorie expenditure as detailed in the previous section and was issued at fixed distance milestones (each 50 meters, totalling 8 feedback messages; content was based on user performance at the time). Task order was randomized for each individual, ensuring a balance between all combinations. There was a 10 minute interval between each task, during which participants were provided with refreshments offered by our team. At the end, we distributed a questionnaire which aimed to assess the subjects’ perceptions on how each type of presence influenced their motivation and competitiveness levels. All answers were given scores using a Likert scale from 1 (lowest) to 5 (highest). 4.3. Tools & Equipment Subjects were provided with an HTC Desire Android smart-phone loaded with the ExodUS application and a set of headphones to hear all feedback being conveyed. All users opted to carry it in their pockets. Lastly, they were offered with 4.2. Participants 15 participants (engineering course students; 12 male; 3 female; Age M = 27.7; Age SD = 7.3) volunteered to partake in these sessions. All participants exercised regularly and approximately 80% had had contact with exergames before. 4.1. Methodology For this milestone of the research we opted to employ subjective assessment techniques using questionnaires to retrieve participants’ perceptions. 4.4. Procedure The experiment began with a profiling period to characterize each subject. Participants were provided with information concerning ExodUS (how it works, what type of data it infers, since subjects did not directly interact with it). The experiment then required each participant to carry out three tasks: 4.5. Results The questionnaire’s results can be observed in Figure 2 and Figure 3. 308 A Preliminary Assessment of Physical & Virtual Presence in Exergames Duarte, Ribeiro, Rodrigues, Guerreiro, Carriço A Preliminary Assessment of Physical & Virtual Presence in Exergames Duarte, Ribeiro, Rodrigues, Guerreiro, Carriço Figure 2 – Perceived motivation: XX axis – number of respondents; YY axis – Likert scale. influence. While cheering / booing sounds do not perform poorly, opinions are mixed regarding its motivational value. These results are in line with Fogg’s persuasion theories in which it is stated that the usage of natural language approaches can more easily reach users (Fogg 2002). Figure 4 – Perceived motivation impact from written messages and cheering / booing sound cues for virtual audiences: XX axis – number of respondents; YY axis – Likert scale.. Figure 2 – Perceived motivation: XX axis – number of respondents; YY axis – Likert scale. ure 2 – Perceived motivation: XX axis – num respondents; YY axis – Likert scale. When asked “How much did the exercising with a real / virtual partner affect your motivation”, users stated that the presence of the real partner (RP) had the most positive impact, with 7 participants stating it greatly motivated them. The usage of a virtual partner (VP), albeit slightly less positive, still manages to produce a noteworthy motivational impact on our subjects. Virtual audiences (VA) had the least positively impacting influence, with two users even stating they felt less motivated with the presence of that feature. Figure 4 – Perceived motivation impact from written messages and cheering / booing sound cues for virtual audiences: XX axis – number of respondents; YY axis – Likert scale.. 5.1. Real VS Virtual Considering all of our research on this topic, the virtual ghost partner was expected to be the playing mode that created more impact on the players´ motivation levels, since it is intrinsically linked with exercising alone and creating competition (most times against oneself). The results, although levelled, show that for this sample, exercising with a real world partner impacted players´ motivation further. However, practicing alongside a virtual peer instilled a more emphasized competitive edge on athletes. Another interesting characteristic regarding the two is that running alongside a real partner made three participants feel more relaxed while that did not happen with the ghost challenge for any of the participants. Figure 3 – Perceived Competitiveness: XX axis – number of respondents; YY axis – Likert scale.. When questioned about “How much did the exercising with a real / virtual partner affect your competitiveness”, the most influencing presence types are switched. The virtual partner garners the choice of our subjects as the most impacting type of presence for their perceived competition level. Real partners also obtain a respectable approval rating. The usage of a virtual audience still fails to capture an overwhelmingly positive response from our population. 5. DISCUSSION We will now address a few topics that are important to discuss not only within the context of this research, but also for possible future endeavours. Figure 3 – Perceived Competitiveness: XX axis – number of respondents; YY axis – Likert scale.. 8. REFERENCES While the retrieved data is valuable on its own, not only to improve ExodUS’ design, but also for other researchers and developers to learn more about how determined features affect a user’s motivation and competitiveness levels, its scope is limited due to the preliminary nature of this stage of research. Chittaro, L. et al (2012). Turning the classic Snake mobile game into a location-based exergame that encourages walking. In Proceedings of PERSUASIVE, 2012. Chittaro, L. et al (2013). Exploring audio storytelling in mobile exergames to affect the perception of physical exercise. In Proceedings of Pervasive Health '13. The most important limitation is the design of the study itself. While we defend that giving almost no time to rationalize on their opinions is valuable to obtain honest answers, a longitudinal study would be more adequate. That option, however, also presents its limitations: it requires users who are willing to use the application regularly (or reward them to do so, increasing the cost of research). This leaves a slice of exergames potential user base out, since these applications are also developed aiming at users who do not exercise regularly and utilize the features we assessed in this article as a way to motivate them into doing so. Duarte. L. et al (2014). Defining a Design Space for Persuasive Cooperative Interactions in Mobile Exertion Applications. In Proceedings of the 20th Conference on Collaboration and Technology, Santiago, Chile. Fogg, BJ. (2002). Persuasive Technology: Using Computers to Change What We Think and Do. In Ubiquity Magazine. Gerling, K. et al (2012). Full-body Motion-based Game Interaction for Older Adults. In Proceedings of the SIGCHI Conference on Human Factors in Computing Systems (CHI 2012). ACM, New York. The number of participants is another caveat, aggravated by performing a single lap. Still, despite these caveats, we stand by this initial assessment paving the way for a more robust study in the future, given that the foundations for the next steps have hereby been validated. Lindley, Set al (2008). Stirring Up Experience Through Movement in Game Play: Effects on Engagement and Social Behavior. In Proceedings of the SIGCHI Conference on Human Factors in Computing Systems (CHI 2008). ACM, New York. 5.3. Creating an Enticing Audience Our virtual audience did not encompass any text- to-speech technology. Instead, we opted to use cheering and booing sounds to evoke feelings of having a good or poor performance, respectively. A possible research direction involves the assessment of which types of sounds are most adequate to motivate users. Analysing the crowd size (number of people cheering / booing) can also be taken into account here. 7. ACKNOWLEDGEMENTS This work was partially supported by Fundação para a Ciência e Tecnologia (FCT) through multiannual funding to the LaSIGE research unit and Individual Scholarship SFRH/BD/39496/2007. 5.2. Adjusting the Ghost Even though the impact of the ghost participant in both the users’ motivation and perceived competitiveness is commendable, we believe there is room for improvement. The ghost used in the task stemmed from a performance run that one of the researchers had previously recorded. Inspired by the results reported by Mandryk (2004), we question whether players would feel more engaged if exercising against a friend’s ghost rather than that of an unknown person. At the same time, we also dispute if different types of During the virtual audience trial, the audience members sent an average of 6 encouraging written messages (WM), 7 cheers and 5 boos to each participant (C/B). Figure 4 displays the perceived motivation impact results for the usage of written messages and cheering / booing sound cues. Written messages present an overall better appreciation concerning their motivational 309 A Preliminary Assessment of Physical & Virtual Presence in Exergames Duarte, Ribeiro, Rodrigues, Guerreiro, Carriço A Preliminary Assessment of Physical & Virtual Presence in Exergames Duarte, Ribeiro, Rodrigues, Guerreiro, Carriço intonations or voice types could have an effect on the perceived motivation and competition levels. intonations or voice types could have an effect on the perceived motivation and competition levels. These findings were already incorporated into the latest ExodUS version. In the future we want to expand this research by conducting larger longitudinal studies to assess the long term effect of these presence types of user performance, motivation and social activity. These findings were already incorporated into the latest ExodUS version. In the future we want to expand this research by conducting larger longitudinal studies to assess the long term effect of these presence types of user performance, motivation and social activity. 6. CONCLUSION & FUTURE WORK Mandryk, R. et al (2004). Physiological indicators for the evaluation of co-located collaborative play. In Proceedings of CSCW 2004. In this preliminary assessment addressing the effects of different types of presence in mobile exertion applications we showed that virtual entities can have a similar influence as their real counterparts. Despite user sense of motivation being lower with virtual entities, the perceived competitiveness conveyed by virtual opponents was observed to be higher in the latter’s case. Furthermore, we showed that virtual audiences, while less impactful, can still be a relevant feature in this type of applications. The type of feedback conveyed by those audiences is also capable of affecting users differently. Mueller, F., (2009). Exertion in Networked Games. In Proceedings of the 4th International Conference on Foundation of Digital Games. Mueller, F. et al (2010). Jogging over a distance between Europe and Australia. In Proceedings of UIST '10. ACM, 189-198. Mueller, F. et al (2011). Designing Sports: A Framework for exertion Games. In Proceedings of CHI 2011. 310
https://openalex.org/W4313410078	https://www.businessperspectives.org/images/pdf/applications/publishing/templates/article/assets/17452/PPM_2022_04_Santos.pdf	English	null	Burnout in hostels: How it shapes workers’ perceptions of human resources practices	Problems and perspectives in management/Problems & perspectives in management	2,022	cc-by	8,551	“Burnout in hostels: How it shapes workers’ perceptions of human resources practices” AUTHORS Magda Estevez Santos Álvaro Dias Mafalda Patuleia ARTICLE INFO Magda Estevez Santos, Álvaro Dias and Mafalda Patuleia (2022). Burnout in hostels: How it shapes workers’ perceptions of human resources practices. Problems and Perspectives in Management, 20(4), 382-395. doi:10.21511/ppm.20(4).2022.29 DOI http://dx.doi.org/10.21511/ppm.20(4).2022.29 RELEASED ON Friday, 09 December 2022 RECEIVED ON Saturday, 17 September 2022 ACCEPTED ON Thursday, 01 December 2022 LICENSE This work is licensed under a Creative Commons Attribution 4.0 International License JOURNAL "Problems and Perspectives in Management" ISSN PRINT 1727-7051 ISSN ONLINE 1810-5467 PUBLISHER LLC “Consulting Publishing Company “Business Perspectives” FOUNDER LLC “Consulting Publishing Company “Business Perspectives” NUMBER OF REFERENCES 47 NUMBER OF FIGURES 1 NUMBER OF TABLES 3 © The author(s) 2023. This publication is an open access article. © The author(s) 2023. This publication is an open access article. © The author(s) 2023. This publication is an open access article. businessperspectives.org businessperspectives.org businessperspectives.org Problems and Perspectives in Management, Volume 20, Issue 4, 2022 Magda Estevez Santos (Portugal), Alvaro Dias (Portugal), Mafalda Patuleia (Portugal) JEL Classification JEL Classification M10, M14 Alvaro Dias, Ph.D., Full Professor, Lusófona University; Higher Institute of Labour and Business Sciences, University Institute of Lisbon (ISCTE- IUL), Portugal. (Corresponding author) BUSINESS PERSPECTIVES www.businessperspectives.org LLC “СPС “Business Perspectives” Hryhorii Skovoroda lane, 10, Sumy, 40022, Ukraine This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Conflict of interest statement: Author(s) reported no conflict of interest Abstract There are scarce results on burnout in such organizations as hostels and how it influ- ences workers’ perceptions of their companies. Therefore, this study aims to analyze burnout of hostel workers and assess how it influences their perceptions of the type of leadership, the kind of support, the existence of feedback sessions, the organizational climate, and the quality of T&D programs. Data were collected from 96 employees of Portuguese hostels; existing validated measures were adapted from relevant literature. A pilot test was conducted to ensure the accuracy of the measures according to the tar- get population. A PLS analysis of survey data provided the following results. Burnout plays a negative role in employees’ overall acknowledgment of the organization and their sense of satisfaction and loyalty toward it. Specifically, the most substantial nega- tive effects of burnout were on feedback quality, manager support, and organizational climate (β = –0.557; –0.549; –0.542, p < 0.001, respectively). Furthermore, the indirect and negative effect of burnout on employee loyalty and employee satisfaction was also verified (β = –0.415; –0.418, p < 0.001, respectively). Therefore, managers can elabo- rate well-designed and applied practices based on these factors. Finally, this paper also discusses future research recommendations. Received on: 17th of September, 2022 Accepted on: 1st of December, 2022 Published on: 9th of December, 2022 Received on: 17th of September, 2022 Accepted on: 1st of December, 2022 Published on: 9th of December, 2022 © Magda Estevez Santos, Alvaro Dias, Mafalda Patuleia, 2022 Magda Estevez Santos, MsC., Researcher, Higher Institute of Labour and Business Sciences, University Institute of Lisbon (ISCTE-IUL), Portugal. Magda Estevez Santos, MsC., Researcher, Higher Institute of Labour and Business Sciences, University Institute of Lisbon (ISCTE-IUL), Portugal. hostels, empowering leadership, feedback quality, manager support, organizational climate © Magda Estevez Santos, Alvaro Dias, Mafalda Patuleia, 2022 Magda Estevez Santos, MsC., Researcher, Higher Institute of Labour and Business Sciences, University Institute of Lisbon (ISCTE-IUL), Portugal. Mafalda Patuleia, Ph.D., Associate Professor, ECEO, Lusófona University, Portugal; GOVCOPP, Universidade de Aveiro, Portugal. Alvaro Dias, Ph.D., Full Professor, Lusófona University; Higher Institute of Labour and Business Sciences, University Institute of Lisbon (ISCTE- IUL), Portugal. (Corresponding author) Keywords JEL Classification M10, M14 1. LITERATURE REVIEW AND HYPOTHESES tant decisions about the daily activities of a com- pany. For instance, it can be through supplying in- formation about strategic or operational goals. As a result, employees will see the value of their work, increasing their sense of meaningfulness, self-de- termination, and impact, along with more active participation (Courtright et al., 2015). Over the last decades, burnout syndrome has been studied, and on January 1, 2022, it was included in the International Classification of Diseases 11th Revision (ICD-11). Although it is not considered a disease, the World Health Organization (WHO) defined it as a condition that may affect people’s health (WHO, 2019). An excellent organizational climate, employee satisfaction, and employee loy- alty are the result of the good use of the practices referred to above (Locke, 1976). Accordingly, this study aims to explore burnout among hostel work- ers and assess how it influences their perceptions of the type of leadership, the kind of support, the existence of feedback sessions, the organizational climate, and the quality of T&D programs. Manager support is crucial for the development and motivation of employees (Bodner et al., 2011). Moreover, it increases job satisfaction and commit- ment levels (Aquino et al., 1997). Moreover, when workers feel they have a good relationship with their supervisor, they also feel they have the free- dom to increase their innovative and creative be- havior, contributing to a company with suggestions without the fear of rejection (Anderson et al., 2014). Besides, showing empathy and care opens a door for employees to feel more comfortable sharing worries or problems that may jeopardize their work life. However, on the other hand, sharing this type of information creates an opportunity for super- visors to be aware of what is happening with their workers, making it easier to spot gaps and problems and search for solutions (Darvishmotevali, 2019). Burnout is a relatively recent concept that has gained visibility over the years. Maslach (1982) de- fined it as a syndrome of emotional exhaustion, loss of capacity for empathy and impersonal response to customers, reduced professional accomplishment, unproductive work, and fatigue. It is a multifactori- al process where social, environmental, and organi- zational factors influence individual characteristics. Maslach et al. (1997) perceive high workload, lack of self-control, insufficient rewards, breakdown in the community (relationships on the job), absence of fairness, and value conflicts as the organizational risk factors for burnout. INTRODUCTION Hostels are an ever-growing accommodation type that provides not only the accommodation itself but all the experience of meeting new people among fellow guests and even workers due to its relaxed and open atmosphere (Andrade, 2014). However, when this concept is brought up, it is difficult to a priori connect it with the exhaustion felt when workers experience burnout (Maslach, 1982). Mafalda Patuleia, Ph.D., Associate Professor, ECEO, Lusófona University, Portugal; GOVCOPP, Universidade de Aveiro, Portugal. The literature stated that companies could adopt different HR practic- es to influence their employees’ feelings toward their organization. It is also essential to determine what type of leadership is used as well as whether manager support is felt and feedback sessions exist (Bandura, 1991). A worker will feel motivated to do the tasks in case these practices are frequent. Similarly, investing in training and development (T&D) of employees’ skills will improve their performance (Wentland, 2003). Although burnout has been studied in other industries, in tourism and particularly in hostels, it is a virtually unexplored phenomenon. Nevertheless, knowing the causes and consequences of this problem is of particular relevance. Professionals in this type of business have been exposed to high levels of stress and workload, which has been enhanced in the post-pandemic context due to the labor shortage. 382 http://dx.doi.org/10.21511/ppm.20(4).2022.29 http://dx.doi.org/10.21511/ppm.20(4).2022.29 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 Although there is extensive research on each of the points separately, there is a need to explore the con- nections between these concepts. Similarly, there is very little research on the hostels’ environment regarding burnout or HR practices. By providing knowledge about the causes and consequences of burnout, this study provides an essential tool for mitigating this problem and creating more sustainable organizations with better service. Although there is extensive research on each of the points separately, there is a need to explore the con- nections between these concepts. Similarly, there is very little research on the hostels’ environment regarding burnout or HR practices. By providing knowledge about the causes and consequences of burnout, this study provides an essential tool for mitigating this problem and creating more sustainable organizations with better service. http://dx.doi.org/10.21511/ppm.20(4).2022.29 1. LITERATURE REVIEW AND HYPOTHESES The study developed an in- strument to measure this syndrome – the Maslach Burnout Inventory (MBI). However, despite all definitions and research presented, there still exists controversy on the context-dependency of burnout (Bianchi et al., 2014). The key to understanding organizational climate is the word “climate.” Glisson (2007) stated that climate is the collective perceptions of employees about their work environment. This means organ- izational climate exists when workers in the same organization share the same perceptions of their company’s atmosphere and how that affects them. Being a shared perception, it is both the result and the determinant factor of employee behavior with- in the organization, originating in the actions of a company and its internal policies, practices, and conditions for work (D’Alleo & Santangelo, 2011). As a result, it will affect staff motivation, turnover rates, and service quality and outcomes. Since the first definitions of empowering leader- ship, sharing power and delegating responsibili- ties have been the fundamental characteristics of this type of leadership (Burke, 1986). Managers transfer power to employees, who become more and more autonomous and able to make impor- Training and development (T&D) is considered an HR practice that has more influence over em- ployee job satisfaction and performance, reflected 383 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 good supervision, and training and career devel- opment (Chi & Gursoy, 2009) as some of the prin- cipal elements that condition workers’ perception of satisfaction. in the company’s outcomes. Katz and Kahn (1978) referred to T&D as a “safeguarding subsystem” to ensure organizational effectiveness. In fact, this is supported by Aguinis and Kraiger (2009), who con- clude that this practice aims to develop and enhance employee skills, abilities, and knowledge toward or- ganizational effectiveness. This way, it is also a key component of leadership development (Collins & Holton, 2004). Hence, the employee perception that a company is investing in their career and personal development increases their job satisfaction and re- tention due to the feeling of obligation to repay this investment (Lee & Bruvold, 2003). Employee loyalty is connected to employee satis- faction. Loyalty is the inclination of the employ- ee to continue with his/her company (Solomon, 1992). Guillon and Cezanne (2014) refer to it as a multidimensional phenomenon influenced by em- ployees’ identification, attachment, commitment, and trust toward the organization, resulting in in- creased satisfaction emerging from internal evolu- tion and met expectations. 1. LITERATURE REVIEW AND HYPOTHESES This means that loyalty is a reciprocate process – to expect high levels of loyalty from their workers, companies must show similar or higher levels of loyalty toward them. Employee satisfaction is one of the most exam- ined topics in the literature observed. According to Locke (1976), job satisfaction can be defined as “a pleasurable or positive emotional state resulting from the appraisal of one’s job or job experience.” Similarly, several studies show that employee sat- isfaction is often seen as the worker’s assessment of the overall job quality (Prajogo & Cooper, 2010). In contrast, it is also possible to relate it to one’s perception of the degree of fit between individu- al and organizational values (Chi & Gursoy, 2009). Many factors can influence the level of employee satisfaction. For example, they include work con- ditions, wage structure, feeling of security and Based on the literature review and the connections observed between concepts, this study explores the possibility of burnout in hostel workers. Moreover, it assesses how it influences their perceptions of leadership, the kind of support, the existence of feedback sessions, the organizational climate, and the quality of T&D programs. On the other hand, it aims to explore the effects of these perceptions on employees’ sense of satisfaction and loyalty. Lastly, it is relevant to evaluate how the existence 384 http://dx.doi.org/10.21511/ppm.20(4).2022.29 Figure 1. Conceptual model Burnout Employee Loyalty Employee Satisfaction Empowering Leadership Feedback Quality Manager Support Organizational Climate Training & Development H1a H1b H1c H1d H1e H2a H2a H3a H3b H4a H4b H5a H5b H6a H6b H7a H7b Employee Satisfaction Figure 1. Conceptual model 384 http://dx.doi.org/10.21511/ppm.20(4).2022.29 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 H6a: The perception of training and development is positively related to employee loyalty. of burnout influenced workers’ satisfaction and loyalty. Accordingly, a conceptual model can be proposed (Figure 1) to guide this study, contain- ing all hypotheses meant to be tested. H6b: The perception of training and development is positively related to employee satisfaction. The conceptual model considers the following hypotheses: The conceptual model considers the following hypotheses: H7a: The possibility of burnout is negatively relat- ed to employee loyalty. H1a: The possibility of burnout is negatively relat- ed to employees’ perception of empowering leadership. H7b: The possibility of burnout is negatively relat- ed to employee satisfaction. H1b: The possibility of burnout is negatively re- lated to employees’ perception of feedback quality. 2.1. Participants H1c: The possibility of burnout is negatively re- lated to employees’ perception of manager support. The target population was employees from hos- tels in mainland Portugal, working in the man- agement, reception, housekeeping, bar/kitchen, and other areas to verify the results amongst var- ious working positions. The study found no age interval or gender or nationality preference. The sample hostels were identified on a search on the Hostelworld platform because it is the leading hos- tel booking platform (Hostelgeeks, 2022). H1d: The possibility of burnout is negatively relat- ed to employees’ perception of organizational climate. H1e: The possibility of burnout is negatively relat- ed to employees’ perception of training and development. The study used a non-probability sampling meth- od with convenience sampling, given that all workers from each hostel had the same chance to answer the surveys. However, only a part of them decided to contribute. The sampling frame of this study consisted of 96 answers from workers of 13 different hostels, from a total population of 42 hostels contacted. All hostels approached were rat- ed between 8 and 10 (out of 10) on the Hostelworld. H2a: The perception of empowering leadership is positively related to employee loyalty. H2b: The perception of empowering leadership is positively related to employee satisfaction. H3a: The perception of feedback quality is posi- tively related to employee loyalty. H3b: The perception of feedback quality is posi- tively related to employee satisfaction. H3b: The perception of feedback quality is posi- tively related to employee satisfaction. H3b: The perception of feedback quality is posi- tively related to employee satisfaction. This study’s sample comprises 58% female and 40% male respondents. There is no relevant differ- ence given that the hostel work is not stereotyped as primarily for men or women. The results also show that 2% of the participants consider them- selves as having “Other” gender. Regarding the age range, the results concluded that most par- ticipants are between 18 and 30 years (60%). This can be justified by the fact that hostels are mainly an environment for young people, and companies prefer to have staff with which their customers will identify themselves. A minority of workers are 40 or older (9%), and 32% of the participants range from 30 to 40. H4a: The perception of manager support is posi- tively related to employee loyalty. H4a: The perception of manager support is posi- tively related to employee loyalty. http://dx.doi.org/10.21511/ppm.20(4).2022.29 2.1. Participants H4a: The perception of manager support is posi- tively related to employee loyalty. H4b: The perception of manager support is posi- tively related to employee satisfaction. H5a: The perception of organizational climate is positively related to employee loyalty. H5b: The perception of organizational climate is positively related to employee satisfaction. 385 http://dx.doi.org/10.21511/ppm.20(4).2022.29 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 Regarding the geographic location, there is a sig- nificant group of participants from hostels in Lisbon, 64%. It is easy to justify as most hostels that answered this study’s questionnaire are from Portugal’s capital. Nevertheless, 18% of answers were from Porto, followed by 9% from Coimbra, and, finally, Faro, Braga, Évora, and Leiria with 2% each. of feedback present in the scale (source credibil- ity, feedback quality, feedback delivery, favorable feedback, unfavorable feedback, source availabili- ty, and promotion of feedback seeking). The scale used was composed of nine items. Manager support was measured using SE-SC8 scale (Gonsalvez, 2021). A scale of five items was used related to clusters of manager support, and they were measured using a five-point Likert scale ranging from “Never” to “Always.” To measure organizational climate, the scale chosen was the working environment scale (Røssberg et al., 2004), composed of ten items that assess how workers feel at their workplace. The participants rated each item on a five-point Likert scale ranging from “Never” to “Always.” The work areas were divided into management, reception, housekeeping, bar/kitchen, or other. Reception and management were the areas with the most percentage of workers answering (42% and 31%, respectively). This can be explained by the fact that these workers received the email sent for the study and might have not passed it to oth- er team members. 16% of the answers are from housekeeping teams, followed by 7% correspond- ing to “Other,” where the only mention of the area was “Marketing.” Bar/Kitchen had reduced partic- ipation by only 4%. Training and development six-items scale was extract- ed from a scale constructed by Nanjundeswaraswamy et al. (2020), originally meant to evaluate the qual- ity of work life of employees in mechanical manu- facturing small and medium enterprises (SMEs) in Karnataka, India. Employee loyalty was measured using an adapted scale from Homburg and Stock (2004). This scale measures the level of loyalty work- ers feel toward their company. It is composed of five items. 2.1. Participants To measure employee satisfaction, the paper chose a scale from Homburg and Stock (2004), as- sessing the general level of satisfaction. The scale was composed of six items that participants rated on a five-point Likert scale. The same scale was used for the two previous constructs. 2.2. Variables In this study, the dependent variable is burnout, and the antecedents are empowering leadership, feed- back quality, manager support, organizational cli- mate, and training and development. Furthermore, the paper evaluated how these variables influenced the independent variables: employee loyalty and satisfaction. The study also considered sociodemo- graphic factors such as age, gender, geographic lo- cation, and work position. Finally, validated scales were used to measure all variables. To measure burnout levels, the Portuguese ver- sion of the Maslach Burnout Inventory (MBI) was employed (Maslach et al., 1996), translated and adapted by Melo et al. (1999). The adapted scale was shortened from 22 items to 18 items after an analysis of the main components (Melo et al., 1999). Participants rated each item on a five-point Likert scale. Using Van Dierendonck and Nuijten (2011) meas- urement model, empowerment leadership was ana- lyzed. This scale assesses the level of empowerment leaders give to employees in a particular organiza- tion. It is composed of seven items, and the partic- ipants rated each item on a five-point Likert scale ranging from “Never” to “Always.” http://dx.doi.org/10.21511/ppm.20(4).2022.29 2.3. Data collection and analysis To measure feedback quality, the feedback envi- ronment scale (FES) was used (Levy & Steelman, 2004). This scale was developed to assess how feed- back is provided from the supervisor and co-work- er sources. In this study, the main goal was to evaluate the supervisor source; it is the reason for discarding co-worker source. Therefore, a selec- tion was made amongst the different components Before launching the questionnaire to the selected companies, a pilot test was conducted on a con- venience sample of three Master students and three hostel workers. It was requested to highlight any possible problematic questions or misspelled words, and respondents were encouraged to share 386 http://dx.doi.org/10.21511/ppm.20(4).2022.29 http://dx.doi.org/10.21511/ppm.20(4).2022.29 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 any other feedback regarding the survey. All com- ments were considered to adapt the questionnaire and close the final version. filled the threshold for reliability, as well as being significant at p < 0.001 (Hair et al., 2017). The sur- vey results showed that the standardizing factor loadings of all items were indeed above 0.5 (val- ues range from 0.922 to 50.594) and were all sig- nificant at p < 0.001. When it comes to internal consistency reliability, the goal is to assess the con- sistency of the results across items related to the same variable, measured by the composite relia- bility (CR) values and the Cronbach’s alpha values – both should surpass the cut-off of 0.7 (Hair et al., 2017). The results confirmed internal consistency reliability, as shown in Table 1. The final version of the survey was sent in a link by email to 42 different hostels located in Portugal rated from 8 to 10 (out of 10) on the Hostelworld platform (Appendix A). Although the email ex- plained the purpose of the study, when opening the link, there was again a brief introduction be- fore starting to answer, and it provided the re- searcher’s contact for questions that might have arisen. In addition, the questionnaire had a ver- sion in Portuguese and another in English so that it could be more inclusive. Convergent validity was also assessed to under- stand to which extent an item correlates positively with other items related to the same variable (Hair et al., 2017), being evaluated and confirmed on three key points. The first point is that all items have positive and significant loads. 2.3. Data collection and analysis Second, all items had CR values higher than 0.7. Lastly, the average variance extracted (AVE) met the value of 0.5 for all items (Bagozzi & Yi, 1988), as shown in Table 1. Similarly, the paper used a two-step ap- proach to evaluate discriminant validity. This as- pect shows to which extent each item is truly dis- tinct from the others by empirical standards. The conceptual model for this study was tested by using structural equation modeling (SEM), respectively, partial least squares (PLS), which is a variance-based structural equation modeling technique, through SmartPLS 3 software (Ringle et al., 2015). The reliability and validity of the measurement model were evaluated to analyze and interpret the results, and then the structural model was assessed. http://dx.doi.org/10.21511/ppm.20(4).2022.29 3. RESULTS ception of the companies’ HR practices to study, namely empowering leadership, feedback quali- ty, manager support, organizational climate, and training and development (β = –0.510, p < 0.001; β = –0.557, p < 0.001; β = –0.549, p < 0.001; β = –0.542, p < 0.001; β = –0.325, p < 0.05, respectively). This means that H1a, H1b, H1c, H1d, and H1e are all supported. Before evaluating the structural model, a check for collinearity was conducted (Hair et al., 2017). Kock and Lynn (2012) state that “two or more var- iables are said to be collinear if they measure the same attribute of an object.” This was assessed by checking the VIF values, which should all be be- low the critical value of five (Hair et al., 2017). The values ranged from 1.00 to 4.81, which indicates no collinearity between variables. Although hypotheses H2a to H6b are not support- ed by the results, p-values are all superior to 0.05. However, it is vital to refer that there exist some critical relationships. For example, empowering leadership practices positively influence employee satisfaction (β = 0.304, n.s.). Similarly, when feed- back quality levels are high, the organizational climate is perceived as good, and when there are adequate programs for training and developing employees’ capacities, the satisfaction of workers appears to increase (accordingly, β = 0.181, n.s.; β = 0.297, n.s.; β = 0.056, n.s.). On the other hand, em- ployee loyalty seems to be increased by feedback quality, manager support, and training and devel- opment programs (β = 0.486, n.s.; β = 0.288, n.s.; β = 0.195, n.s.). Moreover, empowering leadership seems to negatively influence employee loyalty (β = –0.252, n.s.), just as manager support relation- ship with employee satisfaction (β = –0.031, n.s.), and organizational climate and employee loyalty (β = –0.192, n.s.). Lastly, it was possible to observe two significant indirect negative relations sup- ported by the results. First, the results showed that To assess the structural model, the predictive ac- curacy was assessed by checking the magnitude of R2 value, also known as the coefficient of de- termination, and the Stone-Geisser Q2 values as a measure of the model’s predictive relevance (Hair et al., 2017). 3. RESULTS On the one hand, the Fornell and Larcker criteri- on was used, which requires that an item’s square root of AVE is greater than its highest correlation with any other item (Fornell & Larcker, 1981). The diagonal in bold refers to the square root of AVE and the numbers below the relevant correlations. On the other hand, the heterotrait-monotrait cri- terion (HTMT) was tested, where it is required that HTMT ratios meet the threshold value of 0.85 (Henseler et al., 2015; Hair et al., 2017). Therefore, as In order to evaluate the quality of the measure- ment model, individual indicators of reliability, internal consistency reliability, convergent va- lidity, and discriminant validity were considered (Hair et al., 2017). Indicators of reliability show the suitability and capability of items generated for a certain variable to answer the main research question. For example, an item with a standard- izing factor loading above 0.5 is held to have ful- Table 1. Composite reliability, average variance extracted, correlations, and discriminant validity Variables α CR AVE 1 2 3 4 5 6 7 8 Burnout 0.851 0.881 0.543 0.737 0.463 0.617 0.675 0.507 0.487 0.526 0.335 Empowering leadership 0.916 0.934 0.673 –0.510 0.821 0.710 0.715 0.891 0.786 0.826 0.586 Employee loyalty 0.727 0.879 0.784 –0.560 0.604 0.885 0.775 0.821 0.832 0.732 0.573 Employee satisfaction 0.857 0.899 0.691 –0.673 0.698 0.664 0.831 0.666 0.641 0.744 0.455 Feedback quality 0.936 0.946 0.665 –0.557 0.860 0.712 0.679 0.815 0.917 0.868 0.470 Manager support 0.926 0.945 0.774 –0.549 0.745 0.690 0.616 0.864 0.880 0.882 0.504 Organizational climate 0.856 0.896 0.634 –0.542 0.770 0.594 0.682 0.795 0.796 0.796 0.603 Training and development 0.953 0.962 0.808 –0.325 0.579 0.491 0.474 0.487 0.483 0.568 0.899 Note: α – Cronbach alpha; CR – Composite reliability; AVE – Average variance extracted. Bolded numbers are the square roots of AVE. Below the diagonal elements are the correlations between the constructs. Above the diagonal elements are the HTMT ratios. Note: α – Cronbach alpha; CR – Composite reliability; AVE – Average variance extracted. Bolded numbers are the square roots of AVE. Below the diagonal elements are the correlations between the constructs. Above the diagonal elements are the HTMT ratios. 387 http://dx.doi.org/10.21511/ppm.20(4).2022.29 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 confirmed in Table 1 by observing the values above the diagonal in bold, there is discriminant validity. http://dx.doi.org/10.21511/ppm.20(4).2022.29 4. DISCUSSION The results show that all the hypotheses that state that burnout negatively influences the perception of company practices and environment (empow- ering leadership, feedback quality, manager sup- port, organizational climate, and training and de- velopment) are supported (namely, H1a to H1e). Two significant indirect relations were also re- vealed by H7a and H7b. Thus, employee loyalty can be a result of employee satisfaction. When workers’ satisfaction levels are high, there are more chances to remain loyal to a company and the other way around (Chi & Gursoy, 2009). Therefore, burnout negatively influences both concepts. On the one hand, according to Freudenberg (1974) and Maslach (1982), burnout is precisely a condi- tion of exhaustion that makes the individuals with that condition develop a sense of an unhealthy work environment, supporting H1d, as well as a negative perception of most of the company’s prac- tices. On the other hand, organizational climate is defined as the employees’ construct of the or- ganizational internal policies, practices, and con- ditions for work. This perception of an unhealthy environment will lead to poor individual perfor- mance (Barth, 1974), which turns the process in- to a negative snowball. Although not supported by the study, it is also vital to highlight that H4b showed a positive relationship between organiza- tional climate and employee satisfaction. Employee satisfaction is seen as the evaluation of workers of the general quality of their current job (Hsu & Wang, 2008; Prajogo & Cooper, 2010; Jung & Yoon, 2015 cited in Amin et al., 2017) and the extent to which employees’ expectations are ful- filled (Abraham, 2012). Therefore, as proved by the results, a worker experiencing the condition of burnout is doubtfully unsatisfied with his work, supporting H7b. Once more, despite not being confirmed by this study, empowering leadership, feedback quality, and training and development (H2b, H3b, and H6b) showed a positive relation- ship with employee satisfaction. On the other hand, there are some risk factors appointed as the triggers of this condition: high workload, insufficient rewards, and conflicts (Freudenberg, 1974; Maslach, 1982). This is rel- evant for this study as these factors relate to the practices analyzed in the sense that they can all be prevented. 3. RESULTS With regards to the predictive accu- racy, the R2 value for all endogenous variables em- powering leadership, employee loyalty, employee satisfaction, feedback quality, manager support, organization climate, and training and develop- ment reached the minimum value of 10% (respec- tively 26%, 62%, 54.5%, 31.1%, 30.1%, 29.3%, and 10.6%). Regarding Q2 values, all of them surpassed the minimum value of zero (0.16, 0.41, 0.30, 0.18, 0.22, 0.17, and 0.07, respectively), which indicates the predictive relevance of the structural model. Table 2 concludes that burnout in hostel workers has a significantly negative influence on their per- Table 2. Structural model assessment Table 2. Structural model assessment Path Path Coefficient Standard Deviation T Statistics P Values Burnout → Empowering leadership –0.510 0.117 4.345 0.000 Burnout → Feedback quality –0.557 0.117 4.748 0.000 Burnout → Manager support –0.549 0.119 4.630 0.000 Burnout → Organizational climate –0.542 0.132 4.100 0.000 Burnout → Training and development –0.325 0.145 2.249 0.025 Empowering leadership → Employee loyalty –0.252 0.261 0.967 0.334 Empowering leadership → Employee satisfaction 0.304 0.221 1.375 0.170 Employee satisfaction → Employee loyalty 0.371 0.219 1.697 0.090 Feedback quality → Employee loyalty 0.486 0.267 1.820 0.069 Feedback quality → Employee satisfaction 0.181 0.282 0.641 0.522 Manager support → Employee loyalty 0.288 0.176 1.639 0.102 Manager support → Employee satisfaction –0.031 0.287 0.107 0.915 Organizational climate → Employee loyalty –0.192 0.183 1.047 0.296 Organizational climate → Employee satisfaction 0.297 0.221 1.343 0.180 Training and development → Employee loyalty 0.195 0.134 1.449 0.148 Training and development → Employee satisfaction 0.056 0.133 0.419 0.675 388 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 Table 3. Bootstrap results for indirect effects Path Original Sample Standard Deviation T Statistics P Values Burnout → Employee loyalty –0.415 0.109 3.822 0.000 Burnout → Employee satisfaction –0.418 0.109 3.853 0.000 the higher the possibility of burnout among work- ers, the lower their loyalty (β = –0.415, p < 0.001) or satisfaction with the work environment (β = –0.418, p < 0.001), as presented in Table 3. ries that may be threatening their performance at work (Anderson et al., 2014), as well as keep devel- oping themselves, alongside with training and de- velopment programs. Finally, if an employee feels support and investment, he/she is most likely to develop a healthy relationship with the organiza- tion (Lee & Bruvold, 2003). 3. RESULTS All the latter practices mentioned, if applied, prevent burnout in organi- zations since, according to WHO (2019), this syn- drome results from workplace stress not adequate- ly managed, and support accordingly H1a, H1b, H1c, and H1e. http://dx.doi.org/10.21511/ppm.20(4).2022.29 CONCLUSION This study aimed to analyze burnout in hostel workers and assess how it influences their perceptions of the type of leadership, the kind of support, the existence of feedback sessions, the organizational climate, and the quality of T&D programs. The existence of burnout is a possibility amongst workers in many different areas and organizations. Hostels are no exception despite their apparently relaxed work envi- ronment. Therefore, this study aimed to see to which extent the possibility of burnout shaped employees’ perceptions of their company, specifically across HR practices and the climate felt in the organization. Furthermore, it also identified how employee loyalty and satisfaction are influenced by burnout or the practices mentioned. The present study concluded that the possibility of burnout in hostel workers is negatively related to their perception of empowering leadership, feedback quality, manager support, organizational climate, and training and development. Likewise, the negative influence of burnout on employee loyalty and satis- faction is confirmed. However, the results also showed positive relationships between empowering lead- ership and employee satisfaction, feedback quality and employee loyalty/employee satisfaction, manager support and employee loyalty, organization climate and employee satisfaction, and training and devel- opment and employee loyalty/employee satisfaction. Thus, if the perception of employees of one of the variables is positive, the perception of the other is also positive. Although supported by the literature, these connections were not supported by the results obtained, re- quiring further studies. Besides, unexpected associations were observed, such as the negative influence of empowering leadership on employee loyalty, manager support on employee satisfaction, and organi- zational climate on employee loyalty, contradicted by the literature. From a managerial point of view, some valuable insights can be highlighted to improve not only hostels but any business. First, it is expected that this study has raised awareness for a condition that may be more common than what managers think – burnout syndrome. Since it is a condition caused exclusive- ly by work stress (WHO, 2019), managers play an essential role in preventing the risk or mitigating the situation when it is already spotted. This analysis shows that burnout lowers the perception of quality from the employee toward the company, and both the individual and the company can be jeopardized. In addition, it is recommended that companies invest in acceptable practices, such as empowering lead- ership, feedback quality, manager support, organizational climate, and training and development. 4. DISCUSSION For instance, if a company provides its employees with leaders who believe in the work- ers as individuals, value their work, and encour- age them to participate actively (Courtright et al., 2015), the chances of employees developing a sense of helplessness are lower. In comparison, if there is constant feedback and manager support, workers will feel motivated to share the problems or wor- Furthermore, if loyalty is the will of an employ- ee to remain in the organization due to the sat- isfaction felt toward it (Solomon, 1992), it is clear that the syndrome will erase that inclination, given support to H7a. This negative relationship between burnout and satisfaction and loyalty is most likely to bring companies higher turnover rates and lower their competitive advantage and work efficiency (Kim et al., 2016; Rice et al., 2017). Moreover, although the hypotheses were not vali- dated, employee loyalty was increased by feedback 389 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 quality, manager support, and training and devel- opment (H3a, H4a, and H6a, respectively). to communicate problems so they can be solved, which should increase job satisfaction and, con- sequently, loyalty. Regarding manager support, the paper indicates that a good relationship with supervisors increases the sense of support, de- veloping employees’ motivation and satisfaction (Aquino et al., 1997; Zhao & Zhou, 2008). Finally, if the organizational climate is the way workers perceive the quality of the work environment, the better perception, the more inclination to stay (Glisson, 2007). Further research should discover the reasons behind these results. Lastly, results show three negative relationships that the literature review did not note, such as em- powering leadership and employee loyalty (H2a), manager support and employee satisfaction (H4b), and organizational climate and employee loyalty (H5a). This contradicts the literature. For example, according to Birch (2020), empowering leadership builds a culture of trust in companies where it is practiced. It also provides employees with space CONCLUSION All these practices, some more than others and each one in a different way, benefit the sense of satisfaction that leads to loyalty. Besides, they are also ways of preventing burnout because they are ways of fol- lowing up with the employees and not letting their worries become extreme. In conclusion, this study 390 http://dx.doi.org/10.21511/ppm.20(4).2022.29 http://dx.doi.org/10.21511/ppm.20(4).2022.29 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 analyzed how variables affect each other and ways to prevent burnout by focusing on the development of the remaining ones. Looking back at the process of the present study, it is possible to reflect on limitations and come up with suggestions for future research to complete the findings. Firstly, the chosen population limited the data collection approaches. The survey was sent either to a company’s general email or to a manager’s email. Thus, the answers were subject to the willingness of the person to pass the email to other team members. Secondly, the quantitative method chosen, using a questionnaire, can lead to bias, although it was stated that the answers were anonymous and strictly used for academic purposes. Concerning future research, there is much space to develop since it is not a broadly studied subject. On the one hand, it could be helpful if the data collection methods were amplified, proceeding not only with questionnaires but with more objective ways of collecting information. In addition, since this study only analyzed the effects of burnout on the perceptions of the organizations’ practices, a study focused on analyzing the level of burnout itself on workers would complement this paper. Furthermore, the hy- potheses where the results contradicted the literature should be objected to in further research. Finally, it could be interesting to explore to which extent the practices are applied in hostels and what are their outcomes. AUTHOR CONTRIBUTIONS Conceptualization: Magda Estevez Santos, Alvaro Dias. Conceptualization: Magda Estevez Santos, Alvaro Dias. p g Data curation: Magda Estevez Santos, Alvaro Dias. Data curation: Magda Estevez Santos, Alvaro Dias. Formal analysis: Alvaro Dias. Funding acquisition: Mafalda Patuleia. Funding acquisition: Mafalda Patuleia. Investigation: Magda Estevez Santos. Investigation: Magda Estevez Santos. g g Methodology: Magda Estevez Santos. Methodology: Magda Estevez Santos. gy g Resources: Mafalda Patuleia. gy g Resources: Mafalda Patuleia. Software: Alvaro Dias. Supervision: Alvaro Dias. Supervision: Alvaro Dias. Validation: Magda Estevez Santos, Mafalda Patuleia. Validation: Magda Estevez Santos, Mafalda Patuleia. Visualization: Magda Estevez Santos, Alvaro Dias, Mafalda Patuleia. Visualization: Magda Estevez Santos, Alvaro Dias, g Writing – original draft: Magda Estevez Santos, Mafalda Patuleia. Writing – original draft: Magda Estevez Santos, M Writing – review & editing: Mafalda Patuleia. 3. Amin, M., Aldakhil, A. M., Wu, C., Rezaei, S., & Cobanoglu, C. (2017). The structural relation- ship between TQM, employee satisfaction and hotel perfor- ACKNOWLEDGMENT This study was financially supported by the research unit on Governance, Competitiveness and Public Policy (UIDB/04058/2020) + (UIDP/04058/2020), funded by national funds through FCT – Fundação para a Ciência e a Tecnologia. Review of Psychology, 60, 451-474. https://doi.org/10.1146/annurev. psych.60.110707.163505 http://dx.doi.org/10.21511/ppm.20(4).2022.29 Review of Psychology, 60, 451-474. https://doi.org/10.1146/annurev. psych.60.110707.163505 net/publication/256187156_De- velopment_of_Employee_Engage- ment_Programme_on_the_ba- sis_of_Employee_Satisfaction REFERENCES 1. Abraham, S. (2012). Develop- ment of employee engagement programme on the basis of employee satisfaction survey. Journal of Economic Development, Management, IT, Finance and Marketing, 4(1), 27-37. Retrieved from https://www.researchgate. 1. Abraham, S. (2012). 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Swamy, D. R., Nanjundeswaraswa- my, T. S., & Rashmi, S. (2015). Quality of work life: scale develop- ment and validation. International Journal of Caring Sciences, 8(2), 281-300. Retrieved from https:// www.internationaljournalofcar- ingsciences.org/docs/6_swamy.pdf 36. Melo, B. T., Gomes, A. R., & Cruz, J. F. A. (1999). Desenvolvimento e adaptação de um instrumento de avaliação psicológica do “burnout” para os profissionais de Psicologia [Development and adaptation of a psychological evaluation instrument of burnout to Psychology professionals]. In A. P. Soares, S. Araújo, & S. an “occupational phenomenon”: International Classification of Dis- eases. Retrieved October 15, 2021, from https://www.who.int/news/ item/28-05-2019-burn-out-an- occupational-phenomenon-inter- national-classification-of-diseases REFERENCES Caires (Eds.), Avaliação psicológica: formas e contextos (Vol. VI, pp. 596-603). Braga: APPORT (Associação dos Psicólogos Portugueses) – Universidade do Minho. 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The effect of people- 393 http://dx.doi.org/10.21511/ppm.20(4).2022.29 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 http://dx.doi.org/10.21511/ppm.20(4).2022.29 APPENDIX A SATISFACTION CRITERIA Organizational Climate (Friis, 1981) My tasks give me a chance to see how good my abilities are My tasks help me to have more confidence I feel nervous at work I feel anxious about going to work I feel I have the support I need when I am in troubles I can use my knowledge at work Problem-solving is complicated by conflicts among staff members It is easy to conciliate loyalty toward my team with loyalty toward my profession The number of tasks imposed is acceptable I have the feeling I have to be in several places at the same time Manager Support (Gonsalvez, 2020) My supervisor is approachable, caring, and supportive My supervisor advises me in an effective way Supervision goals are designed to match my developmental needs Supervision sessions are thoughtfully structured and goal-driven My supervisor helps me to understand my patterns of emotional response (e.g., in contact with the clients) Empowering Leadership (Van Dierendonck & Nuijten, 2011) Management gives me the information I need to do my work well Management gives me the authority to make decisions Management trusts me to solve problems instead of just telling me what to do Management encourages staff to come up with new ideas Management offers staff abundant opportunities to learn new skills Management encourages me to use my talents Management helps me to develop myself Feedback Quality (Levy & Steelman, 2004) My supervisor is familiar with my performance on the job My supervisor gives me helpful feedback about my job performance The feedback I receive helps me to have a better performance I value the feedback I receive My supervisor considers my feelings when giving feedback When I do a good job, my supervisor praises my performance When I do not meet the goals, my supervisor lets me know My supervisor is available if I request performance feedback I feel comfortable asking my supervisor for feedback on my performance Training and Development (Nanjundeswaraswamy et al., 2020) Management offers me opportunities to develop my abilities The goals of the training programs are well directed The existing training programs are efficient There is training focused on interpersonal competencies The existing training programs are sufficient The existing training programs are executed frequently SATISFACTION AND LOYALTY Satisfaction (Homburg & Stock 2004) Overall, I am satisfied with my job I intend to look for a job in another company I like my job There are things I would change in my job I like my job more than many employees of other companies I would choose this company again Loyalty (Homburg & Stock, 2004) I speak positively about my company when talking to customers I speak positively about my company when talking to friends and relatives I recommend the services of my company to others SATISFACTION CRITERIA Organizational Climate (Friis, 1981) 394 http://dx.doi.org/10.21511/ppm.20(4).2022.29 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 Problems and Perspectives in Management, Volume 20, Issue 4, 2022 Burnout (Melo et al., 1999) I feel emotionally unsatisfied with my job I feel tired when I get up in the morning to go to work I can easily understand how my clients feel toward my company’s service I feel I deal with some clients as if they were objects Working with people every day is a pressure for me I deal with problems in an efficient way I feel tired from my job I feel I am positively influencing other people’s lives I have become crueler with people since I have had this job I feel frustrated with my job I feel like I am working too much I do not care what happens to some of my clients I can easily create a relaxed environment with my clients Working directly with people causes me too much stress In this job, I have accomplished outstanding achievements I feel I am at the limit of my capacities In my job, I deal calmly with emotional problems I feel my clients blame me for the bad things that happen to them 395 http://dx.doi.org/10.21511/ppm.20(4).2022.29
https://openalex.org/W2801094656	https://lilloa.univ-lille.fr/bitstream/20.500.12210/17305/1/Continuous%20positive%20airway%20pressure.pdf	English	null	Continuous Positive Airway Pressure Reduces Night-Time Blood Pressure and Heart Rate in Patients With Obstructive Sleep Apnea and Resistant Hypertension: The RHOOSAS Randomized Controlled Trial	Frontiers in neurology	2,018	cc-by	8,855	Clinical Trial published: 08 May 2018 doi: 10.3389/fneur.2018.00318 Reviewed by: Miguel Angel Martinez-Garcia, Hospital Universitari i Politècnic La Fe, Spain Manuel Sánchez-de-la-Torre, Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), Spain Francisco Campos-Rodriguez, Hospital Universitario de Valme, Spain 1 HP2 Laboratory, INSERM, University Grenoble Alpes, Grenoble, France, 2 Department of Physiology and Sleep, Grenoble Alpes University Hospital (CHU de Grenoble), Grenoble, France, 3 Cardiology Department, Grenoble Alpes University Hospital (CHU de Grenoble), Grenoble, France, 4 Biochemistry, Toxicology and Pharmacology Department, Grenoble Alpes University Hospital (CHU de Grenoble), Grenoble, France, 5 Cardiology Department, Poitiers University Hospital (CHU de Poitiers) – La Millétrie, Poitiers, France, 6 Vascular Medicine and Hypertension Department, Lille University Hospital (CHU de Lille), Lille, France Objective: Most patients with resistant hypertension (RH) have obstructive sleep apnea (OSA). We aimed to determine the impact of OSA and continuous positive airway pres- sure (CPAP) treatment on the leptin profile and blood pressure (BP) in patients with RH. Correspondence: Marie Joyeux-Faure mjoyeuxfaure@chu-grenoble.fr Correspondence: Marie Joyeux-Faure mjoyeuxfaure@chu-grenoble.fr †These authors have contributed equally to this work. Methods: After an initial case-control study (RH with and without OSA), we performed a randomized, single blind study in OSA + RH patients receiving either sham CPAP (3 months) followed by active CPAP (6 months) or 6 months of active CPAP. The primary outcome was the comparison of leptin levels between groups of RH patients with or without OSA. Secondary outcomes were the comparison of metabolic parameters, bio- markers of sympathetic activity, and BP indices between the two groups of RH patients with or without OSA. The same outcomes were then evaluated and compared before and after sham and effective CPAP intervention. Specialty section: This article was submitted to Sleep and Chronobiology, a section of the journal Frontiers in Neurology Received: 02 March 2018 Accepted: 23 April 2018 Published: 08 May 2018 Results: Sixty-two patients (60 ± 10 years; 77% men) with RH (24-h daytime systolic BP (SBP)/diastolic BP: 145 ± 13/85 ± 10 mmHg, 3.7 antihypertensive drugs) were included. The 37 RH patients exhibiting OSA (60%) were predominantly men (87 vs 64% for non-OSA patients), with a greater prevalence of metabolic syndrome and higher cre- atininemia. Their leptin concentrations were significantly lower than in non-OSA patients [9 (6; 15) vs 17 (6; 29) ng/mL] but increased after 6 months of CPAP. Three months of effective CPAP significantly decreased night-time SBP by 6.4 mmHg and heart rate (HR) by 6.0 bpm, compared to sham CPAP. Edited by: David Gozal, University of Chicago, United States Edited by: David Gozal, University of Chicago, United States Edited by: David Gozal, University of Chicago, United States Marie Joyeux-Faure1,2†, Jean-Philippe Baguet3†, Gilles Barone-Rochette3, Patrice Faure1,4, Philippe Sosner5, Claire Mounier-Vehier6, Patrick Lévy1,2, Renaud Tamisier1,2 and Jean-Louis Pépin1,2 Marie Joyeux-Faure1,2†, Jean-Philippe Baguet3†, Gilles Barone-Rochette3, Patrice Faure1,4, Philippe Sosner5, Claire Mounier-Vehier6, Patrick Lévy1,2, Renaud Tamisier1,2 and Jean-Louis Pépin1,2 CONDENSED ABSTRACT markers in RH patients with or without OSA, followed by a study of the OSA + RH patients who were randomized to receive either sham CPAP or active CPAP. The association between obstructive sleep apnea and resistant hypertension corresponds to a specific, predominately male phenotype with a higher burden of metabolic syndrome and higher creatininemia. Active continuous positive airway pressure decreases night-time blood pressure and heart rate. Citation: Joyeux-Faure M, Baguet J-P, Barone-Rochette G, Faure P, Sosner P, Mounier-Vehier C, Lévy P, Tamisier R and Pépin J-L (2018) Continuous Positive Airway Pressure Reduces Night-Time Blood Pressure and Heart Rate in Patients With Obstructive Sleep Apnea and Resistant Hypertension: The RHOOSAS Randomized Controlled Trial. Front. Neurol. 9:318. doi: 10.3389/fneur.2018.00318 Conclusion: The association between OSA and RH corresponds to a specific, pre- dominately male phenotype with a higher burden of metabolic syndrome and higher May 2018 \| Volume 9 \| Article 318 Frontiers in Neurology \| www.frontiersin.org 1 Joyeux-Faure et al. CPAP for OSA Patients With RH creatininemia but there was no significant difference between OSA and non-OSA patients regarding BP indices, and the number of antihypertensive drugs used. Active CPAP could be efficient at decreasing night-time BP and HR, but there was no dif- ference between CPAP and sham CPAP groups for all metabolic and SNS markers (NCT00746902 RHOOSAS). Keywords: obstructive sleep apnea, resistant hypertension, leptin, blood pressure, continuous positive airway pressure Keywords: obstructive sleep apnea, resistant hypertension, leptin, blood pressure, continuous positive airway pressure INTRODUCTION Obstructive sleep apnea (OSA) syndrome is characterized by recurrent episodes of upper airway obstruction during sleep, causing intermittent hypoxia (IH) and impaired sleep continuity and quality (1). OSA is recognized as an important and independ- ent risk factor for hypertension (2), coronary heart disease (3), and stroke (4). Resistant hypertension (RH) is defined as blood pressure (BP) that remains above goal in spite of the concurrent use of three antihypertensive agents of different classes (5), and is associated with adverse cardiovascular and renal outcomes and increased mortality (6). OSA is one of the most common causes of RH (7) and OSA patients exhibit a fivefold higher risk of suffering from RH than the general population (8). Setting and Patients Consecutive, potentially eligible patients were recruited by the Cardiology departments of 3 University Hospitals (Grenoble, Poitiers and Lille, France) between May 2010 and November 2013. Subjects over 18, with RH and naive of CPAP treatment were eli- gible. RH was defined as the failure to achieve the target BP (office SBP/diastolic BP (DBP) ≥140/90 or ≥130/80 mmHg for diabetic patients) with three classes of antihypertensive drugs (including diuretics), and 24-h daytime SBP/DBP >135/85 mmHg and/or night-time SBP/DBP >120/70 mmHg. In RH the adipokine leptin is associated with lack of BP control primarily mediated by leptin sympathoexitatory effects and renin–angiotensin–aldosterone activation (9, 10) that are also seen in sleep apnea. Adiponectin has been recognized for its antiinflammatory, antiproliferative, and antiatherogenic proper- ties. It has been shown that uncontrolled RH patients have higher leptin and lower adiponectin levels suggesting that abnormal adipokine serum levels contribute to the difficulty in controlling BP in RH (11–13). Patients unable to give written consent or presenting any of the following criteria were not included: known reason for antihyper- tensive treatment resistance (such as iatrogenic RH, high alcohol consumption, etc.), history of severe renal or cardiac failure or transplantation, Parkinson’s disease, dysautonomia, atrial fibrilla- tion and frequent extrasystoles, severe hypertension (office SBP/ DBP ≥180/110 mmHg), daytime alveolar hypoventilation, severe sleepiness defined as the risk of a traffic accident estimated at discretion of investigators, pregnancy, or lactation. Patients with secondary RH were excluded before inclusion. Continuous positive airway pressure (CPAP) treatment is the first line therapy for OSA. Recently, two meta-analyses (14, 15) showed that CPAP significantly reduced 24-h BP as well as nocturnal BP in RH patients with OSA (16–19). To date, only one RCT has been sham controlled in a double-blind manner; it confirmed a significant reduction of 24-h systolic BP (SBP) in patients with RH and moderate OSA, after 8 weeks of CPAP treat- ment (20). The HIPARCO study (17) is by far the largest RCT in the field and has the strength of being multicenter, which allows better generalization of the conclusions. Interestingly, even in the population of CPAP compliers, a significant subgroup did not respond to CPAP treatment (21). This raises the need for a better understanding of the mechanisms underlying RH in this specific OSA population. Design and Ethicst After a baseline case-control study (RH with OSA vs RH without OSA), we performed a multicenter, randomized, single blind study of the patients with OSA + RH only. Patients were rand- omized to either sham CPAP (for 3 months) than active CPAP (for 6 months) or active CPAP for 6 months only. The study was conducted in accordance with good clinical practice require- ments in Europe, French law, ICH E6 recommendations, and the Helsinki Declaration (1996 and 2000). The protocol was approved by an independent Ethics Committee (Comité de Protection des Personnes, Grenoble, France, IRB0006705) and registered on the ClinicalTrials.gov site (NCT00746902 RHOOSAS). All patients gave their written informed consent. Frontiers in Neurology \| www.frontiersin.org Statistical Analysis Analysis was performed with SAS software (version 9.4, SAS Institute Inc.). All randomized patients were included in the intention-to-treat (ITT) analysis. The per-protocol population was defined as patients who completed all the visits without any protocol deviation. In the ITT analysis, missing data were replaced by imputation at the median, using the minimum bias method for baseline data and the maximum bias method for data after 3 months of CPAP or sham CPAP treatments. Study design and data are reported here in accordance with the CONSORT criteria (32). Office BP Measurement Pressure values were obtained using a mercury sphygmoma- nometer. The mean of three measurements was calculated on three occasions, in line with European Society of Hypertension– European Society of Cardiology guidelines (27). Office SBP, DBP, and MABP and HR were assessed at baseline for all patients. Treatments 24-h ambulatory BP monitoring (ABPM), which was performed with a Spacelabs 90207 device (Spacelabs Healthcare, Redmond, WA), was measured at baseline for all patients. Measurements were made every 15 min over 24 h. The following ABPM vari- ables were studied: mean HR, SBP, DBP, and MABP [calculated as DBP + 1/3(SBP–DBP)] over the 24 h and over the day (07:00–22:00) and night (22:00–07:00). Patients with a nocturnal reduction in BP compared to average daytime BP of less than 10% were classed as non-dippers and those with more than 10% were classed as dippers. 24-h ambulatory BP measurements with 15 min intervals is the methodology that has been used by the majority of the studies assessing the impact of CPAP on BP in randomized controlled trials (17, 24, 25). We did not make a beat by beat assessment of BP (26) because although it has the advantage of assessing complementary information regarding BP variability this is at the cost of deterioration in sleep quality and duration, and moreover needs to be recorded in the hospital in artificial conditions were different from those of real life. Active CPAP was provided with an auto-titrating device (Autoset Spirit®, ResMed®, UK or Remstar Auto®, Philips Respironics®, Murrysville, PA, USA). Patients receiving sham CPAP had a simi- lar machine delivering a pressure that was too low to maintain the pharynx open. This procedure has been previously validated as an appropriate placebo for CPAP treatment (31). Compliance to active and sham CPAP was recorded by the device. CPAP follow- up was done by experienced homecare providers with nurses making home visits at CPAP initiation and on demand in case of CPAP side effects. Renin–Angiotensin System (RAS) Markers Renin Angiotensin System (RAS) Markers Aldosterone was measured in a blood sample using a liquid chromatography–tandem mass spectrometry technique, as previ- ously described (30). Active renin was measured using a radio- immunometric sandwich assay (Renin III generation kit, Cisbio).t Sample Size A sample size calculation was not performed for this study. We included consecutive, eligible, consenting patients with both RH and OSA presenting at the three Cardiology departments between May 2010 and November 2013. Non-OSA RH patients were age matched with patients presenting with OSA. We recog- nize that we had some difficulties in recruiting eligible patients, resulting in a relatively small sample size despite the long period of inclusion. Outcomesh The goal of the case-control study was to compare the adipokine profiles in RH patients with or without sleep apnea. This might provide interesting mechanistic insights regarding disease asso- ciations and responses to treatment. Other variables compared To address these issues, we performed a case-control study comparing adipokines and rennin–angiotensin system (RAS) May 2018 \| Volume 9 \| Article 318 2 CPAP for OSA Patients With RH Joyeux-Faure et al. were BP (office and 24-h), and metabolic, SNS, and RAS biomark- ers. In the randomized part of the study, outcomes were evalu- ated after 3 months of effective CPAP vs sham CPAP, and after 6 months of active CPAP. (Dimension Vista 1500, Siemens). Leptin and adiponectin were measured using a radio-immunometric sandwich assay (MI-HL- 81HK kit for leptin and MI-HADP-61HK kit for adiponectin, Millipore). Metabolic syndrome was defined in accordance with the International Diabetes Federation recommendations (29). Study Procedure and Variables Measured At the baseline visit (D0), patients underwent an overnight sleep study as described in Ref. (22) in which apnea–hypopnea index (AHI), mean nocturnal SaO2, and time spent with <90% of SaO2 were monitored in order to characterize sleep apnea severity, scored according to international guidelines (23). On waking a fasting peripheral blood sample was drawn. The Epworth sleepi- ness scale was completed and arterial blood gases analysis was performed to exclude obesity hypoventilation syndrome. Patients diagnosed as having OSA (AHI >15/h) were randomized to either CPAP or sham CPAP. Randomization was performed by an independent statistician using a computer-generated rand- omization. Only patients (but not the study team) were blinded to treatment allocation. Study Procedure and Variables Measured Sympathetic Nervous System Biomarkers Methoxylated derivatives of norepinephrine (normetepinephrine) and epinephrine (metepinephrine) were measured in a single blood sample using a high-performance liquid chromatography technique (Clinrep kit, Recipe) coupled with electrochemical detection (colorimetry detector, ESA). Sympathetic Nervous System Biomarkers Renin–Angiotensin System (RAS) Markers Aldosterone was measured in a blood sample using a liquid chromatography–tandem mass spectrometry technique, as previ- ously described (30). Active renin was measured using a radio- immunometric sandwich assay (Renin III generation kit, Cisbio). All markers were measured in duplicate at baseline, after 3 months (3M) of treatment and at the end of the study (M6 or M9). The study flow chart is shown in Figure 1. The study flow chart is shown in Figure 1. Metabolic Markerst After peripheral blood sampling, plasma glucose and serum triglyc- eride concentrations were measured automatically (Dimension Vista 1500, Siemens). Serum insulin was measured using a radio- immunometric sandwich assay (CIS bio international). Serum creatinine was measured using an automated enzymatic system (Dimension Vista 1500, Siemens). The modification of diet in renal disease (MDRD) index was determined according to the literature (28). Ultra-sensitive C-reactive protein (us-CRP) was measured using an automated immunonephelometry technique Baseline data were compared by a Student or a Mann–Whitney test for continuous data (depending on the validity of the nor- mality of distribution) and by a Chi2 or Fishers exact test for May 2018 \| Volume 9 \| Article 318 Frontiers in Neurology \| www.frontiersin.org 3 CPAP for OSA Patients With RH Joyeux-Faure et al. categorical data. A Spearman-rank correlation was used to assess the relationship between leptin concentrations and office DBP.i categorical data. A Spearman-rank correlation was used to assess the relationship between leptin concentrations and office DBP.i results were presented as non-transformed data. This is why differences in variables (CPAP-sham CPAP effect) are presented as mean (95% confidence interval). Treatment effect (after 3 and 6 months) was also tested after adjustment to compliance and the interaction between treatment and compliance was analyzed. fi A p-value <0.05 was considered statistically significant. Data are presented as mean ± SD or median [25th; 75th percentiles] while differences (CPAP sham-CPAP active) are presented as mean (95% confidence interval). i For the analysis of changes in values between baseline and 3 months of CPAP or sham CPAP treatment, an analysis of covariance including baseline measurements and the treatment (CPAP/sham CPAP) was performed. The intragroup differences from baseline to 3 months were evaluated with a paired t-test or a Wilcoxon test (depending on the validity of the normality of distribution). Logistic regression modeling the probability to be a BP dipper or not was performed.t DISCUSSION The originality of our study was to address both the hormonal and metabolic characteristics of the combination of OSA and RH and the effect of CPAP in a randomized sham CPAP controlled trial. OSA patients with RH exhibited distinct characteristics compared to non-OSA RH. They were more often men with a higher prevalence of metabolic syndrome and significantly lower leptin concentrations than in non-OSA patients. Three months of active CPAP significantly decreased night-time SBP by 6.4 mmHg (p = 0.033) and HR by 6.0 bpm (p = 0.010) compared to sham CPAP. The presence of OSA in 60% of the included RH patients is in line with previous studies (7, 8, 34). The negative impact of OSA on renal function has now been demonstrated both in animal models and in epidemiological studies (35), and accord- ingly our study found higher levels of creatinine in the group with OSA and RH. Thus, systematic screening for sleep apnea is one of the more prevalent and treatable secondary causes of RH are now recommended by the European society of Hypertension (36). Effect of 6 Months of Active CPAP Effect of 6 Months of Active CPAP Treatment in RH Patients With OSA In ITT analysis, night-time BP significantly decreased after 6 months of active CPAP treatment [SBP: −4.7 (8.4; −0.9) mmHg (p = 0.016) and DBP: −2.3 [−4.5; −0.2] mmHg (p = 0.036)]. Moreover, dipper profile was improved by active CPAP [56.8 vs 29.7% (p = 0.004) for SBP, and 64.9 vs 48.7% (p = 0.058) for DBP]. After 6 months of active CPAP treatment, blood leptin concentra- tions increased significantly, independently of the patient’s weight [Leptin/weight: 0.04 (0.00; 0.07) ng/mL/kg (p = 0.025)].h i There was no significant difference between OSA and non- OSA patients regarding other baseline demographic data, medical history, baseline office BP, 24-h BP, and the number of antihypertensive drugs used (Table 1). Antihypertensive medica- tion in the different patient groups is reported in Table 2.i f In patients with RH, we observed significantly lower leptin concentrations in the presence of OSA, independent of the patients’ weight (Table 1) and OSA severity (data not shown). An inverse correlation between leptin concentrations and office DBP (−0.349, p = 0.006) and MABP (−0.277, p = 0.029) was found for the whole group. The amount of missing data was between 5 and 16% for data before and after CPAP treatment. Fasting glucose, triglycerides, and creatinine levels were significantly higher in RH patients with OSA (Table 1). All other metabolic, SNS and RAS markers were comparable between OSA and non-OSA patients. BP Analysis TT l In ITT analysis, 3 months of CPAP treatment significantly lowered night-time SBP and heart rate (HR) compared to sham CPAP (Table 3). Moreover, CPAP significantly decreased night-time DBP [by 8.10 (−13.91; −2.29) mmHg, p = 0.009] and MABP [−9.09 (−16.75; −1.43) mmHg, p = 0.023], compared to sham CPAP, but only in patients with baseline values ≥ the median. Finally, office BP, 24-h BP and dipper profile were not modified by 3 months of CPAP treatment, compared to sham CPAP. The results were the same after adjustment for CPAP compliance, which was compara- ble between arms. Results were similar in a per-protocol analysis. High leptin levels are associated with impaired BP control and poor long-term prognosis (39, 40). There remains a controversy as to the relationship between circulating leptin levels and OSA, since several studies have demonstrated higher leptin levels in subjects with OSA compared with BMI-matched control subjects, suggesting a relative leptin-resistant state in OSA (41–44), whereas in other studies the relationship was suppressed after adjustment for obesity (45, 46). At baseline, we found counter-intuitive lower leptin concentrations (independent of patient weight) in OSA patients with RH, that were inversely correlated with office DBP and MABP. OSA patients were predominantly males (87% compared Comparison Between 3 Months of Active CPAP and Sham CPAP Treatment in RH Patients With OSA After Randomization and Before CPAP Treatment After randomization and before CPAP treatment, there was no sig- nificant difference between the CPAP (n = 19) and the sham CPAP (n = 18) groups regarding all demographic data, medical history, respiratory and biological markers, BP (Table 3), and the number of antihypertensive drugs used. In both groups, two patients discon- tinued because they refused to use the treatment (but not because they felt the treatment was not effective). There are many previous studies using a sham CPAP design that did report a placebo effect in the sham CPAP arm (33). After 3 months, mean treatment compli- ance was comparable between CPAP [3.90 (0.60; 5.82) h/night] and sham CPAP [1.86 (0.60; 6.75) h/night] groups, as well as the number of compliant patients/group (use >4 h/night, Figure 1). We observed that compared to non-OSA RH patients, meta bolic syndrome was more prevalent in those with OSA, with significantly higher triglyceridemia and fasting glucose levels. OSA is known to be associated with a ninefold higher risk of metabolic syndrome after adjustment for confounders (37, 38). As metabolic syndrome and renal function are major predictors of late cardiovascular events this suggests that the association of OSA and RH forms a specific high-risk subgroup. Patient Characteristics Key demographics for the entire study population were: age: 60 ± 10 years, 77% male, mean body mass index (BMI): 29.6 ± 3.9 kg/m2, with essential RH (24-h daytime SBP/DBP: 145 ± 13/85 ± 10 mmHg) treated by a mean of 3.7 antihy- pertensive drugs. Unfortunately, the recruitment rate was lower than expected due to the small number of RH patients assessed at the different cardiology departments and willing to participate. In some centers, the link between sleep centers and cardiology units failed. Also some competitive studies with medications or interventional cardiologic procedures were prioritized. For the analysis of the evolution in values after 6 months of active CPAP treatment, a mixed model with two factors (fixed factor: group; random factor: time) was performed. A McNemar test was used for dipper probability analysis.f For the treatment effect analysis (with mixed models and Ancova), variables were log transformed when normality was not observed. Because of the similarity of the results between transformed and non-transformed data (and the low values of the residual skewness statistics for the non-transformed data), Figure 1 \| Study flow-chart. Abbreviations: CPAP, continuous positive airway pressure, OSA, obstructive sleep apnea. Figure 1 \| Study flow-chart. Abbreviations: CPAP, continuous positive airway pressure, OSA, obstructive sleep apnea. May 2018 \| Volume 9 \| Article 318 Frontiers in Neurology \| www.frontiersin.org CPAP for OSA Patients With RH Joyeux-Faure et al. Case-Controlled Study Comparing OSA vs Non-OSA Patients With RH In the 37 apneic patients, 12 had moderate OSA (15/h ≤ AHI < 30/h), 25 severe OSA (AHI ≥ 30/h), and 13 were obese (BMI ≥ 30). Among the 25 non-OSA patients, 11 were obese. Table 1 show that RH patients with OSA were predominantly men and exhib- ited a significantly higher prevalence of metabolic syndrome.hif Case-Controlled Study Comparing OSA vs Non-OSA Patients With RH significantly increased after 3 months of sham CPAP compared with active CPAP, whereas renin was not modified (Table 4). These results were the same in ITT analysis adjusted for CPAP compliance and per-protocol analysis (before and after adjust- ment for CPAP compliance and gender). In the 37 apneic patients, 12 had moderate OSA (15/h ≤ AHI < 30/h), 25 severe OSA (AHI ≥ 30/h), and 13 were obese (BMI ≥ 30). Among the 25 non-OSA patients, 11 were obese. Table 1 show that RH patients with OSA were predominantly men and exhib- ited a significantly higher prevalence of metabolic syndrome.hif Biological Markerst After 3 months of treatment, there was no difference between CPAP and sham CPAP groups for all metabolic and SNS mark- ers, with the exception of aldosterone serum levels that were May 2018 \| Volume 9 \| Article 318 Frontiers in Neurology \| www.frontiersin.org 5 CPAP for OSA Patients With RH Joyeux-Faure et al. Table 1 \| Baseline characteristics of patients with resistant hypertension (RH). Biological Markerst Non obstructive sleep apnea (OSA) patients OSA patients P-value n 25 37 Age (years) 58 ± 11 60 ± 9 0.45 Male [% (n)] 64.0 (16) 86.5 (32)* 0.038 BMI (kg/m2) 29.4 [26.5; 31.2] 28.6 [27.1; 31.0] 0.55 Waist circumference (cm) 104 [97; 110] 105 [99; 113] 0.59 History Smoking [% (n)] 52 (13) 57 (21) 0.50 Alcohol [% (n)] 32 (8) 30 (11) 0.85 Metabolic syndromea [% (n)] 60 (15) 84 (31)* 0.036 Dyslipidemia [% (n)] 44 (11) 38 (14) 0.63 MI [% (n)] 4 (1) 8 (3) 0.64 Stroke/TIA [% (n)] 4 (1) 14 (5) 0.39 Sleep studies AHI (n/h) 8.2 [5.4; 13.2] 37.6 [25.4; 51.8]* <0.001 Mean SaO2 (%) 93.5 ± 1.9 92.7 ± 2.0 0.11 SaO2 < 90% (%) 0.4 [0.1; 1.3] 5.2 [1.6; 19]* <0.001 Epworth sleepiness scale 9.0 ± 4.4 8.2 ± 4.2 0.53 Office BPb SBP (mmHg) 150 [143; 161] 156 [145; 161] 0.56 DBP (mmHg) 89 ± 14 92 ± 11 0.49 MABP (mmHg) 111 ± 14 112 ± 10 0.63 HR (pbm) 63 [57; 72] 68 [60; 75] 0.12 24-h BP SBP (mmHg) 140 [135; 148] 139 [133; 150] 0.70 DBP (mmHg) 81.1 ± 9.4 82.8 ± 10 0.50 MABP (mmHg) 101.6 ± 8.7 102.2 ± 9.0 0.79 HR (bpm) 71.0 ± 11.1 69.8 ± 10.3 0.67 Dipper for SBP (%) 32.0 (8) 32.4 (12) 0.97 Dipper for DBP (%) 48.0 (12) 51.4 (19) 0.80 Antihypertensive drugs 0.75 3 drugs used [% (n)] 44 (11) 51 (19) 4 drugs used [% (n)] 36 (9) 32 (12) 5 drugs used [% (n)] 16 (4) 16 (6) Good compliance [% (n)] 72 (18) 76 (28) 0.67 Metabolic markers Total cholesterol (mmol/L) 5.17 [4.14; 5.69] 5.17 [4.40; 5.95] 0.29 LDL cholesterol (mmol/L) 2.84 ± 0.76 3.10 ± 1.03 0.26 HDL cholesterol (mmol/L) 1.29 [1.03; 1.55] 1.03 [1.03; 1.29] 0.06 Triglycerides (mmol/L) 1.24 [1.02; 1.92] 1.69 [1.24; 2.37]* 0.032 Fasting glucose (mmol/L) 5.2 [4.9; 6.2] 5.9 [5.4; 7.2]* 0.018 Insulinemia (μIU/mL) 10.6 [5.7; 29.0] 12.1 [8.8; 30.8] 0.36 HbA1c 6.0 [5.7; 6.6] 6.0 [5.7; 7.2] 0.72 us-CRP (mg/L) 2.2 [1.0; 4.2] 2.8 [2.0; 5.4] 0.16 Leptin (ng/mL) 17.0 [6.0; 29.0] 9.0 [6.0; 15.0]* 0.041 Leptin/weight (ng/mL/kg) 0.22 [0.07; 0.30] 0.10 [0.08; 0.19]* 0.029 Adiponectin (ng/mL) 7,820 [4,566; 11,718] 6,393 [4,150; 9,518] 0.26 Creatinine (μmol/L) 78 [67; 86] 90 [82; 104]* 0.006 MDRD index (mL/min/1.73 m2) 87 ± 21.6 77.6 ± 20.9 0.09 SNS markers Normetepinephrine (nmol/L) 10.1 [6.2; 12.3] 10.0 [8.8; 16.6] 0.27 Metepinephrine (nmol/L) 4.0 [2.8; 5.1] 4.6 [3.5; 5.5] 0.42 SRA markers Aldosterone (ng/L) 134 [77; 258] 100 [54; 165] 0.14 Renin (ng/L) 11.8 [5.4; 16.0] 15.3 [6.9; 41.9] 0.17 Data are mean ± SD, median [25th; 75th percentiles] or percentage. Data are mean ± SD, median [25th; 75th percentiles] or percentage. aMetabolic syndrome was defined according to the International Diabetes Federation recommendations (29). bAmbulatory BP monitoring was mandatory to define RH. Office BP values are provided only for readers information. p < 0.05 by Student or Mann–Whitney test. For qualitative data, a Chi-square test or a Fisher test was performed. HbA1c, glycosylated hemoglobin; AHI, apnea–hypopnea index; BMI, body mass index; BP, blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; HR, heart rate; LDL, low-density lipoprotein; MABP, mean arterial BP; MDRD, modification of diet in renal disease; MI, myocardial infarction; RAS, renin–angiotensin system; SaO2, oxygen saturation; SaO2 < 90%, percentage of recording time spent at a SaO2 < 90%; SBP, systolic blood pressure; SNS, sympathetic nervous system; TIA, transient ischemic attack; us-CRP, high-sensitivity C-reactive protein. Bold font indicates the significativity of the results. Frontiers in Neurology \| www.frontiersin.org May 2018 \| Volume 9 \| Article 318 Biological Markerst aMetabolic syndrome was defined according to the International Diabetes Federation recommendations (29). bAmbulatory BP monitoring was mandatory to define RH. Office BP values are provided only for readers information. p < 0.05 by Student or Mann–Whitney test. For qualitative data, a Chi-square test or a Fisher test was performed. Non obstructive sleep apnea (OSA) patients May 2018 \| Volume 9 \| Article 318 May 2018 \| Volume 9 \| Article 318 Frontiers in Neurology \| www.frontiersin.org 6 CPAP for OSA Patients With RH Joyeux-Faure et al. to 64% for non-OSA) and leptin concentrations are known to be lower in males (46), thus the lower leptin concentrations observed in the OSA group could be partly attributed to different sex ratios in the two populations. In healthy humans, Spiegel et al. showed that sleep deprivation lowered leptin levels and blunted their normal diurnal variation (47–50). Sleep deprivation and short sleep duration are common in RH (51) and could contribute toward lower leptin levels. Furthermore, Patel et al. have suggested that OSA may selectively suppress morning secretion of leptin with a relative elevation in leptin level in the evening. Relatively low morning levels may contribute to increased morning appetite and weight gain in individuals with OSA (46). We showed that before treatment, leptin levels were significantly lower in patients with both OSA + RH compared to RH alone. In accordance with Mark et al. (52), in this situation the level of sympathetic activation might be higher inhibiting leptin secretion by the adipocytes. This could be partly reversed by CPAP treatment. Table 2 \| Antihypertensive medications in the different patient groups. Different antihypertensive classes Non obstructive sleep apnea (OSA) patients n = 25 OSA patients Continuous positive airway pressure (CPAP) n = 19 Sham CPAP n = 18 Centrally acting antihypertensive drugs 4 (16) 1 (5) 2 (11) α1-Blockers 7 (28) 5 (26) 2 (11) β-Blockers 13 (52) 8 (42) 11 (61) Diuretics 25 (100) 18 (95) 18 (100) Angiotensin-converting enzyme inhibitors 9 (36) 6 (32) 7 (39) Calcium channel blockers 23 (92) 15 (79) 14 (78) Renin blockers 4 (16) 2 (11) 1 (6) Angiotensin II receptor blockers 10 (40) 12 (63) 10 (56) Peripheral vasodilators 0 (0) 1 (5) 0 (0) Data are number of patients (percentage). Biological Markerst In accordance with a previous study (7), we observed no difference regarding office and 24-h BP, or in dipping pattern, between the OSA and non-OSA patients with RH suggesting that the presence of OSA has a limited impact on BP control in RH patients. However, we found that in these patients 3 months of active CPAP treatment lowered night-time BP (in particular SBP) compared to sham CPAP, without modifying daytime and 24-h BP. This fall in night-time BP was confirmed after 6 months of active CPAP treatment that also improved dipper profile. Our results are in line with a meta-analysis (15) that included four RCTs examining the effect of CPAP vs no CPAP (16–19) and one RCT using sham CPAP (20) and showed that overall CPAP treatment reduces night-time BP, but not daytime BP, in Table 3 \| BP at baseline (D0) and after 3 months (M3) of effective continuous positive airway pressure (CPAP) or sham CPAP treatment in patients with resistant hypertension (RH) and obstructive sleep apnea (OSA). Table 3 \| BP at baseline (D0) and after 3 months (M3) of effective continuous positive airway pressure (CPAP) or sham CPAP treatment in patients with resistant hypertension (RH) and obstructive sleep apnea (OSA). Biological Markerst CPAP (n = 19) Sham CPAP (n = 18) Difference in data change (CPAP-sham CPAP effect)a P-value D0 M3 P-value D0 M3 P-value Office BP SBP (mmHg) 149 [144; 159] 143 [133; 155] 0.11 158 [151; 164] 152 [139; 169] 0.38 −1.18 (−11.37; 9.01) 0.82 DBP (mmHg) 90 [78; 98] 89 [83; 93] 0.19 94 [83; 102] 91 [78; 104] 0.93 −3.08 (−10.41; 4.26) 0.40 MABP (mmHg) 109 [102; 118] 109 [100; 110] 0.08 116 [108; 120] 108 [102; 124] 0.75 −2.15 (−9.45; 5.14) 0.55 HR (bpm) 66 [60; 71] 63 [60; 69]* 0.046 73 [61; 87] 66 [60; 73] 0.12b 0.52 (−4.51; 5.55) 0.83 24-h BP SBP (mmHg) 142 [137; 152] 138 [135; 141]* 0.034 137 [130; 148] 137 [131; 143] 0.59 −1.88 (−7.91; 4.16) 0.53 DBP (mmHg) 84 [78; 90] 80 [74; 86]* 0.011 81 [75; 91] 78 [71; 88] 0.20 −1.69 (−6.16; 2.78) 0.45 MABP (mmHg) 102 [97; 111] 97 [94; 105]* 0.019 98 [94; 109] 98 [91; 103] 0.30 −1.55 (−6.45; 3.35) 0.52 HR (bpm) 73 [64; 76] 70 [62; 72] 0.12 66 [61; 73] 69 [64; 77] 0.24b −3.38 (−7.61; 0.84) 0.11 Daytime BP SBP (mmHg) 145 [139; 155] 141 [138; 148] 0.46 139 [132; 151] 139 [134; 147] 0.56 1.29 (−5.91; 8.48) 0.72 DBP (mmHg) 87 [82; 94] 83 [78; 92] 0.30 84 [77; 94] 83 [73; 90] 0.44 −0.15 (−5.71; 5.41) 0.96 MABP (mmHg) 107 [99; 114] 102 [98; 111] 0.37 103 [95; 111] 102 [97; 106] 0.47 0.33 (−5.70; 6.36) 0.91 HR (bpm) 74 [65; 82] 72 [65; 78] 0.41 69 [63; 82] 71 [66; 81] 0.46 −1.69 (−6.55; 3.17) 0.48 Night-time BP SBP (mmHg) 132 [128; 145] 127 [122; 134]* 0.002 131 [124; 139] 127 [125; 137] 0.82 −6.37 (−12.18; −0.55) 0.033 DBP (mmHg) 75 [72; 83] 70 [66; 76]* 0.002 77.0 [68; 85] 73 [65; 80]* 0.022b −2.50 (−6.91; 1.90) 0.26 MABP (mmHg) 95 [91; 102] 92 [86; 94]* 0.002 96 [85; 103] 91 [85; 98] 0.05b −3.14 (−7.83; 1.56) 0.18 HR (bpm) 65 [59; 70] 63 [57; 66]* 0.016b 59.5 [57; 66] 63 [60; 72] 0.10b −5.98 (−10.45; −1.52) 0.010 Dipper profile for SBP [% (n)] 36.8 (7) 47.4 (9) 0.48 27.8 (5) 33.3 (6) 0.56 1.67 (0.40; 6.94) 0.48 for DBP [% (n)] 52.6 (10) 47.4 (9) 0.74 50.0 (9) 33.3 (6) 0.39 1.88 (0.45; 7.89) 0.39 Data are mean ± SD or median [25th; 75th percentiles] or percentage. [ p ] p g aMean (95% confidence interval), adjusted on baseline values, negative values = higher CPAP effect. p < 0.05 vs data at D0. BP, blood pressure; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; MABP, mean arterial pressure. Bold font indicates the significativity of the results. ests used due to the non-normality of the distribution. at D0. e; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; MABP, mean arterial pressure.i inuous P 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CPAP e MDRD ntinuou P m CPAP n; MDRD the American Heart Association Professional Education Committee of the Council for High Blood Pressure Research. Circulation (2008) 117:e510–26. doi:10.1161/CIRCULATIONAHA.108.189141 Study Limitationsh y The primary outcome was the comparison of leptin levels in the case-control study. We acknowledge that the study essentially pro- vides descriptive data and mechanistic insights that will facilitate the sample size calculation and design of further studies. We did not make an a priori sample size calculation. We recognize that we had some difficulties in recruiting eligible patients, resulting in a relatively small sample size. Second, we did not have access to an objective measurement of adherence to antihypertensive medications, which is a major cause of suboptimal BP control. This poor compliance behavior commonly seen in RH patients is certainly part of the explanation for poor CPAP adherence. Indeed, we acknowledge that CPAP adherence was low both in the effective and in the sham CPAP arms. This is a frequent finding in studies in the OSA field (56). This reflects the real life situation and is accounted for by the intention to treat and per-protocol analyses. Our data give an indication of the range of BP reduction that can be expected in an unselected population with OSA plus RH. Lastly, as some patients were characterized by respiratory polygraphy, we were not able to determine the effect of active CPAP on the arousal index. We speculate that RH itself is associated with persistent sympathetic over-activity that conceals the CPAP effect. Another limitation is that sympathetic nervous system biomarkers have been evaluated in a single blood sample and not in a 24-h urine sample as recommended. Finally, the 6-month effective CPAP study was observational and we acknowledge a potential heterogeneity as some patients were truly CPAP naïve, whereas other patients had been treated with sham CPAP (3 months). FUNDING Grenoble Alpes University Hospital, the “Fondation de Recherche sur l’Hypertension Arterielle” and the “AGIR pour les maladies chroniques” endowment fund provided unrestricted funding for the study. This work was also supported by the French National Research Agency in the framework of the “Investissements d’avenir” program (ANR-15-IDEX-02). Role of sponsors: The spon- sors had no role in the realization of the study (beyond financial support). In conclusion, the association between OSA and RH corresponds to a specific phenotype with male predominance, a higher burden of metabolic syndrome and higher levels of creatinine. Active CPAP could be efficient at decreasing night-time BP and HR. ETHICS STATEMENT The study was conducted in accordance with good clinical prac- tice requirements in Europe, French law, ICH E6 recommenda- tions, and the Helsinki Declaration (1996 and 2000). The protocol was approved by an independent Ethics Committee (Comité de Protection des Personnes, Grenoble, France, IRB0006705) and registered on the ClinicalTrials.gov site (NCT00746902 RHOOSAS). All patients gave their written informed consent. AUTHOR NOTE patients with OSA and RH. Indeed, nocturnal BP levels are a bet- ter predictor of cardiovascular risk than daytime BP levels (53). Therefore, nocturnal BP should be controlled to reduce the risk of cardiovascular disease. We also showed that 3 months of CPAP treatment lowered night-time HR, compared to sham CPAP, in OSA patients with RH. As ambulatory HR is a predictor of car- diovascular mortality in hypertensive patients and that lowering HR is essential for prevention (54, 55), CPAP treatment would provide additional benefits for RH patients. All authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation. AUTHOR CONTRIBUTIONS MJ-F and J-LP: designed the study, collected data, contributed to discussion, wrote the manuscript, and reviewed/edited the manuscript. J-PB, GB-R, PF, PS, CM-V, PL, and RT: included patients, collected data, and or performed analyses, contributed to the discussion, and reviewed/edited the manuscript. Biological Markerst aMean (95% confidence interval), adjusted on baseline values, negative values = higher CPAP effect. bNon-parametric tests used due to the non-normality of the distribution. p < 0.05 vs data at D0. BP, blood pressure; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; MABP, mean arterial pressure. May 2018 \| Volume 9 \| Article 318 Frontiers in Neurology \| www.frontiersin.org 7 ension e in da am CP (−0.2 (−0.0 (−0.0 (−0.7 (−1.9 (−4.4 (−0.3 (−3.2 (−7.0 (−5.0 (−0.0 (−741 (−3.8 (−0.6 (−101 (−14. s syste continuous positive airway pressure (CPAP) or sham CPAP treatment for patients with resistant hype Sham CPAP (n = 18) Differen (CPAP-s P-value D0 M3 P-value 0.35 5.95 [4.11; 6.05] 5.35 [4,47; 5.82] 0.82 0.0 0.89b 3.36 [2.59; 3.62] 3.10 [2.59; 3.62] 0.34 0.0 0.28 1.19 [0.88; 1.34] 1.19 [1.01; 1.37] 0.17b 0.0 0.20 1.99 [1.29; 2.57] 1.79 [1.10; 2.52] 0.80b −0.3 0.37 6.0 [5.9; 7.3] 6.2 [5.2; 7.3] 0.43b 0.1 0.12 11.4 [8.8; 30.1] 9.5 [7.7; 14.9] 0.018 1.7 0.71 6.0 [5.8; 7.6] 5.9 [5.8; 6.7] 0.59 0.2 ] 0.22b 87 [76; 109] 91 [77; 98] 0.84b 4.6 0.59b 77 [62; 93] 76 [59; 90] 0.81 −0.5 0.20 10.0 [8.0; 15.0] 12.0 [9.0; 14.0] 0.40b −0.0 0.31 0.10 [0.08; 0.19] 0.13 [0.10; 0.19] 0.38b −0.0 5] 0.80 5,134 [3,846; 9,276] 7,104 [4,428; 8,409]* 0.030 −276 0.05 10.0 [8.8; 19.1] 9.2 [7.4; 14.4] 0.12b −0.3 0.69b 4.4 [3.8; 5.1] 4 [2.4; 4.4] 0.15b 0.6 0.54b 103 [72; 154] 140 [101; 215]* 0.038b −58 0.34 16.3 [6.6; 58.9] 9.7 [4.9; 39.7] 0.06 3.6 am CPAP effect. ein; MDRD, Modification of Diet in Renal Disease; RAS, renin–angiotensin system; SNS, sympathetic nervo Joyeux-Faure et al. CPAP for OSA Patients With RH ACKNOWLEDGMENTS The authors are grateful to Hélène Pierre (Cardiology Department) for patient management, to Anne-Sophie Gauchez (Biochemistry Department) for biological analysis, to Nathalie Arnol (EFCR Laboratory) for statistical analyses, and to Dr. Alison Foote (Publications unit) for revision and critical editing of the manu- script; all from Grenoble Alpes University Hospital, France. 6. Rimoldi SF, Messerli FH, Bangalore S, Scherrer U. Resistant hypertension: what the cardiologist needs to know. Eur Heart J (2015) 36:2686–95. doi:10.1093/ eurheartj/ehv392 7. Pedrosa RP, Drager LF, Gonzaga CC, Sousa MG, de Paula LKG, Amaro ACS, et al. Obstructive sleep apnea: the most common secondary cause of hyper- tension associated with resistant hypertension. Hypertension (2011) 58:811–7. doi:10.1161/HYPERTENSIONAHA.111.179788 9. de Faria AP, Modolo R, Fontana V, Moreno H. Adipokines: novel players in resistant hypertension. J Clin Hypertens (Greenwich) (2014) 16:754–9. doi:10.1111/jch.12399 8. Gonçalves SC, Martinez D, Gus M, de Abreu-Silva EO, Bertoluci C, Dutra I, et al. Obstructive sleep apnea and resistant hypertension: a case-control study. Chest (2007) 132:1858–62. doi:10.1378/chest.07-1170 the American Heart Association Professional Education Committee of the Council for High Blood Pressure Research. Circulation (2008) 117:e510–26. doi:10.1161/CIRCULATIONAHA.108.189141 6. Rimoldi SF, Messerli FH, Bangalore S, Scherrer U. Resistant hypertension: what the cardiologist needs to know. Eur Heart J (2015) 36:2686–95. doi:10.1093/ eurheartj/ehv392 7. Pedrosa RP, Drager LF, Gonzaga CC, Sousa MG, de Paula LKG, Amaro ACS, et al. Obstructive sleep apnea: the most common secondary cause of hyper- tension associated with resistant hypertension. Hypertension (2011) 58:811–7. doi:10.1161/HYPERTENSIONAHA.111.179788 8. Gonçalves SC, Martinez D, Gus M, de Abreu-Silva EO, Bertoluci C, Dutra I, et al. Obstructive sleep apnea and resistant hypertension: a case-control study. Chest (2007) 132:1858–62. doi:10.1378/chest.07-1170 9. de Faria AP, Modolo R, Fontana V, Moreno H. Adipokines: novel players in resistant hypertension. J Clin Hypertens (Greenwich) (2014) 16:754–9. doi:10.1111/jch.12399 Frontiers in Neurology \| www.frontiersin.org REFERENCES 1. Lévy P, Kohler M, McNicholas WT, Barbé F, McEvoy RD, Somers VK, et al. Obstructive sleep apnoea syndrome. Nat Rev Dis Primers (2015) 1:15015. doi:10.1038/nrdp.2015.15 1. Lévy P, Kohler M, McNicholas WT, Barbé F, McEvoy RD, Somers VK, et al. Obstructive sleep apnoea syndrome. Nat Rev Dis Primers (2015) 1:15015. doi:10.1038/nrdp.2015.15 2. Peppard PE, Young T, Palta M, Skatrud J. Prospective study of the association between sleep-disordered breathing and hypertension. N Engl J Med (2000) 342:1378–84. doi:10.1056/NEJM200005113421901 2. Peppard PE, Young T, Palta M, Skatrud J. 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Friedman O, Bradley TD, Ruttanaumpawan P, Logan AG. Independent associ- ation of drug-resistant hypertension to reduced sleep duration and efficiency. Am J Hypertens (2010) 23:174–9. doi:10.1038/ajh.2009.220 Copyright © 2018 Joyeux-Faure, Baguet, Barone-Rochette, Faure, Sosner, Mounier- Vehier, Lévy, Tamisier and Pépin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Neurology \| www.frontiersin.org REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 52. Mark AL, Rahmouni K, Correia M, Haynes WG. A leptin-sympathetic-leptin feedback loop: potential implications for regulation of arterial pressure and body fat. 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https://openalex.org/W3180011454	https://link.springer.com/content/pdf/10.1007/s42081-021-00130-2.pdf	English	null	Correction to: Inference on high-dimensional mean vectors under the strongly spiked eigenvalue model	Japanese Journal of Statistics and Data Science/Japanese journal of statistics and data science	2,021	cc-by	468	Japanese Journal of Statistics and Data Science (2021) 4:887–888 https://doi.org/10.1007/s42081-021-00130-2 Japanese Journal of Statistics and Data Science (2021) 4:887–888 https://doi.org/10.1007/s42081-021-00130-2 CORRECTION CORRECTION Aki Ishii1 · Kazuyoshi Yata2 · Makoto Aoshima2 Published online: 7 July 2021 © The Author(s) 2021 Published online: 7 July 2021 © The Author(s) 2021 Published online: 7 July 2021 © The Author(s) 2021 Correction to: Inference on high-dimensional mean vectors under the strongly spiked eigenvalue model Aki Ishii1 · Kazuyoshi Yata2 · Makoto Aoshima2 The original article can be found online at https://doi.org/10.1007/s42081-018-0029-z. 1 Department of Information Sciences, Tokyo University of Science, Chiba 278-8510, Japan 2 Institute of Mathematics, University of Tsukuba, Ibaraki 305-8571, Japan Correction to: Japanese Journal of Statistics and Data Science (2019) 2:105–128 https://doi.org/10.1007/s42081-018-0029-z The article “Inference on high-dimensional mean vectors under the strongly spiked eigenvalue model”, written by Aki Ishii · Kazuyoshi Yata and Makoto Aoshima was originally published Online First without Open Access. After publication in volume 2 , issue 1, page 105–128 the author decided to opt for Open Choice and to make the article an Open Access publication. Therefore, the copyright of the article has been changed to © The Author(s) 2021 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (https:// creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, dis- tribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The original article has been corrected. OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included The original article can be found online at https://doi.org/10.1007/s42081-018-0029-z. B Aki Ishii a.ishii@rs.tus.ac.jp Kazuyoshi Yata yata@math.tsukuba.ac.jp Makoto Aoshima aoshima@math.tsukuba.ac.jp 1 Department of Information Sciences, Tokyo University of Science, Chiba 278-8510, Japan 2 Institute of Mathematics, University of Tsukuba, Ibaraki 305-8571, Japan 1 Department of Information Sciences, Tokyo University of Science, Chiba 278-8510, Japan 2 Institute of Mathematics, University of Tsukuba, Ibaraki 305-8571, Japan 123 Japanese Journal of Statistics and Data Science (2021) 4:887–888 888 in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 123
https://openalex.org/W2901551938	http://journal.umpo.ac.id/index.php/istawa/article/download/167/151	Indonesian	null	IMPLEMENTASI KURIKULUM 2013 PADA MATA PELAJARAN PENDIDIKAN AGAMA ISLAM DI SD MUHAMMADIYAH TERPADU PONOROGO	Istawa	2,016	cc-by	7,248	52 52 Oleh: Nuraini (Dosen Fakultas Agama Islam Universitas Muhammadiyah Ponorogo) M. Fata Muhtarima (Alumni Fakultas Agama Islam Universitas Muhammadiyah Ponorogo) IMPLEMENTASI KURIKULUM 2013 PADA MATA PELAJARAN PENDIDIKAN AGAMA ISLAM DI SD MUHAMMADIYAH TERPADU PONOROGO Oleh: Nuraini (Dosen Fakultas Agama Islam Universitas Muhammadiyah Ponorogo) Abstract Curriculum plays a very important role in realizing the generation of reliable, creative, Innovative and personal responsibility. Therefore, the curriculum must always be prepared and perfected in accordance with the times. Associated with changes in the curriculum, the various parties to analyze and see the need for a competency-based curriculum implemented at once a character-based (competency and character-based curriculum), which can provide students with a wide range of attitudes and capabilities in accordance with the demands of the times and the demands of technology. Character and competency based curriculum is expected to solve the problems of the nation, especially in the field of pedidikan to prepare learners through planning, implementation and evaluation of the education system in an effective, efficient and useful. Curriculum change from time to time regarding structural change and conceptual change and now we are also introduced to the new curriculum is the curriculum applied by the government in 2013 and was passed by Parliament. According to Muhammad Nuh curriculum 2013 is designed as an effort to prepare the next generation of Indonesia in 2045 is exactly 100 years of Indonesian independence, while utilizing the productive age population numbers are very abundant in order to become a demographic bonus and not a demographic disaster. Character education in the curriculum in 2013 aimed at improving the quality of the process and outcomes of education that leads to the formation of character and noble character of learners as a whole, integrated, and balanced in accordance with competency standards in each educational unit. Through the implementation of competency-based ISTAWA: Jurnal Pendidikan Islam 53 curriculum in 2013 at the same time with a character-based thematic and contextual approach is expected that learners are able to independently increase and using the knowledge, study and internalize and personalize the character values and noble character so manifest in everyday behavior. Keywords: Curriculum change, Character education, Curriculum 2013 1 Slameto. Proses Belajar Mengajar dalam Sistem Kredit Semester, (Jakarta: Bumi Aksara, 1991), v 2 Zaenal Arifin, Konsep dan Model Pengembangan Kurikulum, (Bandung:PT. Remaja Rosdakarya, 2011) hal.2 2 Zaenal Arifin, Konsep dan Model Pengembangan Kurikulum, (Bandung:PT. Zaenal Arifin, Konsep dan Model Pengembangan Kurikulum, (Bandung:PT. Remaja Rosdakarya, 2011) hal.2 , p Remaja Rosdakarya, 2011) hal.2 Pendahuluan Belajar adalah proses memanusiakan manusia, dimana hanya melalui belajar manusia menemukan dirinya dalam relasinya dengan sesama, lingkungan dan juga dengan Sang Pencipta. Melalui belajar manusia mengaktualisasikan diri dan lingkungannya sedemikian sehingga kualitas hidup dan penghidupan ini menjadi lebih baik.Dalam situasi formal disekolah, belajar tidak akan bisa lepas dengan kegiatan mengajar, siswa belajar karena guru mengajar demikian juga sebaliknya. Bagaimana siswa belajar banyak ditentukan oleh bagaimana guru dalam mengajar. Salah satu usaha untuk mengoptimalkan hasil belajar adalah memperbaiki pengajaran yang dalam hal ini banyak ditentukan oleh guru. Karena pengajaran itu adalah suatu sistem maka perbaikannyapun harus mencakup keseluruhan komponen dalam sistem (pengajaran) tersebut. Komponen-komponen yang terpenting antara lain adalah kurikulum, tujuan, materi dan evaluasi.1 Dari beberapa aspek dalam pembelajaran, kurikulum memainkan peran yang sangat penting dalam mewujudkan generasi yang handal, kreatif, inovatif dan menjadi pribadi yang bertanggug Volume 1, Nomor 2, Januari-Juni 2016 Volume 1, Nomor 2, Januari-Juni 2016 54 jawab. Ibarat tubuh, kurikulum merupakan jantungnya pendidikan. Kurikulum menentukan jenis dan kualitas pengetahuan dan pengalaman yang memungkinkan orang atau seseorang mencapai kehidupan dan penghidupan yang lebih baik. Oleh karena itu kurikulum harus selalu disusun dan disempurnakan sesuai dengan perkembangan zaman. Kurikulum harus bersifat dinamis, artinya kurikulum selalu mengalami perubahan sesuai dengan perkembangan zaman, ilmu pengetahuan dan teknologi, tingkat kecerdasan, kultur, sistem nilai, serta kebutuhan masyarakat. Oleh sebab itu, para pengembang kurikulum termasuk guru harus memiliki wawasan yang luas dan mendalam tentang hal tersebut.Kurikulum harus selalu dimonitoring dan dievaluasi untuk perbaikan dan penyempurnaan.Setiap kali penyempurnaan kurikulum belum tentu meghasilkan sesuatu yang baik karena kurikulum bersifat hipotesis.Maksudnya, baik tidaknya kurikulum akan dapat diketahui setelah dilaksanakan dilapangan.2 Perubahan kurikulum dari masa ke masa menyangkut perubahan struktural dan perubahan konsepsional dan kini kita juga dikenalkan dengan kurikulum baru yang diterapkan pemerintah yaitu kurikulum 2013 dan telah disahkan oleh DPR. Menurut Muhammad Nuh kurikulum 2013 dirancang sebagai upaya menyiapkan generasi Indonesia 2045 mendatang yaitu tepatnya 100 tahun Indonesia merdeka, sekaligus memanfaatkan populasi usia produktif yang jumlahnya sangat melimpah agar menjadi bonus demografi dan tidak menjadi bencana demografi. 3 Mulyasa. H.E.,Pengembangan Implementasi Kurikulum 2013,(PT. Remadja Rosdakarya, 2013), 7 3 Mulyasa. H.E.,Pengembangan Implementasi Kurikulum 2013,(PT. Remadja 3 Mulyasa. H.E.,Pengembangan Implementasi Kurikulum 2013,(PT. Remadja Rosdakarya, 2013), 7 Volume 1, Nomor 2, Januari-Juni 2016 Pendahuluan ISTAWA: Jurnal Pendidikan Islam ISTAWA: Jurnal Pendidikan Islam 55 Berkaitan dengan perubahan kurikulum, berbagai pihak menganalisa dan melihat perlunya diterapkan kurikulum berbasis kompeteni sekaligus berbasis karakter (competency and character based curriculum), yang dapat membekali peserta didik dengan berbagai sikap dan kemampuan yang sesuai dengan tuntutan perkembangan zaman dan tuntutan teknologi.Kurikulum berbasis karakter dan kompetensi diharapkan mampu memecahkan berbagai masalah bangsa, khususnya dalam bidang pedidikan dengan mempersiapkan peserta didik melalui perencanaan, pelaksanaan dan evaluasi terhadap sistem pendidikan secara efektif, efisien dan berdaya guna.Oleh karena itu merupakan langkah yang posiitif jika pemerintah merevialisasi pendidikan termasuk dalam pengembangan kurikulum 2013. Kurikulum 2013 lebih ditekankan pada pendidikan karakter terutama pada tingkat dasar yang akan menjadi pondasi bagi tingkat berikutnya. Melalui pengembangan kurikulum 2013 yang berbasis karakter dan berbasis kompetensi, kita berharap bangsa ini menjadi bangsa yang bermartabat dan masayarakatnya memiliki nilai tambah dan nilai jual yang bisa ditawarkan kepada bangsa lain, sehingga kita bisa bersaing, bersanding bahkan bertanding dengan bangsa-bangsa lain dalam pencaturan global. Hal ini dimungkinkan jika implementasi kurikulum 2013 betul-betul dapat menghasilkan insan yang produtif, kreatif, inovatif dan berkarakter.3 Pendidikan berkarakter dalam kurikulum 2013 bertujuan untuk meningkatkan mutu proses dan hasil pendidikan yang mengarah pada pembentukan budi pekerti dan akhlak mulia peserta didik secara utuh, 3 Mulyasa. H.E.,Pengembangan Implementasi Kurikulum 2013,(PT. Remadja Rosdakarya, 2013), 7 Volume 1, Nomor 2, Januari-Juni 2016 Volume 1, Nomor 2, Januari-Juni 2016 56 terpadu, dan seimbang sesuai dengan standar kompetensi lulusan pada setiap satuan pendidikan. Melalui implementasi kurikulum 2013 yang berbasis kompetensi sekaligus berbasis karakter dengan pendekatan tematik dan kontekstual diharapkan peserta didik mampu secara mandiri meningkatkan dan menggunakan pengetahuannya, mengkaji dan menginternalisasi serta mempersonalisasi nilai-nilai karakter serta akhlak mulia sehingga terwujud dalam perilaku sehari-hari. Dalam pelaksanaan kurikulum 2013, pendidikan berkarakter dapat diintegrasikan dalam seluruh materi pembelajaran pada setiap bidang studi yang terdapat dalam kurikulum. Materi pembelajaran yang berkaitan dengan norma atau nilai pada setiap bidang studi perlu dikembangkan, dieksplisitkan, dihubungkan dengan konteks kehidupan sehari-hari. Dengan demikian, pendidikan nilai dan pembentukan karakter tidak hanya dilakukan pada tataran kognitif, tetapi menyentuh internalisasi, dan pengalaman nyata dalam kehidupan sehari-hari. Pendidikan karakter pada tingkat satuan pendidikan mengarah pada pembentukan budaya sekolah madrasah, yaitu nilai-nilai yang melandasi perilaku, tradisi, kebiasaan sehari-hari serta simbol-simbol yang dipraktikkan oleh semua warga sekolah/madrasah, dan masyarakat sekitarnya. ISTAWA: Jurnal Pendidikan Islam y g g p 5 Dwifantya Aquina dan Daru Waskita, "Kurikulum 2013 Waktu Pelajaran Agama Ditambah", http://nasional.news.viva.co.id/news/read/413090, (diakses pada 09 Juni 2013 pukul 20:29:47 WIB) 4 Mulyasa. H.E.,Pengembangan Implementasi Kurikulum 2013,9 5 Pendahuluan Budaya sekolah/madrasah merupakan ciri khas, karakter, dan citra sekolah/madrasah tersebut di mata masyarakat luas.Pada umumnya pendidikan karakter menekankan pada keteladanan, penciptaan lingkungan dan pembiasaan melalui berbagai tugas keilmuan dan kegiatan kondusif. Dengan demikian, apa yang dilihat, didengar, dirasakan dan dikerjakan oleh peserta didik dapat membentuk karakter mereka. Selain menjadikan keteladanan dan pembiasaan sebagai ISTAWA: Jurnal Pendidikan Islam ISTAWA: Jurnal Pendidikan Islam 57 metode pendidikan utama, penciptaan iklim dan budaya serta lingkungan yang kondusif juga sangat penting, dan turut membentuk karakter peserta didik.4 Waktu pembelajaran Pendidikan Agama Islam yang semula dilaksanakan dua jam perminggu sekarang menjadi tiga jam perminggu. Menteri Pendidikan dan Kebudayaan Mohammad Nuh menilai penambahan waktu pelajaran agama ini sangat tepat.5 Mengenai penambahan jam pelajaran PAI yang menjadi tiga jam ini juga bukan menjadi masalah yang besar, justru penambahan jam tersebut dirasa sangat berguna. Berdasarkan pengamatan peneliti SD Muhammadiyah Terpadu adalah salah satu sekolah yang menerapkan kurikulum 2013 sebagai kurikulum baru pengganti dari kurikulum sebelumnya. Pada penelitian terdahulu, ditemukan bahwa para guru di SD Muhammadiyah Terpadu sudah mempersiapkan untuk menyongsong implementasi Kurikulum 2013 seperti mengikuti diklat kurikulum dalam rangka untuk lebih memahami penerapan kurikulum 2013, mengingat perangkat pembelajaran dan administrasi kurikulum 2013 yang banyak jumlahnya. Para guru tidak hanya diam dan menunggu dari pemerintah, tetapi mereka juga aktif mencari informasi dan pedoman pelaksanaan kurikulum 2013, seperti browsing di internet dan bertanya kepada pakar kurikulum yang lebih paham tentang kurikulum 2013. Volume 1, Nomor 2, Januari-Juni 2016 58 Proses pembelajaran Pendidikan Agama Islam di SD Muhammadiyah Terpadu tidak hanya di dalam kelas saja. Setiap pagi siswa datang, mereka selalu melakukan salam-salaman kepada para guru dan masuk kelas masing-masing. Proses belajar mengajar tidak langsung dilakukan, tetapi diajarkan terlebih dahulu tentang hafalan (tahfidz) al-quran dan tartil al-quran. Sekolah juga menentukan standar batasan dalam penguasaan membaca Alquran kepada peserta didiknya. Sholat dhuha dilakukan setiap harinya oleh siswa dan guru pada jam istirahat yang di-imami langsung oleh peserta didik dan guru bertindak sebagai pembimbing untuk membina peserta didik dalam melakukan gerakan dan bacaan sholat jika ada yang perlu dibenarkan. Peneliti merasa tertarik untuk memilih SD Muhammadiyah Terpadu Ponorogo karena pendidikan karakter sudah diterapkan, mengingat kurikulum 2013 adalah pendidikan yang terfokus pada pendidikan karakter. Disamping itu PAI diajarkan secara mendetail dan langsung dipraktekkan sehingga siswa bisa memahami dan mempraktekkannya secara langsung.Model pembelajaran yang dipakai SDMT berbeda dengan sekolah lain dengan pendekatan yang lebih kontekstual terhadap materi yang memudahkan siswa dalam memahami materi. Implementasi Kurikulum 2013 Pada Mata Pelajaran Pendidikan Agama Islam di SD Muhammadiyah Terpadu Ponorogo Dalam melakukan pembelajaran setiap hari tentunya setiap sekolah mempunyai konsep dan cara masing-masing, namun perlu adanya kurikulum yang mewadahi semua tujuan tersebut sebagai acuan agar kegiatan pembelajaran menjadi satu arah dan linier. ISTAWA: Jurnal Pendidikan Islam 59 Keberadaan kurikulum tidak akan bisa dilepaskan dari kegiatan belajar mengajar di manapun berada, karena pentingnya keberadaan kurikulum maka kurikulum harus disusun untuk bisa menjawab tuntutan zaman yang terus berkembang dengan semua kemajuan- kemajuan yang ditawarkan baik dalam bidang teknologi, kesehatan bahkan dalam pendidikan sehingga kurikulum harus disusun sesuai dengan zaman dan bisa berubah tidak konstan ataupun paten. Dalam perkembangannya Indonesia sudah melakukan perubahan kurikulum beberapa kali dan yang diterapkan terakhir adalah tentang kurikulum 2013.Kurikulum ini menawarkan bahwa pendidikan karakter dan pendidikan sikap menjadi fokus dalam pendidikan sehingga dalam pembelajaran karakter dan sikap harus selalu disampaikan.Disamping itu kurikulum 2013 berdasarkan tematik integratif yang artinya bahwa kurikulum ini antara pelajaran satu dengan palajaran lainnya saling terkait dan saling mendukung.Seperti diungkapkan oleh Ustadz.Aziz bahwa kurikulum 2013 ini sangat fleksibel dan memberikan ruang yang luas untuk guru. Adapun hasil wawancaranya adalah sebagai berikut: “Setuju, karena kurikulum 2013 sangat fleksibel dan memberikan ruang yang lebih luas untuk guru dalam mengeksplor materi karena sifatnya yang tematik dan terintegrasi dengan semua mata pelajaran, namun terlalu detail dan rumit juga terlalu banyak admnistrasinya” “Setuju, karena kurikulum 2013 sangat fleksibel dan memberikan ruang yang lebih luas untuk guru dalam mengeksplor materi karena sifatnya yang tematik dan terintegrasi dengan semua mata pelajaran, namun terlalu detail dan rumit juga terlalu banyak admnistrasinya” Dari pemaparan Ustadz. Aziz dapat disimpulkan bahwa SD Muhammadiyah terpadu Ponorogo setuju dan mendukung dalam implementasi kurikulum 2013 karena materi yang ditawarkan secara tematik integratif bisa membuat pembelajaran saling terkait. Hal senada juga disampaikan oleh Ustadz Imam Saiful Bahri yang Volume 1, Nomor 2, Januari-Juni 2016 Volume 1, Nomor 2, Januari-Juni 2016 60 menyetujui implementasi kurikulum 2013 dalam wawancara sebagai berikut: “Setuju, lebih fleksibel dan lebih bisa integrasi. Tetapi kalo saya punya usul bahwa kurikulum 2013 telalu detail dan terkesan ribet dengan berbagai macam jenis administrasinya. Kurikulum 2013 terlalu menekan dan menjerat sekolah karena dari pusat sudah dipatenkan KI-nya sehingga tidak memberikan keleluasaan sekolah untuk bereksplorasi sesuai kebutuhan sekolah akibatnya sekolah tidak bisa memberikan image baru kepada masyarakat. SDMT bukan sekolah negeri jadi kita harus mampu beinovasi guna bersaing dengan sekolah-sekolah pemerintah” “Setuju, lebih fleksibel dan lebih bisa integrasi. Implementasi Kurikulum 2013 Pada Mata Pelajaran Pendidikan Agama Islam di SD Muhammadiyah Terpadu Ponorogo Tetapi kalo saya punya usul bahwa kurikulum 2013 telalu detail dan terkesan ribet dengan berbagai macam jenis administrasinya. Kurikulum 2013 terlalu menekan dan menjerat sekolah karena dari pusat sudah dipatenkan KI-nya sehingga tidak memberikan keleluasaan sekolah untuk bereksplorasi sesuai kebutuhan sekolah akibatnya sekolah tidak bisa memberikan image baru kepada masyarakat. SDMT bukan sekolah negeri jadi kita harus mampu beinovasi guna bersaing dengan sekolah-sekolah pemerintah” Dari pemaparan Ustadz Imam Saiful Bahri dapat disimpulkan bahwa beliau menyetujui implementasi kurikulum 2013 di SDMT, namun dengan beberapa catatan kekurangan yang harus diperbaiki dalam kurikulum 2013 dimana kurikulum yang terlalu sulit diterapkan dengan banyaknya administrasi yang harus dipersiapkan.Hal senada juga disampaikan oleh Ustadz. Farid tentang kurikulum 2013 yaitu: “Sangat setuju sekali, karena pembelajaran karakter dan sesuai dengan moto sekolah “Character Building School” dan mengedepankan “Tauhid Life Skill”. Maka dapat dimbil kesimpulan bahwa SDMT setuju dengan implementasi kurikulum 2013 karena pembelajaran berbasis karakter menjadi fokus pembelajaran. Pemaparan lain mengungkapkan tentang bagaimana pandangan guru SDMT tentang kurikulum 2013 seperti yang diungkapkan oleh Ustadz Aziz tentang kurikulum 2013 adalah sebagai berikut. “Maksudnya bagus, kurikulum yang terintegrasi dengan semua mata pelajaran. Penilaiannyapun mencakup berbagai aspek baik dari keilmuan, akhlak, sikap semua bisa terangkum.” ISTAWA: Jurnal Pendidikan Islam ISTAWA: Jurnal Pendidikan Islam 61 Dari pemaparan ustadz Aziz dapat disimpulkan bahwa kurikulum 2013 mempunyai maksud dan tujuan yang baik dengan terintegrasikannya semua mata pelajaran sehingga guru dalam eksplorasi materi pembelajaran lebih luas dan bisa dibawa sesuai kebutuhan. Disamping itu dalam kurikulum 2013 mencakup penilaian yang lebih detail sehingga semua aspek bisa terangkum dalam penilaian. Pemaparan lain dari ustadz Imam Saiful Bahri adalah sebagai berikut. ”Memang kurikulum 2013 bagus dan detail, maksudnya juga bagus tetapi kurang efektif. Jadi begini, bahwa kurikulum 2013 ini terlalu menekan anak. Anak seakan dipaksa untuk tahu didepan dan buku-bukunya pun masih belum dipegang oleh anak. Dari sisi penilaian juga merepotkan. Aspek sikap itu harusnya disikapi, bukan diberikan nilai dengan angka” ”Memang kurikulum 2013 bagus dan detail, maksudnya juga bagus tetapi kurang efektif. Jadi begini, bahwa kurikulum 2013 ini terlalu menekan anak. Anak seakan dipaksa untuk tahu didepan dan buku-bukunya pun masih belum dipegang oleh anak. Dari sisi penilaian juga merepotkan. Volume 1, Nomor 2, Januari-Juni 2016 6 Tim Kualita Pendidikan Indonesia,Modul Pelatihan Pembelajaran Tematik Kurikulum 2013..., hal. 35 Implementasi Kurikulum 2013 Pada Mata Pelajaran Pendidikan Agama Islam di SD Muhammadiyah Terpadu Ponorogo Aspek sikap itu harusnya disikapi, bukan diberikan nilai dengan angka” Dari pemaparan ustadz Imam Saiful Bahri dapat disimpulkan bahwa kurikulum 2013 sangat baik dan detail dengan semua perangkat-perangkatnya, namun terdapat kekurangan di mana jika kurikulum 2013 ini diterapkan secara penuh akan menekan siswa dan dari segi penilaian yang rumit dan merepotkan. Pendidikan Agama Islam (PAI) dan Budi Pekerti adalah pendidikan yang memberikan pengetahuan dan keterampilan serta membentuk sikapdan kepribadian peserta didik dalam mengamalkan ajaran agama Islam. Pendidikan Agama Islam dan Budi Pekerti dilaksanakan melalui mata pelajaran pada semua jenjang pendidikan, yang pengamalannya dapat dikembangkan dalam berbagai kegiatan baik yang bersifat kurikuler maupun ekstrakurikuler. Proses pembelajaran dapat dipadankan dengan suatu proses ilmiah, karena itu kurikulum 2013 mengamanatkan esensi pendekatan Volume 1, Nomor 2, Januari-Juni 2016 Volume 1, Nomor 2, Januari-Juni 2016 62 ilmiah dalam pembelajaran. Pendekatan ilmiah diyakini sebagai titian emas perkembangan dan pengembangan sikap, keterampilan dan pengetahuan peserta didik. Dalam pendekatan atau proses kerja yang memenuhi kriteria ilmiah, para ilmuan lebih mengedepankan penalaran induktif (inductive reasoning) dibandingkan dengan penalaran deduktif (deductive reasoning)6. Langkah-langkah pembelajaran dengan metode ini adalah dengan mengamati, menanya, mencoba/mengumpulkan informasi, menalar/mengasosiasi dan mengkomunikasikan. Ditinjau dari penilaian, kurikulum 2013 menggunakan penilaian autentik yang bermakna penilaian yang signifikan atas hasil belajar peserta didik dalam ranah sikap, keterampilan, dan pengetahuan. Jenis-jenis penilaian autentik dapat diklasifikasikan antara lain : penilaian kinerja, penilaian proyek, penilaian portofolio, penilaian tertulis dan penilaian produk. Hal ini dirangkum dalam ranah kompetensi dengan diklasifikasikan KI 1 dan KI 2 (spiritual dan sikap), KI 3 (pengetahuan) KI 4 (keterampilan). Kurikulum 2013 seharusnya memberikan wacana baru tentang dunia pendidikan di Indonesia. SDMT menerapkan kurikulum 2013 sudah sejak lama dan jika dilihat dari sudut pandang PAI SDMT mengaku sudah meninggalkan esensi kurikulum 2013 seperti disampaikan oleh ustadzah St. Rohmatin Kusbah sebaga berikut: “Diterapkan sudah melebihi kurikulum 2013dari dulu hingga sekarang, karena kurikulum 2013 mengedepankan karakter yang baru-baru ini didengungkan, tetapi di SDMT sudah diberlakukan sejak dulu dengan contoh jika pagi pada waktu ISTAWA: Jurnal Pendidikan Islam 63 siswa datang masuk dengan mengucapkan salam dan berjabat tangan dengan ustadz dan ustadzah, setelah itu jam 8 pagi kita budayakan untuk sholat dhuha.” Dari pemaparan ustadzah St Rohmatin Kusbah dapat disimpulkan bahwa SDMT sudah menerapkan kurikulum 2013 sejak berdirinya SDMT dengan pendekatan karakternya khususnya di bidang PAI dan pembinaan akhlak siswa. Implementasi Kurikulum 2013 Pada Mata Pelajaran Pendidikan Agama Islam di SD Muhammadiyah Terpadu Ponorogo Hal senada juga diungkapkan oleh ustadz Farid sebagai berikut : “Kurikulum 2013 di PAI sudah diterapkan di beberapa kelas, dan penerapannya sama dari apa yang dulunya kita sampaikan sebelum ada kurikulum 2013 ini. Jika saya mengajar PAI disekolah ini, yang saya terapkan adalah pendekatan yang dipakai dalam kurikulum 2013 dengan menggunakan asas tematik. Jadi saya mengajar PAI itu memberikan contohnya adalah dengan menggunakan misalnya keagungan Allah yang saya analogikan dengan bagaimana penciptaan alam ini, atau bagaimana alam ini bisa bergerak sehingga ini sudah menjadi contoh bahwa kami menerapkan pembelajaran integratif. Tetapi menurut saya kurikulum 2013 lamban dan ketinggalan jika diterapkan di SDMT khususnya di PAI karena kita sudah berjalan jauh tetapi kurikulum 2013 baru merangkak. Maksudnya begini, di SDMT kita sudah mengajarkan misal di kelas 4 ada tema dikurikulum 2013 tentang membahas surah Al-Fil tetapi di SDMT tema itu sudah dibahas di kelas 2 dan anak sudah mampu menghafalnya. Dalam kurikulum 2013 tidak ada indikator kemampuan menghafal dan membaca Alquran, padahal ini menjadi indikator dan modal kami di sekolah ini.” Dari pemaparan ustadz Farid dapat disimpulkan bahwa pembelajaran PAI di SDMT sudah menggunakan pendekatan dari kurikulum 2013 yang mengintegrasikan beberapa mata pelajaran kepada pelajaran yang disampaikan.Ustadz Farid juga menyampaikan bahwa tema yang ditawarkan oleh kurikulum 2013 sudah tertinggal Volume 1, Nomor 2, Januari-Juni 2016 Volume 1, Nomor 2, Januari-Juni 2016 64 jauh dari pembelajaran yang dipakai sebelumnya oleh SDMT. Pemaparan lain diungkapkan tentang implementasi kurikulum 2013 dari sudut pandang implementasi secara umum di SDMT oleh Ustadz Aziz sebagai berikut : Aziz sebagai berikut : Aziz sebagai berikut : “Dicoba diimplementasikan dengan baik, tetapi dengan beberapa kendala – kendala yang dihadapi, meski dengan keterbatasan tetap diimplementasikan. SDMT memang mencoba untuk mengimplementasikan kurikulum 2013 tetapi juga masih menggunakan kurikulum KTSP. Disamping itu SDMT juga mempunyai kurikulum sendiri sesuai kebutuhan SDMT antara lain dalam menanamkan karakter siswa di bidang agama, SDMT menerapkan sholat dhuha dan dzuhur berjamaah di masjid dan memberikan latihan khusus membaca Al-Quran untuk siswa.” “Dicoba diimplementasikan dengan baik, tetapi dengan beberapa kendala – kendala yang dihadapi, meski dengan keterbatasan tetap diimplementasikan. SDMT memang mencoba untuk mengimplementasikan kurikulum 2013 tetapi juga masih menggunakan kurikulum KTSP. Disamping itu SDMT juga mempunyai kurikulum sendiri sesuai kebutuhan SDMT antara lain dalam menanamkan karakter siswa di bidang agama, SDMT menerapkan sholat dhuha dan dzuhur berjamaah di masjid dan memberikan latihan khusus membaca Al-Quran untuk siswa.” Dari pemaparan ustadz Aziz dapat disimpulkan bahwa SDMT mencoba untuk mengimplementasikan kurikulum 2013 dengan kendala-kendala yang dihadapi dan keterbatasannya, namun dalam menunjang pembelajaran SDMT juga menggunakan kurikulum KTSP. Di samping itu SDMT memiliki kurikulum sendiri dalam pendidikan karakter untuk memenuhi kebutuhan sekolah. Hal senada juga disampaikan oleh Ustadz Imam Saiful Bahri sebagai berikut : “Kami mencoba mengimplementasikan kurikulum 2013, tetapi yang kita pakai adalah ruh dari kurikulum 2013 ini. Maksudnya adalah tidak murni kurikulum 2013 karena jika kita terapkan kita tidak akan punya branded di mata masyarakat mengingat hanya mengikuti pemerintah saja. Dalam metode dan struktur kurikulum kita beda dengan kurikulum 2013, tetapi dalam materi kita mengikuti yang disampaikan kurikulum 2013. Kurikulum 2013 kan banyak administrasinya, jika kita mengikuti murni menurut saya malah akan memberatkan guru dan siswa, maka kita pakai ruh dari kurikulum 2013 ini saja. SDMT menggunakan kurikulum yang sesuai untuk lingkungan sekolah SDMT sendiri dengan “Kami mencoba mengimplementasikan kurikulum 2013, tetapi yang kita pakai adalah ruh dari kurikulum 2013 ini. Maksudnya adalah tidak murni kurikulum 2013 karena jika kita terapkan kita tidak akan punya branded di mata masyarakat mengingat hanya mengikuti pemerintah saja. Dalam metode dan struktur kurikulum kita beda dengan kurikulum 2013, tetapi dalam materi kita mengikuti yang disampaikan kurikulum 2013. Kurikulum 2013 kan banyak administrasinya, jika kita mengikuti murni menurut saya malah akan memberatkan guru dan siswa, maka kita pakai ruh dari kurikulum 2013 ini saja. Aziz sebagai berikut : SDMT menggunakan kurikulum yang sesuai untuk lingkungan sekolah SDMT sendiri dengan ISTAWA: Jurnal Pendidikan Islam ISTAWA: Jurnal Pendidikan Islam 65 mengerucut pada analisa kebutuhan siswa dan pada zamannya.” Dari pemaparan Ustadz Imam Saiful Bahri dapat disimpulkan bahwa SDMT memulai untuk menerapkan kurikulum 2013, tetapi yang diterapkan adalah ruh dan esensi dari kurikulum 2013 karena menurut beliau penerapan kurikulum 2013 di SDMT tidak murni penuh kurikulum 2013, maksudnya adalah SDMT mempunyai pendekatan sendiri terkait penbinaan karakter siswa dan jika kurikulum 2013 diterapkan penuh di SDMT maka akan memberatkan guru dan siswa dengan banyaknya administrasi yang harus dipersiapkan. SDMT juga mempunyai metode dan struktur kurikulum yang berbeda dari kurikulum 2013 untuk memenuhi kebutuhan siswa, namun secara materi, dalam pembelajaran SDMT mengikuti materi kurikulum 2013 dari pemerintah. Dalam pendidikan karakter dan pengembangan sikap anak didik, SDMT mengakui menggunakan metode sendiri yang disesuaikan dengan kebutuhan dengan mengerucut pada ruh kurikulum 2013 bahwa kurikulum 2013 menggunakan pendekatan andragogi dan anak dituntut selalu aktif dalam pembelajaran tidak hanya sebagai objek. Hal itu tercermin dalam penerapan kebiasaan di sekolah seperti pada trankrip observasi sebagai berikut : “Kemudian mereka langsung berjabat tangan dengan guru- guru yang sudah bersiap menjadi penerima tamu di depan pintu gerbang dan berlari menuju kelas untuk menaruh tas-tas mereka. Pada jam 07.00 bel masuk sekolah berbunyi, peneliti menunggu sejenak. Tak berselang beberapa menit, banyak siswa SDMT keluar kelas dan berbondong-bondong menuju ke masjid, ternyata mereka melaksanakan sholat dhuha yang dipimpin oleh temannya sendiri secara terjadwal dan guru Volume 1, Nomor 2, Januari-Juni 2016 66 menjadi pembina dan pembimbing jika ada anak yang kurang benar baik dari bacaan sholat maupun gerakan sholatnya.” Dari hasil observasi yang dipaparkan dapat disimpulkan bahwa dalam membina akhlak siswa dan memberikan pemahaman yang luas serta pembiasaan agama SDMT sudah menerapkan pembiasaan berjabat tangan dengan guru pengasuh sebagai penanaman rasa hormat kepada guru.Di samping itu pembinaan sholat dilakukan dengan sholat dhuha sebagai wahana belajar siswa dan guru sebagai pembinanya. Pemaparan lain yang senada juga ditemui pada hasil observasi selanjutnya sebagai berikut. “Pada saat pembelajaran berlangsung di kelas, suasana yang ditawarkan oleh guru sangat menarik. Pertama guru masuk ke kelas dan mengucapkan salam. Kemudian melakukan absensi dengan diselingi canda tawa kepada siswa yang membuat kelas sejenak keluar suara tawa yang riuh dan ramai.Sebelum dilanjutkan pembelajaran para siswa diharuskan untuk tahsin terlebih dahulu. Aziz sebagai berikut : Barulah setelah itu pembelajaran dilanjutkan dengan diawali guru bertanya pelajaran apa yang sudah dibahas kemarin dan dilanjutkann anak disuruh untuk membuka buku-buku mereka. Di kelas yang lain guru PAI menerapkan tugas penelitian kepada anak didik untuk mencari tahu pengertian tentang sebuah kata dan ditanyakan kepada dewan guru di sekolah. Pada kegiatan ini guru yang ditanya sama sekali tidak merasa terbebani atau menjadi masalah untuk sejenak melayani anak yang bertanya. Pada saat menjelaskan guru tidak memberitahu artinya secara langsung, namun memberi pengarahan tentang pengertian arti tersebut dan peserta didik sendiri yang membuat kalimat yang tepat degan bermodal penjelasan dari guru informan.” Pemaparan di atas dapat disimpulkan bahwa dalam suasana pembelajaran SDMT sudah mengacu pada kurikulum 2013 dengan guru bertanya terlebih dahulu terhadap murid kemudian dilanjutkan dengan penjelasan guru.Dalam pembelajaran SDMT sudah ISTAWA: Jurnal Pendidikan Islam 67 menggunakan metode penelitian kepada siswa dengan siswa diberi tugas untuk mencari tahu dan mendefinisikan arti sebuah kata dengan narasumber dari ustadz dan ustadzah yang mereka temui.Pada kesempatan ini anak dituntut untuk menggunakan nalar kritis mereka dalam mengasosiasi dan mendefinisikan arti. Dilihat dari aspek sikap anak dituntut untuk mencari tahu cara dalam berkomunikasi dan menerapkan sopan santun terhadap guru sebagai informan untuk mencari data. Kemudian setelah itu barulah siswa diharuskan presentasi hasil dari penelitian dan mengkomunikasikan hasil kepada teman satu kelasnya. Dalam hasil observasi lain ditemukan bahwa SDMT mempunyai cara tersendiri dalam mengembangkan kompetensi PAI di lingkungan sekolah dan mewadahi minat dan bakat siswa khususnya PAI dengan memberikan ekstrakurikuler pembinaan membaca Al Quran, Tartil Al Quran dan Qiroah seperti dalam hasil observasi sebagai berikut : “Ketika suasana sudah reda saya masuk ke sekolah, ternyata masih banyak siswa yang belum pulang. Setelah saya tanya ternyata mereka mengikuti kegiatan extra. Kegiatan extra yang berlangsung pada saat itu adalah pembinaan iqro, tartil Al- Quran dan robotik. Pembinaan Iqro dilakukan untuk menunjang cara membaca Al-Quran kepada peserta didik yang memerlukannya karena anak didik yang tidak bisa membaca akan ketinggalan dari teman sebayanya mengingat salah satu indikator keberhasilan sekolah adalah setiap peserta didik mampu membaca Al-Quran dengan baik dan tartil.” “Ketika suasana sudah reda saya masuk ke sekolah, ternyata masih banyak siswa yang belum pulang. Setelah saya tanya ternyata mereka mengikuti kegiatan extra. Kegiatan extra yang berlangsung pada saat itu adalah pembinaan iqro, tartil Al- Quran dan robotik. Aziz sebagai berikut : Pembinaan Iqro dilakukan untuk menunjang cara membaca Al-Quran kepada peserta didik yang memerlukannya karena anak didik yang tidak bisa membaca akan ketinggalan dari teman sebayanya mengingat salah satu indikator keberhasilan sekolah adalah setiap peserta didik mampu membaca Al-Quran dengan baik dan tartil.” Dari hasil observasi ini dapat disimpulkan bahwa SDMT sangat peduli terhadap PAI dengan menyelenggarakan pembinaan Iqro bagi yang membutuhkan dan pembinaan tartil Al Quran untuk yang berminat. Dalam kurikulum 2013 pendidikan agama dan budi Volume 1, Nomor 2, Januari-Juni 2016 Volume 1, Nomor 2, Januari-Juni 2016 68 pekerti memberikan indikator bahwa siswa mampu memahami Islam, mempu mengamalkannya dalam kehidupan sehari-hari, mampu menghayatinya dan mampu menghargai orang lain dalam artian mempunyai budi pekerti yang luhur sehingga jika dikorelasikan dengan pemaparan hasil observasi di atas SDMT mampu mewadahi semua yang terangkum dalam kurikulum 2013 khususnya dalam bidang PAI. Berdasarkan hasil penelitian yang sudah tersebut di atas dapat disimpulkan bahwa SD Muhammadiyah terpadu ponorogo sudah menerapkan kurikulum 2013 pada mata pelajaran PAI dengan pendekatan dan metode yang dipakai oleh SDMT sendiri yang sepaham dengan kurikulum 2013 ditinjau dari tujuan kurikulum 2013 yaitu terbentuknya peserta didik yang memiliki akhlak yang mulia (budi pekerti yang luhur), yang merupakan misi utama diutusnya Nabi Muhammad saw di dunia. SDMT mengakui bahwa belum menerapkan sepenuhnya kurikulum 2013 secara penuh karena ditinjau dari sisi administrasi dan penilaian yang di rasa ribet dan membebani guru dan siswa sehingga yang diimplementasikan dari kurikulum 2013 adalah ruh dari kurikulum 2013 yaitu tematik integratif khususnya pada mata pelajaran PAI. Hal ini dibuktikan dengan hasil temuan observasi peneliti bahwa SDMT menggunakan pendekatan pembelajaran andragogi yang diterapkan dalam penugasan kepada siswa dalam bentuk penelitian/wawancara kepada ustadz dan ustadzah yang bila disimpulkan terdapat muatan-muatan pokok kurikulum 2013 yang dipakai yaitu mengamati, menanya, mengumpulkan, menalar/mengasosiasi, dan mengkomunikasikan. Akan tetapi dalam melakukan penilaian masih menggunakan penilaian berbasis KTSP, ISTAWA: Jurnal Pendidikan Islam ISTAWA: Jurnal Pendidikan Islam 69 terbukti dengan rapor dari SD Muhammadiyah Terpadu yang masih belum menggunakan penilaian autentik seperti pada kurikulum 2013. Penilaian yang dilakukan masih bersifat global dan belum spesifik. Ponorogo Dalam Implementasi kurikulum yang masih baru tentunya ada berbagai kendala dan dampak dari implementasinya seperti yang dilakukan SDMuhammadiyah Terpadu Ponorogo dalam penerapan kurikulum 2013. Ustadz Aziz dalam wawancara dengan peneliti mengatakan bahwa kurikulum 2013 masih perlu penyempurnaan dan banyak kendala yang dihadapi seperti pada hasil wawancara sebagai berikut: “Kurikulum 2013 itu juga kurikulum baru dan masih diujicobakan, tentunya juga masih perlu penyempurnaan dan banyak kendala antara lain kesiapan guru dan kemampuan guru dalam memahami kurikulum 2013 sendiri juga masih kurang. Bagaimana menerapkan penuh jika paham saja belum. Disamping itu perangkat-perangkat yang digunakan juga masih simpang siur belum kami terima secara utuh, jadi guru harus aktif mencari sendiri bahan untuk kurikulum 2013” “Kurikulum 2013 itu juga kurikulum baru dan masih diujicobakan, tentunya juga masih perlu penyempurnaan dan banyak kendala antara lain kesiapan guru dan kemampuan guru dalam memahami kurikulum 2013 sendiri juga masih kurang. Bagaimana menerapkan penuh jika paham saja belum. Disamping itu perangkat-perangkat yang digunakan juga masih simpang siur belum kami terima secara utuh, jadi guru harus aktif mencari sendiri bahan untuk kurikulum 2013” Dari pemaparan ustadz Aziz dapat disimpulkan bahwa kendala yang dihadapi dari SDMT adalah mengenai pemahaman guru terhadap kurikulum 2013 masih kurang sehingga masih kesulitan jika diterapkannya kurikulum 2013. Disamping itu dilihat dari segi perangkat yang masih belum jelas dan simpang siurnya informasi mengenai kurikulum 2013 karena buku pedoman dari pemerintah yang belum diterima menambah kesulitan dalam implementasi Volume 1, Nomor 2, Januari-Juni 2016 Volume 1, Nomor 2, Januari-Juni 2016 70 kurikulum yang mengharuskan guru untuk aktif dalam mencari pedoman dan bahan kurikulum 2013. Hal senada juga diungkapkan oleh ustadz Imam Saiful Bahri sebagai berikut : “Pola kurikulum yang kita pakai menurut kami lebih bisa menjawab tantangan zamandan kita tidak kesulitan dalam menerapkan kurikulum 2013 karena yang kita pakai adalah ruh dari kurikulum 2013 ini dan itu cocok dengan yang kita terapkan. Membahas kurikulum 2013 jika dilihat di SDMT kendalanya adalah tataran operasional yang ribet dengan segudang perangkatnya bahkan sampai pada penilaiannya.” Dari pemaparan ustadz Saiful Bahri dapat disimpulkan bahwa kurikulum 2013 banyak kendala dalam administrasinya sehingga dalam operasionalnya terkesan ribet dan dengan penilaian yang banyak menjadikan kurikulum 2013 seakan penuh dengan keribetan. Ponorogo Hal ini juga diungkapkan oleh guru PAI yaitu ustadz Farid yang mengatakan ribetnya administrasi kurikulum 2013 sebagai berikut : ”Sama sekali tidak ada kendala, hanya perangkatnya yang belum siap dan ribet saja.” Dari uraian ustadz Farid sebagai guru PAI dapat disimpulkan bahwa sebenarnya tidak ada kendala yang dihadapi dalam implementasi kurikulum 2013 di PAI, namun ketidaksiapan perangkat dan kesan ribet menjadikan implementasi kurikulum 2013 belum diimplementasikan secara penuh. Pemaparan hasil wawancara lain juga ditemui hal senada dari ustadzah St. Rohmatin Kusbah,S. HI sebagai berikut : “Karena masih belum begitu paham betul dengan kurikulum 2013, dan yang jelas bukunya belum ada. Di SDMT masih belum diterapkan secara penuh kurikulum 2013 dan kita masih bercampur dengan KTSP dalam kita mengajar anak-anak.” “Karena masih belum begitu paham betul dengan kurikulum 2013, dan yang jelas bukunya belum ada. Di SDMT masih belum diterapkan secara penuh kurikulum 2013 dan kita masih bercampur dengan KTSP dalam kita mengajar anak-anak.” ISTAWA: Jurnal Pendidikan Islam ISTAWA: Jurnal Pendidikan Islam 71 Dari pemaran ustadzah St. Rohmatin Kusbah dapat disimpulkan bahwa SDMT masih mencampurkan kurikulum KTSP dan kurikulum 2013 dalam pembelajarannya karena terkait beberapa kendala yaitu masih kurangnya pemahaman tentang kurikulum 2013 dan dengan belum jelasnya buku dari kurikulum 2013 yang belum diterima dari pemerintah. Hal ini menyulitkan dalam impelementasi kurikulum 2013. Di samping kendala-kendala yang dipaparkan diatas terdapat beberapa dampak dalam implementasi kurikulum 2013 seperti yang diungkapkan oleh ustadz Aziz bahwa kurikulum 2013 memberatkan guru sehingga beban kerja guru menjadi berat dengan segala administrasi kurikulum 2013 yang harus dipersiapkan seperti pada pada paparan hasil wawancara sebagai berikut : “Sebenarnya implementasi kurikulum 2013 tidak berdampak signifikan terhadap guru, tetapi memang guru lebih berat beban kerjanya karena banyaknya perangkat yang harus dipersiapkan sebelum pembelajaran.Penambahan jam pelajaran sangat menguntungkan bagi guru PAI yang 2 jam menjadi 3 jam per minggunya. Disamping itu, guru juga terlalu banyak melakukan penilaian, sehingga sangat membebani guru. Karena menurut saya dalam aspek sikap itu tidak dinilai secara angka, tapi penilaiannya langsung dengan ditegur dan diingatkan sehingga karakter benar-benar terbangun.” “Sebenarnya implementasi kurikulum 2013 tidak berdampak signifikan terhadap guru, tetapi memang guru lebih berat beban kerjanya karena banyaknya perangkat yang harus dipersiapkan sebelum pembelajaran.Penambahan jam pelajaran sangat menguntungkan bagi guru PAI yang 2 jam menjadi 3 jam per minggunya. Disamping itu, guru juga terlalu banyak melakukan penilaian, sehingga sangat membebani guru. Ponorogo Karena menurut saya dalam aspek sikap itu tidak dinilai secara angka, tapi penilaiannya langsung dengan ditegur dan diingatkan sehingga karakter benar-benar terbangun.” Dari hasil wawancara dengan ustadz Aziz dapat disimpulkan bahwa dampak dari implementasi kurikulum 2013 memberikan beban kepada guru pengajar karena banyaknya administrasi pembelajaran yang dipersiapkan namun dengan penambahan jam pelajaran yang diterapkan kurikulum 2013 tidak menjadi masalah justru menguntungkan dengan bertambahnya jam pelajaran yang dalam KTSP dari 2 jam pelajaran sekarang menjadi 3 jam pelajaran. Volume 1, Nomor 2, Januari-Juni 2016 Volume 1, Nomor 2, Januari-Juni 2016 72 Disamping itu dalam kurikulum 2013 terlalu banyak penilaiannya sehingga guru merasa terbebani mengingat penilaian autentik adalah penilaian yang berbasis fakta baik dari ranah sikap, spiritual, pengetahuan, dan keterampilan sehingga siswa dinilai dari semua aspek. Dari pemaparan ustadz Aziz diatas dapat disimpulkan juga bahwa ustadz Aziz tidak sepakat dengan penilain aspek sikap dengan menggunakan angka karena aspek sikap seharusnya tidak dinilai dengan angka namun disikapi dengan teguran dan nasihat kepada siswa yang melakukan kesalahan atau perlu bimbingan dalam budi pekertinya yang kurang baik.Jika ditinjau dari sudut pandang siswa terhadap implementasi kurikulum 2013 terdapat beberapa dampak yang dirasakan seperti yang dipaparkan oleh ustadz Aziz sebagai berikut : “Jika terhadap siswa banyak, mulai dari buku siswa yang belum ada sehingga menyulitkan dalam belajar juga salah satunya adalah menekan siswa. Karena dalam kurikulum 2013 siswa dituntut untuk aktif, sangat aktif baik dari belajar maupun dalam memperoleh nilai. La itu penilainnya yang banyak berarti juga berdampak pada anak untuk memperoleh nilai baik.” Dari paparan ustadz Aziz dapat disimpulkan bahwa siswa kesulitan dalam belajarnya karena buku yang belum ada dan terlebih lagi bahwa dalam kurikulum 2013 siswa dituntut untuk aktif baik dalam belajarnya dan juga dalam memperoleh nilai.Karena dalam kurikulum 2013 terdapat banyak sekali indikator penilaian sehingga konsekuensinya apabila siswa ingin mempunyai nilai yang baik maka siswa dituntut dengan harus mampu menguasai indikator penilaian tersebut secara menyeluruh baik kognitif sampai kepada ISTAWA: Jurnal Pendidikan Islam ISTAWA: Jurnal Pendidikan Islam 73 afektifnya.Dari banyaknya indikator penilaian ini siswa merasa terbebani dan merasa berat. Hal senada juga diungkapkan oleh ustadzah St. Rohmatin Kusbah, S. HI bahwa siswa terbebani dengan banyaknya penilaian dan tugas seperti pada hasil wawancara sebagai berikut : “Lebih membebani siswa, dengan banyaknya penilaian dan tugas maka banyak tugas yang tidak dikerjakan oleh siswa. Dan siswa seperti dipaksa-paksa.”. Volume 1, Nomor 2, Januari-Juni 2016 Ponorogo berdasarkan wawancara tersebut maka peneliti dapat menyimpulkan bahwa dalam kurikulum 2013 terdapat banyak penilaian dan tugas yang diberikan sehingga membebani siswa dan berdampak pada banyak tugas siswa yang tidak dikerjakan. Hal serupa juga dirasakan oleh wali murid terhadap perubahan belajar putra-putrinya seperti pada wawancara berikut : “Anak saya banyak tugas.” Dari pemaparan wali murid dapat disimpulkan bahwa implementasi kurikulum 2013 memberikan beban dengan banyaknya tugas yang dikerjakan siswa. Hal serupa juga dirasakan oleh siswa seperti pada wawancara sebagai berikut :“Banyak tugas dan bukunya belum ada”. Pemaparan lain diungkapkan dalam hasil wawancara peneliti kepada wali murid terhadap dampak implementasi kurikulum 2013 kepada siswa SDMT seperti dalam hasil wawancara sebagai berikut : “Anak menjadi lebih aktif dalam mencari bahan dan selanjutnya berani berdiskusi secara kekeluargaan.” Dari hasil wawancara peneliti dan wali murid dapat disimpulkan bahwa setelah implementasi kurikulum 2013 di SDMT terdapat perubahan terhadap siswa dalam tingkah laku belajarnya. Disampaikan bahwa anak lebih aktif dalam mencari bahan belajarnya dan melakukan diskusi, ini menyatakan bahwa minat belajar dan Volume 1, Nomor 2, Januari-Juni 2016 Volume 1, Nomor 2, Januari-Juni 2016 74 penalaran siswa naik menjadi lebih kritis dan mampu menalar dan mengkomunikasikan apa yang didapatnya. Berdasarkan semua uraian diatas dapat disimpulkan bahwa Implementasi Kurikulum 2013 tidak berdampak signifikan pada pembelajaran di SD Muhammadiyah Terpadu Ponorogo hanya penambahan jam pelajaran yang bertambah menjadi tiga jam pelajaran dalam seminggunya justru menguntungkan mengingat SDMT adalah sekolah yang mengunggulkan pembinaan budi pekerti dan Agama Islam. Namun jika ditinjau dari dampak terhadap guru adalah guru merasa keberatan dan merasa terbebani dalam implementasi kurikulum 2013 dengan jumlah perangkat yang ribet dan banyak yang harus dipersiapkan. Jika ditinjau dari siswa, dampak yang dirasakan adalah siswa menjadi lebih aktif mengingat kurikulum 2013 memposisikan siswa sebagai subjek pembelajaran namun di lain sisi merasa berat dengan banyaknya tugas untuk mencukupi penilaian. Faktor Pendukung dan Penghambat Implementasi Kurikulum 2013 Pada Mata Pelajaran Pendidikan Agama Islam di SD Muhammadiyah Terpadu Ponorogo Implementasi kurikulum 2013 disamping merupakan hal yang baru diterapkan dengan berbagai tantangan dan hambatan namun tentunya ada yang mengakibatkan hal tersebut menjadi halangan dan ada yang menjadi pendukung dalam implementasi kurikulum 2013. Implementasi kurikulum 2013 di SDMT terdapat beberapa kesulitan dan ada pula yang mendukung dalam implementasinya sehingga memudahkan dalam mengimplementasikan kurikulum 2013 dan yang menghambat seperti yang diutarakan oleh ustadz Aziz sebagai berikut: ISTAWA: Jurnal Pendidikan Islam ISTAWA: Jurnal Pendidikan Islam 75 “Jumlah jam pelajaran yang padat, penilaian ribet, disamping itu materi PAI jam nya terlalu sedikit padahal di SDMT PAI itu menjadi idola sekolah sehingga kami tidak menggunakan K13 dengan secara utuh.” Dari pemaparan ustadz Aziz dapat disimpulkan bahwa hal yang menghambat dalam implementasi kurikulum 2013 adalah jumlah jam pada mata pelajaran PAI yang sedikit yang dirasa kurang dalam pembelajaran PAI pada anak padahal PAI adalah ikon sekolah dan menjadi mata pelajaran idola di SDMT. Disamping itu menurut ustadz Aziz bahwa kurikulum 2013 penilaiannya terlalu ribet.Hal senada juga disampaikan oleh ustadz Imam Saiful Bahri dalam wawancara sebagai berikut. “Yang mendukung jumlah jamnya saja yang berubah, yang menghambat adalah perangkatnya yang banyak dan ribet kemudian penilaian yang ribet. Kalo dalam pelajaran PAI menurut kami kurikulum 2013 ketinggalan, karena begini PAI di SDMT menjadi sebuah idola.” “Yang mendukung jumlah jamnya saja yang berubah, yang menghambat adalah perangkatnya yang banyak dan ribet kemudian penilaian yang ribet. Kalo dalam pelajaran PAI menurut kami kurikulum 2013 ketinggalan, karena begini PAI di SDMT menjadi sebuah idola.” Dari hasil wawancara dengan ustadz Imam Saiful Bahri dapat disimpulkan bahwa hal yang mendukung adalah tentang perubahan jam yang dulunya PAI mempunyai jatah 2 jam pelajaran per minggunya sekarang berubah menjadi 3 jam per minggunya. Tetapi yang menghambat adalah perangkat dan penilainnya yang dirasa ribet sehingga memberatkan guru dan siswa dalam implementasi kurikulum 2013.Beliau menambahkan bahwa kurikulum 2013 khususnya PAI jika diterapkan di SDMT dirasa sudah ketinggalan. Dalam pemaparan lain diungkapkan dari ustadz Farid bahwa yang menghambat dalam implementasi kurikulum 2013 adalah keberadaan buku yang masih belum diterima seperti pada wawancara sebagai berikut :“Hanya buku-bukunya sajadan kita sudah punya buku sendiri untuk PAI hasil Volume 1, Nomor 2, Januari-Juni 2016 Volume 1, Nomor 2, Januari-Juni 2016 76 karya dari ustadz-ustadzah SDMT sendiri”. Dari pemaparan ustadz Farid tersebut dapat disimpulkan bahwa SDMT sudah mempunyai buku sendiri untuk mata pelajaran PAI dari hasil karya ustadz- ustadzah SDMT sendiri. ISTAWA: Jurnal Pendidikan Islam Faktor Pendukung dan Penghambat Implementasi Kurikulum 2013 Pada Mata Pelajaran Pendidikan Agama Islam di SD Muhammadiyah Terpadu Ponorogo Pada kesempatan yang sama ustadz Farid mengatakan bahwa implementasi kurikulum 2013 di SDMT sudah diterapkan dengan beberapa hal yang mendukung seperti wawancara sebagai berikut :“Karena memang sudah sepaham dengan kurikulum 2013 maka dampaknya adalah seperti yang kita harapkan”. Dari wawancara tersebut dapat disimpulkan bahwa hal yang mendukung dari implementasi kurikulum 2013 adalah SDMT sudah sepaham dengan kurikulum 2013 khususnya di mata pelajaran PAI dengan pendekatan pengembangan karakter siswa sehingga tidak kesulitan dalam implementasinya. Hal senada juga disampaikan oleh ustadz Imam Saiful Bahri dalam wawancara sebagai berikut : Saiful Bahri dalam wawancara sebagai berikut : “Pola kurikulum yang kita pakai menurut kami lebih bisa menjawab tantangan zaman dan kita tidak kesulitan dalam menerapkan kurikulum 2013 karena yang kita pakai adalah ruh dari kurikulum 2013 ini dan itu cocok dengan yang kita terapkan. Membahas kurikulum 2013 jika dilihat di SDMT kendalanya adalah tataran operasional yang ribet dengan segudang perangkatnya bahkan sampai pada penilaiannya” Dari pemaparan ustadz Imam Saiful Bahri dapat disimpulkan bahwa SDMT tidak kesulitan dalam implementasi kurikulum 2013 karena yang dipakai adalah ruh dari kurikulum 2013 dan itu sepaham dan sejalan dengan apa yang sudah diterapkan di SDMT. Disamping itu jumlah jam pelajaran yang bertambah menjadi menguntungkan untuk SDMT sehingga bisa disimpulkan penambahan jam dalam mata pelajaran PAI menjadi faktor pendukung dalam implementasi ISTAWA: Jurnal Pendidikan Islam ISTAWA: Jurnal Pendidikan Islam 77 kurikulum 2013. Faktor pendukung lain adalah keberadaan internet sebagai lahan belajar dan pusat informasi siswa. Karena dalam implementasi kurikulum 2013 siswa dituntut untuk aktif sehingga harus mencari bahan belajar sendiri. Disamping faktor pendukung adalah adanya faktor penghambat dari implementasi kurikulum 2013 antara lain adalah tentang keberadaan buku yang masih belum diterimakan dari pemerintah. Disamping itu kesiapan guru dalam implementasi kurikulum 2013 masih kurang, baik dari sisi pemahaman dan perangkat pembelajarannya.Pemaparan dari ustadzah St. Rohmatin Kusbah, S.HI mengungkapkan bahwa hal yang menghambat dari implementasi kurikulum 2013 adalah tentang perangkat-perangkat yang terlalu banyak dan dirasa membebani guru seperti dalam wawancara sebagai berikut :“Perangkat-perangkat yang terlalu banyak membebani guru dan buku-buku panduan yang belum lengkap.”Maka dapat disimpulkan bahwa dalam implementasi kurikulum 2013 kendala yang dirasa adalah karena buku-buku panduan yang belum lengkap dan perangkat yang banyak. Wali murid dari siswa SDMT merasakan hal yang menghambat dalam proses belajar putra-putrinya seperti wawancara sebagai berikut:“Faktor yang mendukung yang diperlukan adalah fasilitas. Volume 1, Nomor 2, Januari-Juni 2016 Faktor Pendukung dan Penghambat Implementasi Kurikulum 2013 Pada Mata Pelajaran Pendidikan Agama Islam di SD Muhammadiyah Terpadu Ponorogo Misal internet,karena anak saya jika tidak tahu dia akan mencarinya di Internet.”Dari hasil wawancara dengan wali murid tersebut dapat disimpulkan bahwa diperlukannya fasilitas dalam implementasi kurikulum 2013 yaitu adanya internet untuk menunjang proses belajar siswa karena siswa dituntut untuk aktif dalam mencari bahan belajarnya. Volume 1, Nomor 2, Januari-Juni 2016 Volume 1, Nomor 2, Januari-Juni 2016 78 Penutup Secara umum SD Muhammadiyah Terpadu Ponorogo telah mengimplementasikan pendekatan kurikulum 2013 pada mata pelajaran Pendidikan Agama Islam akan tetapi mengalami kesulitan dalam administrasinya. SD Muhammadiyah Terpadu Ponorogo menggunakan metode dan pendekatan pembelajaran yang diterapkan pada kurikulum 2013 yaitu dengan menggunakan standar : (1) Mengamati (2) Menanya (3) Mengumpulkan (4) Menalar/mengasosiasi (5) Mengkomunikasikan. Pendekatan pembelajaran Pendidikan Agama Islam yang digunakan adalah pembelajaran berbasis pengalaman langsung langsung dengan melakukan dan merasakan proses dari materi yang diajarkan. Pembelajaran Pendidikan Agama Islam menerapkan pembelajaran tematik integratif dengan melakukan integrasi materi Pendidikan Agama Islam terhadap materi pelajaran lain dengan menyesuaikan tema. Sistem Penilaian yang diterapkan SD Muhammadiyah Terpadu belum menggunakan penilaian autentik seperti pada kurikulum 2013, namun masih menggunakan penilaian berbasis kurikulum KTSP. (4) Implementasi kurikulum 2013 terhadap pembelajaran Pendidikan Agama Islam di SD Muhammadiyah Terpadu Ponorogo tidak berdampak signifikan karena sebelumnya SD Muhammadiyah Terpadu telah menerapkan pembelajaran yang mengarah pada kurikulum 2013 sehingga tidak terdapat kesulitan dalam mengimplementasikannya. Perubahan jam pelajaran Pendidikan Agama Islam dari 2 jam pelajaran per minggu menjadi 3 jam pelajaran per minggu, menjadi keuntungan SD Muhammadiyah Terpadu karena basis sekolah yang lebih fokus kepada pendidikan Agama Islam. 116 ISTAWA: Jurnal Pendidikan Islam ISTAWA: Jurnal Pendidikan Islam 79 Faktor pendukung dari implementasi kurikulum 2013 di SDMT pada mata pelajaran PAI adalah karena metode dan esensi dari kurikulum 2013 sepaham dengan kebutuhan pembelajaran di SDMT. Disamping itu jam pelajaran yang bertambah perminggunya menjadi lebih menguntungkan bagi SDMT. Ditinjau dari fasilitas terdapat hal yang mendukung dalam implementasi kurikulum 2013 yaitu keberadaan internet sebagai lahan belajar siswa dalam mencari bahan belajarnya sehingga siswa bisa mandiri dalam belajar dengan menggunakan internet.Ditinjau dari faktor penghambat dalam implementasi kurikulum 2013 di SDMT adalah terkait perangkat yang ribet dan banyak sehingga menjadikan guru keberatan dan membebani.Disamping itu buku-buku yang belum diterima menjadi salah satu penghambat dari implementasi kurikulum 2013 SD Muhammadiyah Terpadu Ponorogo. ISTAWA: Jurnal Pendidikan Islam Daftar Pustaka Aquina, Dwifantya.dkk. 2013.Kurikulum 2013 Waktu Pelajaran Agama Ditambah,http://nasional.news.viva.co.id/news/read/413090, (diakses pada 09 Juni 2013 pukul 20:29:47 WIB). Arifin, Zaenal. 2011. Konsep dan Model Pengembangan Kurikulum, Bandung:PT. Remaja Rosdakarya. Indratno, Fery. (Ed.). 2013. Menyambut Kurikulum 2013, Jakarta: PT Kompas Media Nusantara. Mulyasa. H.E. 2013.Pengembangan Implementasi Kurikulum 2013.Bandung : PT. Remadja Rosdakarya. Muzamiroh, Mida L.Kupas Tuntas Kurikulum 2013 Kelebihan dan Kekurangan Kurikulum 2013,(Kata Pena,2013) Volume 1, Nomor 2, Januari-Juni 2016 80 Slameto. Proses Belajar Mengajar dalam Sistem Kredit Semester. Jakarta: Bumi Aksara Slameto. Proses Belajar Mengajar dalam Sistem Kredit Semester. Jakarta: Bumi Aksara Tim Kualita Pendidikan Indonesia.2013.Modul Pelatihan Pendekatan Saintifik dan Penilaian Kurikulum 2013. Surabaya: Kualita Pendidikan Indonesia. Tim Kualita Pendidikan Indonesia. 2013. Modul Pelatihan Pembelajaran Tematik Kurikulum 2013, Surabaya: Kualita Pendidikan Indonesia.
https://openalex.org/W3200716455	https://repositorio.ipcb.pt/bitstream/10400.11/7742/1/19148-59065-1-PB.pdf	English	null	Power saving MAC protocols in wireless sensor networks: a survey	Telkomnika	2,021	cc-by-sa	6,592	TELKOMNIKA Telecommunication, Computing, Electronics and Control Vol. 19, No. 6, December 2021, pp. 1778~1786 ISSN: 1693-6930, accredited First Grade by Kemenristekdikti, Decree No: 21/E/KPT/2018 DOI: 10.12928/TELKOMNIKA.v19i6.19148 TELKOMNIKA Telecommunication, Computing, Electronics and Control Vol. 19, No. 6, December 2021, pp. 1778~1786 ISSN: 1693-6930, accredited First Grade by Kemenristekdikti, Decree No: 21/E/KPT/2018 DOI: 10.12928/TELKOMNIKA.v19i6.19148 Keywords: Energy efficiency MAC protocols Survey Wireless sensor networks This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. Corresponding Author: Vasco N. G. J. Soares Instituto Politécnico de Castelo Branco Escola Superior de Tecnologia Campus da Talagueira, Avenida do Empresário, 6000-767 Castelo Branco, Portugal Email: vasco.g.soares@ipcb.pt Power saving MAC protocols in wireless sensor networks: a survey Rafael Souza Cotrim1, João M. L. P. Caldeira2, Vasco N. G. J. Soares3, Youcef Azzoug4 1,2,3Instituto Politécnico de Castelo Branco, Castelo Branco, Portugal 2,3Instituto de Telecomunicações, Covilhã, Portugal ç g 4Department of Informatics, University of Science and Technology Houari Boumediene, Algiers, Algeria ABSTRACT In a wireless sensor network, energy is almost always the greatest limitation. Energy sources are restricted in many of the environments where nodes are deployed, limiting them to the use of batteries for power. Therefore, conserving energy is supremely important, however, such a task poses many challenges to hardware and protocol design. One of the greatest problems faced is reducing the energy consumption of the communications systems, which represents a substantial amount of the total consumption. This paper surveys the most recent schemes designed to reduce the communications module energy consumption with a focus on novel MAC protocols for ad-hoc wireless sensor networks. It initially describes the many challenges involved, then it analyses each protocol individually. Finally, the presented protocols are compared and the issues that remain open are raised for further research. Journal homepage: http://journal.uad.ac.id/index.php/TELKOMNIKA 1. INTRODUCTION With the world’s increasing reliance on technology and its integration in every aspect of our lives, subjects relating to internet of things (IoT) [1] have increasingly become more relevant. Wireless sensor networks (WSNs) [2] in particular have been used in a variety of applications such as monitoring patients in real-time inside healthcare facilities [3], enhancing emergency response [4], control lighting in urban scenarios [5], monitoring electric grids, and many others [6], [7]. A WSN is a network comprised of sensor nodes that use radio transmission to communicate with each other and gather data [8]. Such structures allow the collection of data from multiple spatially separated points at once and can be adapted to each specific situation. The properties it can measure include temperature, pressure, sound, pollution, and so on. Most WSN nodes are made to be cheap and small, containing sensors, communications, processing, and power modules. However, the specifics of the environment where they will be deployed significantly affect the node’s composition, capabilities, restrictions, and price. The design of a WSN faces many challenges [9]–[14], however, in general, power consumption is the greatest one [15]. Some applications allow for power generation on-site with solar panels and alike, but these produce a limited amount of power and do not work in all situations. Others require nodes to be fully battery-operated, with some kinds of nodes being discarded once the battery is depleted. Therefore, reducing energy consumption is a priority in WSNs. Data processing requires very little power in general, but the Journal homepage: http://journal.uad.ac.id/index.php/TELKOMNIKA TELKOMNIKA Telecommun Comput El Control 1779  1779   transmission of the data acquired, and especially idle listening consumes a much greater share of the available energy [16]. transmission of the data acquired, and especially idle listening consumes a much greater share of the available energy [16]. There have been many techniques to reduce power consumption developed throughout the years. Duty cycling [17], separate communication channels to wake up nodes in low power mode [18], clustering protocols [19]–[21], reworking the MAC protocol [22], and many others [23]. This paper explores the literature for ways to extend network lifetime with a focus on novel MAC protocols. The rest of this paper is structured as follows: section 2 discusses the challenges involved in improving network lifetime while section 3 explains many of the MAC implementations available and their characteristics. 1. INTRODUCTION Section 4 analyzes the problems that remain unsolved and explore future research paths. Finally, section 5 concludes the paper. 2. INCREASING NETWORK LIFETIME IN WSNS There are many things to consider when trying to increase the lifetime of a WSN [24], but most implementation focuses on the communication system as it consumes the most energy. Transmitting a single bit of data can consume as much energy as thousands of operations [25] while receiving and idle listening are of the same magnitude; thus, such operations should be avoided whenever possible. To that end, many novel MAC protocols have been created. The MAC sublayer is a part of the Open Systems Interconnection Model (OSI) reference model. It controls how devices have access to the transmission medium which, in wireless networks, is shared by all devices, therefore rules need to be set to avoid interference. However, the separation of concerns provided by the OSI model is not ideal for WSNs, where optimization often supersedes modularity. As has been shown by previous research, adding information from different layers to the MAC protocol can greatly reduce energy consumption [26]. Because of that, some of the protocols discussed here may break OSI guidelines. The following are some of the challenges involved in reducing the communications system’s impact on battery life: − Power balancing − Power balancing Another factor to consider when designing a WSN is the fact that nodes consume more energy depending on their location and duties on the network. For example, route optimization causes traffic to be concentrated on certain paths. If this discrepancy is not accounted for, nodes in critical paths are likely to fail before the others, leading to loss of connectivity in entire sections of the network. Ideally, nodes would only fail when the entire network runs out of energy. This is such a concern that some WSN applications [29], [30] use minimum node lifetime as one of their main statistics. Considering the remaining energy available for a node (also called residual energy) when making decisions can slow down the growth of the energy gap between nodes but does not stop it. To achieve a better balance, protocols can shift their overhead between sender and receiver, leading one to spend more energy than the other. − Mobility support − Collision avoidance Interference between transmissions is a concern in all shared medium networks, but they carry an additional penalty in WSNs. Retransmitting data costs energy and reduces the total network lifetime, therefore it must be avoided as much as possible. This is usually implemented by a MAC protocol, but the IEEE 802.11 carrier sense multiple access/collision avoidance (CSMA/CA) implementation requires continuous idle listening. Alternative contention-based MAC implementations focus on reducing the transmission and listening times while keeping collisions to a minimum. Some MAC schemes utilize time-division multiple access (TDMA) to eliminate collision by giving devices specified time frames where they can transmit data. This is challenging to implement in ad-hoc networks since they require regular synchronization to prevent schedules from drifting and it introduces additional latency [16], but it provides great improvements and often can easily be adapted to include a sleep schedule. Other approaches use specialized preambles [27] or modification to the usual expectations of medium access control [28]. Both approaches eliminate the necessity of synchronization between nodes but come with their own set of challenges. − Duty cycling To avoid wasting energy while there is no data being transmitted, nodes should minimize the communications module’s active time. Usually, this is done by putting the transceiver in sleep (low power) mode. However, WSNs require inter-node collaboration to prevent areas of the network from losing connectivity when key nodes are inactive. It is also important to note that transitions between states can have associated costs [17]. In these scenarios reducing the total amount of transitions can further reduce energy consumption. Duty cycling can also increase the latency of the network and reduces maximum throughput, requiring extra added complexity to avoid these issues. − Collision avoidance 3.1. Sensor MAC (S-MAC) S-MAC is a MAC pr S-MAC is a MAC procedure specifically designed for WSNs that includes a sleep schedule. S-MAC divides time into fixed-size frames, each having an active and a sleeping part. Nodes are free to choose their own schedule, but a new node will attempt to follow the same schedule as its neighbors to avoid control overhead. If it can’t find any other nodes, it will choose an arbitrary timetable and broadcast it. Each node keeps a table of its neighbors’ schedules and, when it wants to send data, it simply waits for its destination to be in the active part of the frame. To avoid collisions S-MAC uses the same request to send/clear to send (RTS/CTS) messages present in the IEEE 802.11 specification, forcing nodes to compete for the medium. While this scheme greatly reduces node awake time, the active part of the frame must be big enough to cope with the highest throughput required. This means that S-MAC wastes energy when not working at full capacity. Another problem arises when there is more than one schedule on the network, in this situation virtual clusters are formed and nodes at the edges of each cluster consume more energy due to having to wait long periods to transmit to nodes using other schedules [22]. 3.2. Timeout MAC (T-MAC) T-MAC aims to reduce S-MAC’s energy wastage when not working at full capacity. It does so by dynamically modifying the length of S-MAC’s active portion of the frame. When a node becomes active, it will send all the stored data it has and waits for a short period (TA). If it receives no messages to forward in that time, the node will go back to its sleep phase. The proposed procedure reduces node active time when the network is not working at full capacity and increases it to meet demand during activity spikes. However, the energy consumed during a TA period where it does not receive messages is still wasted [37]. Figure 1 illustrates the differences between T-MAC and S-MAC. Figure 1. Comparison between S-MAC and T-MAC, adapted from [37] Figure 1. Comparison between S-MAC and T-MAC, adapted from [37] 3. MAC PROTOCOLS FOR WSNS Below the studied MAC protocols are briefly described, highlighting their benefits and flaws: − Mobility support Most networks change over time. In a WSN nodes can be added, removed deliberately, removed due to failure or battery depletion. These events are infrequent and represent what is called weak mobility. Most Power saving MAC protocols in wireless sensor networks: a survey (Rafael Souza Cotrim)  1780 ISSN: 1693-6930 wireless MAC protocols are built to handle such occurrences because they are a normal part of a network’s lifecycle [31]. However, that is not enough in many situations. Some applications require support for deliberate and frequently moving nodes with minimal loss of connectivity or delay. This is called strong mobility and protocols that support it not only consider a node's connections to other nodes but also its position, velocity, and other factors depending on the specific situation where they are applied [32]–[36]. wireless MAC protocols are built to handle such occurrences because they are a normal part of a network’s lifecycle [31]. However, that is not enough in many situations. Some applications require support for deliberate and frequently moving nodes with minimal loss of connectivity or delay. This is called strong mobility and protocols that support it not only consider a node's connections to other nodes but also its position, velocity, and other factors depending on the specific situation where they are applied [32]–[36]. 3.3. Berkeley MAC (B-MAC) y ( ) The B-MAC protocol allows nodes to have completely independent sleep/wakeup schedules by using a CSMA-based system to transmit data between themselves. When a node wants to send data, it precedes the package with a preamble slightly longer than the sleep time of the receiver. Eventually, the receiver node will wake up, sample the medium, and, if it notices a preamble, it will remain awake to receive the transmission once the preamble is done. B-MAC offers much better throughput and battery life when compared to S-MAC. However, transmitting the preamble necessary for low power listening (LPL) cost a substantial amount of energy to the sender, and all receivers that sample the medium and find a preamble must stay awake until the ELKOMNIKA Telecommun Comput El Control, Vol. 19, No. 6, December 2021: 1778 - 1786  1781 1781 TELKOMNIKA Telecommun Comput El Control  preamble is finished to determine if they are the recipients of the message, causing a problem known as overhearing [38]. preamble is finished to determine if they are the recipients of the message, causing a problem known as overhearing [38]. 3.4. X-MAC To improve on B-MAC’s preamble approach, X-MAC attempts to mitigate its biggest drawbacks. To reduce the inefficiency caused by overhearing, the extended preamble is broken into smaller preamble packages that contain the ID of the target node. When a node wakes up, samples the medium, and finds a preamble, it checks the ID transmitted with it. If it is not the intended receiver, it will go back to sleep, otherwise, it remains awake. As a result of this, overhearing nodes can go back to their sleep mode much more quickly. Preamble strobing, the periodic insertion of small pauses in the preamble, is used to prevent nodes from having to wait until the end of the preamble to send/receive data. Such pauses allow the receiver to send an early acknowledgment (ACK) message to indicate that the transmission may be initiated. The resulting protocol is much more efficient than B-MAC and has lower latency [27]. Figure 2 compares X-MAC to LPL, the process used by B-MAC. Figure 2. Comparison between LPL and X-MAC, adapted from [27] Figure 2. Comparison between LPL and X-MAC, adapted from [27] 3.5. BoX-MAC BoX-MAC-1 and BoX-MAC-2 are a pair of protocols that build upon B-MAC and X-MAC, respectively. They improve their predecessors by including data from other network layers into their structure. The first uses a predominantly layer 1 (physical) approach: It packetizes B-MAC’s long preamble and adds link-layer information, allowing nodes that are not the intended receiver of the message to return to their sleep mode earlier. The second uses a predominantly layer 2 mechanism that is very similar to X-MAC, but it replaces the package-based receive check with a layer 1 energy-based receive check. The resulting protocols are more energy-efficient than their predecessors, but each is better in specific situations. BoX-MAC-1 is more efficient in high volatility networks with little traffic while BoX-MAC-2 is better at handling high traffic with lower volatility [26].  1782 ISSN: 1693-6930 the sleep period where sender and receiver can communicate without collisions between data packages happening. As a result, DW-MAC has much lower latency and energy consumption, however, it does not specify a synchronization mechanism, requiring it to be implemented separately [16]. the sleep period where sender and receiver can communicate without collisions between data packages happening. As a result, DW-MAC has much lower latency and energy consumption, however, it does not specify a synchronization mechanism, requiring it to be implemented separately [16]. 3.7. Receiver initiated MAC protocol (RI-MAC) The RI-MAC flips the usual expectation of medium access control: The receiver checks for the existence of pending messages by sending a probe message (also known as a beacon). Senders with data to transmit listen for the probe, wait for it to finish, and then starts transmitting. By doing this, RI-MAC avoids having to send lengthy preamble messages like other asynchronous protocols do while using LPL. It also reduces the amount of time a pair of nodes occupy the medium before they reach rendezvous, which allows for more contending nodes to transmit, increasing potential throughput. Finally, overhearing is minimized because the receiver only expects to transmission within a short window after the beacon is sent [28]. 3.10. Asymmetric MAC (Asym-MAC) Asym-MAC was created to deal with the problems posed by asymmetric links to receiver-initiated MAC protocols such as RI-MAC and A-MAC. Asymmetric connections exist when the reliability or throughput of the channel in one direction is smaller than in the other direction. At manageable levels, asymmetry can increase the probability of losing probe messages, leading to delays and a lower package delivery ratio. At its extreme, it can lead to the communications channel essentially becoming unidirectional and completely prevent receiver-initiated MAC from working. Asym-MAC remediates this by employing a hybrid approach. By default, all links are in R-mode (receiver-initiated), however, if a sender fails to receive the probe multiple times, it will become T-mode (sender-initiated) and the sender will transmit its data before going back to R-mode. The receiver is notified of the change in mode using a clear channel assertion (CCA) period added at the end of each probing package. During this period, the receiver briefly checks for potential preamble packages, which signify the mode change. The more asymmetric a connection, the better Asym-MAC is when compared to other receiver-initiated MAC protocols. These improvements to package delivery ratio slightly increase Asym-MAC’s average energy consumption (by around 2.6%) when compared to A-MAC on low asymmetry scenarios, but it greatly improves network reliability and delay times. Asym-MAC is also more energy-efficient than sender-initiated protocols like BoX-MAC [41]. Figure 3 illustrates Asym-MAC’s mode transition. 3.8. Lifetime balance MAC (LB-MAC) LB-MAC allows pairs of nodes to adjust MAC behavior to optimize network lifetime. Neighbor nodes communicate and shift the communication overhead to the one with the longest expected lifetime. The shifting of the overhead is done by controlling the receiver's channel checking period and the sender's retry interval. When the sender has a lower expected lifetime, the receiver increases its medium checking frequency to allow the sender to use a longer retry period while still guaranteeing rendezvous between the nodes. When the receiver has a lower life expectancy, the reverse process happens. The receiver leads the overhead shifting process and embeds the changes it makes to the connection’s attributes in the ACK messages it sends to the sender, avoiding the necessity for extra messages. As a result, the minimal lifetime among neighbors is extended and the total network lifetime may be prolonged [39]. 3.9. A-MAC A-MAC is a receiver-initiated MAC protocol for low power networks. This protocol works similarly to RI-MAC but makes a few changes that improve its performance. First, it optionally allows for the use of multiple channels where one is reserved for beacon messages, and the remaining traffic is dispersed among the others. Secondly, it uses backcasts, a link-layer primitive that allows nodes to probe others in parallel and reliably distinguish between the case of zero replays (indicating no traffic) from the case of one or more replays (indicating pending traffic). This mechanism uses non-destructive interference, making it robust against collisions and overhearing. As a result, it allows the receiver node to make better decisions on whether to go back to sleep or remain awake to receive messages. However, most radios lack the hardware and software support necessary to properly implement A-MAC, requiring workarounds [40]. TELKOMNIKA Telecommun Comput El Control, Vol. 19, No. 6, December 2021: 1778 - 1786 3.6. Demand wakeup MAC protocol (DW-MAC) p p The DW-MAC improves on the S-MAC implementation by fully integrating medium access control to sleep scheduling. Nodes using DW-MAC that want to transmit contend for the medium normally, but they replace the RTS/CTS messages with a scheduling frame (SCH) that is used to schedule a time during the next sleep period where they can send their data. The scheduled period is calculated based on how far into the active period the SCH was received. This process creates a one-to-one mapping between the active (data) period and the sleep period. Lastly, when the scheduled time is finally reached, the receiver and sender wakeup and complete the data transfer before going back to sleep. DW-MAC prevents data transmissions from colliding by exploiting the contention that happens in each active period. Since the mapping between the periods is one-to-one, once a node gains medium control by contention that automatically generates an interval during Power saving MAC protocols in wireless sensor networks: a survey (Rafael Souza Cotrim)  1782 3.11. CSMA/CA and TDMA hybrid MAC protocol (CTh-MAC) Mobility support: Determined by a protocol’s general ability to deal with changes in the network structur − Mobility support: Determined by a protocol’s general ability to deal with changes in the network structure. Table 1. Comparison between MAC protocol Table 1. Comparison between MAC protocol Protocol Year Synchronicity Clustering Power- Balancing Sender or receiver- initiated Impact of asymmetric links Mobility support S-MAC 2002 Synchronous Yes No Sender-initiated None Weak mobility T-MAC 2003 Synchronous Yes No Sender-initiated None Weak mobility B-MAC 2004 Asynchronous No No Sender-initiated None Weak mobility X-MAC 2006 Asynchronous No No Sender-initiated None Weak mobility BoX-MAC 2008 Asynchronous No No Sender-initiated None Weak mobility DW-MAC 2008 Synchronous Yes No Sender-initiated None Weak mobility RI-MAC 2008 Asynchronous No No Receiver-initiated High chance of probe loss Weak mobility LB-MAC 2012 Asynchronous No Yes Sender-initiated None Weak mobility A-MAC 2012 Asynchronous No No Receiver-initiated High chance of probe loss Weak mobility Asym- MAC 2014 Asynchronous No No Receiver-initiated Changes to the sender-initiated mode Weak mobility CTh-MAC 2018 Hybrid Yes No Sender-initiated None Strong mobility Table 1. Comparison between MAC protocol Table 1. Comparison between MAC protocol Table 1. Comparison between MAC protocol 3.11. CSMA/CA and TDMA hybrid MAC protocol (CTh-MAC) The CTh-MAC is designed to improve energy efficiency in 3D mobile WSNs. Nodes are allocated into concentric spherical subsets based on their distance to the network gateway. The allocation algorithm also considers the velocity of each node, predicting its future position to optimize the subset formation. Subsets communicate with the gateway according to a TDMA schedule. Outer subsets go first with inners ones serving as relays to the network sink. When it is the turn of a given subset, its nodes content with one another to access  1783 TELKOMNIKA Telecommun Comput El Control  the gateway in a CSMA/CA fashion. The resulting network has considerably better throughput and longer lifetime when compared to other hybrid approaches. However, this protocol assumes that all sensing nodes are within the transmission range of the base station so that it can gather information on their positions [36]. Figure 3. Asym-MAC mode transition, adapted from [41] Figure 3. Asym-MAC mode transition, adapted from [41] Table 1 summarizes the characteristics of the protocols studied. Below, the columns of the table are briefly discussed: − Synchronicity: Refers to a protocol's necessity to use synchronization mechanisms to prevent drift between node schedules. − Synchronicity: Refers to a protocol's necessity to use synchronization mechanisms to prevent drift between node schedules. − Clustering: Clustering happens when groups of nodes have different properties from one another. In some cases that can lead to nodes at the edges of clusters consuming more energy, but CTh-MAC deliberately causes this kind of division as part of its operations. − Clustering: Clustering happens when groups of nodes have different properties from one another. In some cases that can lead to nodes at the edges of clusters consuming more energy, but CTh-MAC deliberately causes this kind of division as part of its operations. Sender or receiver-initiated: In sender-initiated MAC protocols, the sender initiates the data transf process. The opposite happens in receiver-initiated ones. − Impact of asymmetric links: Describes the problems that may arise when the connection is asymmetric. Sender-initiated protocols are not affected by asymmetric links. − Impact of asymmetric links: Describes the problems that may arise when the connection is asymmetric. Sender-initiated protocols are not affected by asymmetric links. − Mobility support: Determined by a protocol’s general ability to deal with changes in the network structure. 5. CONCLUSION This paper surveys MAC protocols designed for wireless sensor networks, focusing on their capability to extended network lifetime. First, we introduced the challenges involved in the design of such protocols and outlined some common solutions. Studied factors include duty cycling, collision avoidance, power balancing, and mobility support, all of which can heavily impact the network's battery consumption. Then novel MAC implementations were analyzed and compared to determine their strengths and weaknesses. The analyzed protocols were classified according to their synchronicity, power balancing support, and many other characteristics. Finally, the biggest remaining issues and possible paths of future research were raised. ACKNOWLEDGEMENTS This work is funded by FCT/MCTES through national funds and when applicable co-funded EU funds under the project UIDB/50008/2020. The authors would like to acknowledge the company InspiringSci, Lda for the interest and valuable contribution to the successful development of this work. 4. OPEN ISSUES Although the studied protocols have greatly improved energy consumption and reduced the constraints that arise from a restricted power source, there are still issues that remain unsolved. Most notably, power balancing has been absent from most of the examined schemes. It may be possible to adapted LB-MAC’s approach of shifting the overhead of communications between sender and receiver to other asynchronous protocols, including receiver-initiated ones. However, adapting it to be used in synchronous communications presents many challenges because of the rigid schedules employed in this kind of communication. Likewise, its integration with approaches from other layers is still untested [39]. If possible, upper layer schemes such as the improved energy-efficient clustering protocol (IEECP) [20] could be used to balance traffic and reduce transmission distance while LB-MAC balances power on each node pair. Power saving MAC protocols in wireless sensor networks: a survey (Rafael Souza Cotrim  1784 ISSN: 1693-6930 While receiver-initiated MACs are some of the most efficient protocols studied, it is also important to point out that current implementations are limited by the hardware available. A-MAC specifically would benefit from native support of the backcast primitive or processors with memory-mapped radios. The creators of A-MAC estimate that these features could reduce idle listening power by 40% [40]. REFERENCES [1] K. Routh and T. 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Son, “Asym-MAC: A MAC protocol for low-power duty-cycled wireless sensor networks with asymmetric links,” in IEEE Communications Letters, vol. 18, no. 5, pp. 809-812, May 2014, doi: 10.1109/LCOMM.2014.032014.132679. Power saving MAC protocols in wireless sensor networks: a survey (Rafael Souza Cotrim)  1786  ISSN: 1693-6930 BIOGRAPHIES OF AUTHORS BIOGRAPHIES OF AUTHORS Rafael Souza Cotrim is an undergraduate informatics engineering student at the Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal. He has an interest in electronics, networking, and enterprise software development. Rafael Souza Cotrim is an undergraduate informatics engineering student at the Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal. He has an interest in electronics, networking, and enterprise software development. João M. L. P. Caldeira is an Assistant Professor at the Escola Superior de Tecnologia do Instituto Politécnico de Castelo Branco, Portugal, and a Researcher at the Instituto de Telecomunicações, Portugal. He is also Co-Founder of the start-up Inspiring’s, Lda. He received the Ph.D. degree in Computer Science and Engineering from the University of Beira Interior, Covilhã, Portugal and from the University of Haute Alsace, Colmar, France. He received the B.S. degree (Hons. (5-year)) in Electrical and Computer Engineering from the University of Coimbra, Portugal, and the M.Sc. degree in Information Systems and Technologies from the University of Beira Interior, Covilhã, Portugal. He has authored or co-authored more than 40 papers in refereed book chapters, journals, and conferences. He has served as a TPC member and reviewer for a number of international conferences and journals. His current research interests include mobility support for wireless sensor networks, Internet of Things, and smart-cities. Vasco N. G. J. TELKOMNIKA Telecommun Comput El Control, Vol. 19, No. 6, December 2021: 1778 - 1786 REFERENCES Soares is a Professor at the Escola Superior de Tecnologia do Instituto Politécnico de Castelo Branco, Portugal, a Cisco Instructor at the Cisco Academy of the Escola Superior de Tecnologia, and also a Researcher at the Instituto de Telecomunicações, Portugal. He is a Co-Founder of the start-up InspiringSci, Lda. He received a five-year B.Sc. degree (licentiate) in Informatics Engineering from the University of Coimbra, Portugal and the Ph.D. degree in Computer Science and Engineering from the University of Beira Interior, Covilhã, Portugal. He has authored or co-authored more than 60 papers in refereed book chapters, journals, and conferences, and 2 patents. He has served as a TPC member and reviewer for a number of international conferences and journals, and as a publicity chair and web chair for a number of international conferences. He is a member of the editorial board of international journals. Also, he has participated in several national and European research projects related to vehicular delay-tolerant networks. He is an IEEE Senior Member. His current research interests include vehicular networks, delay/disruption tolerant networks, Internet of everything, smart-cities, and technological solutions for the agro-industrial sector. Youcef Azzoug is currently a PhD student in computer science at the University of Sience and Technology Houari Boumediene (USTHB). He received his Masters degree in Software Engineering from the Faculty of Electronic and Informatics (FEI) of USTHB in 2015. His research interests on bio-inspired ad-hoc routing optimization and VANET routing protocols.
https://openalex.org/W2885086760	https://europepmc.org/articles/pmc6082848?pdf=render	English	null	Transcriptional and physiological adaptations in nucleus accumbens somatostatin interneurons that regulate behavioral responses to cocaine	Nature communications	2,018	cc-by	12,022	1 Department of Neuroscience, Friedman Brain Institute, New York 10029 NY, USA. 2 Department of Genetics and Genomic Science, Icahn Institute of Genomics and Multiscale Biology, New York 10029 NY, USA. 3 Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York 10029 NY, USA. 4 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 5 Department of Psychology, McGill University, Québec H3A 1B1 Montreal, Canada. 6 Department of Neural Development and Disease, Korea Brain Research Institute, Daegu 41068, Republic of Korea. Correspondence and requests for materials should be addressed to E.J.N. (email: eric.nestler@mssm.edu) ARTICLE Results g p y g p g Only recently has it been possible to assess cell type-speciﬁc transcriptional mechanisms in D1- and D2-type principal med- ium spiny neurons (MSNs) of NAc that regulate their physiolo- gical responses to psychoactive drugs such as cocaine15. Still, pooling tissue from multiple animals, small sample sizes, and a focus on whole striatum have confounded these studies. In addition, sequencing studies have focused primarily on protein- coding genes by performing poly-A selection prior to sequencing, which enriches libraries with mRNA transcripts. However, polyadenylated transcripts obtained from whole cells represent fully mature transcripts and fail to capture many regulatory transcripts required for gene regulation in mammalian cells. There is therefore a need to perform total (as opposed to poly-A selected) RNA-sequencing on brain tissue to identify genome- wide transcriptional changes induced by a psychoactive drug in a speciﬁc cell type of an individual mouse. We used this approach to ﬁrst test the effect of stimulation or suppression of NAc somatostatin interneurons on cocaine- induced locomotor activation over 5 days. During each day of testing, the mice were allowed to habituate to the testing chambers for 5 min before laser stimulation commenced. Mice were injected with 7.5 mg/kg cocaine and locomotor activity was recorded for 30 min with simultaneous optogenetic stimulation or suppression. We found that NAc somatostatin interneurons bidirectionally control cocaine-induced locomotor activation (Fig. 1b, c), with stimulation of the cells promoting locomotor responses and suppression reducing such responses, ﬁndings that are in line with previous pharmacological studies10,13. We did not observe any differences in locomotor activity during habituation across the three groups, suggesting that the effects of NAc somatostatin interneuron activity are dependent on cocaine’s effects on the reward circuitry (see next section). In this study, we ﬁrst use optogenetics to determine the func- tional role of NAc somatostatin interneurons in regulating beha- vioral responses to cocaine. We ﬁnd that stimulation of these cells potentiates the effects of cocaine, while blocking their activity attenuates cocaine’s effects on behavior. We next record from EYFP-tagged somatostatin interneurons in NAc slices from cocaine- or saline-treated mice and found that repeated cocaine administration decreases the intrinsic excitability of these neurons. To characterize transcriptional alterations that cocaine induces in these cells, we perform cell type-speciﬁc RNA-sequencing on FACS-isolated nuclei of somatostatin interneurons and identiﬁed 1100 DETs enriched for processes related to neural plasticity. Results NAc somatostatin interneurons and cocaine-elicited behaviors. To investigate the functional role of NAc somatostatin inter- neurons in cocaine-elicited behaviors, we used optogenetics to bidirectionally control the activity of these cells in awake, freely moving mice (Fig. 1a). We injected an adeno-associated virus (AAV) vector expressing channelrhodopsin-2 (AAV-DIO-CHR2- EYFP)16, or EYFP alone as a control (AAV-DIO-EYFP), into NAc of adult Sst-Cre mice and allowed the vectors to express for 3 weeks. Immunohistochemistry for EYFP and SST protein conﬁrmed the cell type-speciﬁcity of viral expression: all EYFP(+) cells also expressed SST (Supplementary Fig. 1). We validated the bidirectional optogenetic manipulation of NAc somatostatin interneurons in brain slices, achieving stimulation (473 nm, 20 Hz frequency, 1 burst of 10 pulses, 4 ms pulse width, every 10 s) versus suppression (473 nm, 20 Hz frequency, 1 burst of 20 pul- ses, 49 ms pulse width) of the neurons adapted from previous studies17 (Supplementary Fig. 2) (see Methods). Our ﬁnding that our suppression protocol completely silences ~50% of somatos- tatin interneurons and that the ﬁring rate of the other half is reduced from 20 Hz to less than 4 Hz, is comparable to other groups’ ﬁndings using halorhodopsin or Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) for cortical somatostatin interneurons18,19. Both groups achieved reduced action potential ﬁring, but neither fully silenced somatostatin interneurons. Further validation of our stimulation and sup- pression protocols was obtained by C-FOS immunohistochem- istry: we see >2-fold more C-FOS + MSNs after suppression of somatostatin interneurons than after their stimulation (Supple- mentary Fig. 2F–H). These results are consistent with the scheme that somatostatin interneurons control the excitability of princi- pal neurons via distal dendritic inhibition in the striatum20. A predominating theory of somatostatin interneuron function is that they control the ﬂow of information into and out of a brain region by rhythmically inhibiting—via GABA and somatostatin release—the excitability of the distal dendrites of principal neu- rons and thereby their bursting5–7. There is pharmacological evidence concerning somatostatin signaling in NAc and dorsal striatum, showing that locomotor responses to amphetamine require somatostatin receptor activation, and that somatostatin peptide infusion into these regions rapidly induces dopamine release via presynaptic facilitation8–14. However, it is unclear to what extent the gene expression and physiology of somatostatin interneurons in NAc are altered by cocaine and what role these changes play in altering behavioral responses to the drug. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 A A major challenge in neuroscience is to characterize the contributions of a single cell type to the transcriptional proﬁle and functioning of a brain region of interest. This challenge is especially prominent for GABAergic interneurons, since they often account for small fractions of cells within a given brain region. Despite their scarcity, the role of somatostatin (SST)-expressing GABAergic interneurons in regulating cortical plasticity has been well established over the last decade and highlights the profound impact that a miniscule population of neurons can have on plasticity of neural circuits in vivo. For example, a reduced number of somatostatin interneurons in neocortical and hippocampal regions studied postmortem has been reported for several neuropsychiatric disorders and this reduction is proposed to underlie some common pathological changes in circuit function across numerous syndromes1,2. However, somatostatin interneurons in the nucleus accumbens (NAc), a critical brain reward region implicated in drug addic- tion, remain poorly understood due to the fact that these cells represent less than < 1% of all NAc neurons, which makes their isolation and study technically challenging3. As somatostatin interneurons in NAc, cerebral cortex, and hippocampus originate from the same Nkx2.1(+) embryonic lineage (medial ganglionic eminence)4, this cell type might be important in regulating NAc plasticity as seen for the other forebrain regions. differentially expressed target gene that regulates cocaine action. Finally, we use cell type-speciﬁc viral-mediated gene transfer to show that JUND function in NAc somatostatin interneurons reg- ulates a subset of behavioral responses to cocaine. Together, our results identify a cell type-speciﬁc transcriptional mechanism underlying cocaine-induced alterations in NAc. NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunications Transcriptional and physiological adaptations in nucleus accumbens somatostatin interneurons that regulate behavioral responses to cocaine Transcriptional and physiological adaptations in nucleus accumbens somatostatin interneurons that regulate behavioral responses to cocaine Efrain A. Ribeiro1, Marine Salery1, Joseph R. Scarpa2, Erin S. Calipari1, Peter J. Hamilton1, Stacy M. Ku1,3, Hope Kronman1, Immanuel Purushothaman1, Barbara Juarez3, Mitra Heshmati1, Marie Doyle1, Casey Lardner1, Dominicka Burek1, Ana Strat1, Stephen Pirpinias1, Ezekiell Mouzon1, Ming-Hu Han1,3, Rachael L. Neve4, Rosemary C. Bagot1,5, Andrew Kasarskis2, Ja Wook Koo1,6 & Eric J. Nestler 1 The role of somatostatin interneurons in nucleus accumbens (NAc), a key brain reward region, remains poorly understood due to the fact that these cells account for < 1% of NAc neurons. Here, we use optogenetics, electrophysiology, and RNA-sequencing to characterize the transcriptome and functioning of NAc somatostatin interneurons after repeated exposure to cocaine. We ﬁnd that the activity of somatostatin interneurons regulates behavioral responses to cocaine, with repeated cocaine reducing the excitability of these neurons. Repeated cocaine also induces transcriptome-wide changes in gene expression within NAc somatostatin interneurons. We identify the JUND transcription factor as a key regulator of cocaine action and conﬁrmed, by use of viral-mediated gene transfer, that JUND activity in somatostatin interneurons inﬂuences behavioral responses to cocaine. Our results identify alterations in NAc induced by cocaine in a sparse population of somatostatin interneurons, and illustrate the value of studying brain diseases using cell type-speciﬁc whole tran- scriptome RNA-sequencing. 1 Department of Neuroscience, Friedman Brain Institute, New York 10029 NY, USA. 2 Department of Genetics and Genomic Science, Icahn Institute of Genomics and Multiscale Biology, New York 10029 NY, USA. 3 Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York 10029 NY, USA. 4 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 5 Department of Psychology, McGill University, Québec H3A 1B1 Montreal, Canada. 6 Department of Neural Development and Disease, Korea Brain Research Institute, Daegu 41068, Republic of Korea. Correspondence and requests for materials should be addressed to E.J.N. (email: eric.nestler@mssm.edu) 1 NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunications Results Using this strategy, we identify the transcription factor JUND as a We proceeded to test the function of these cells on reward learning by use of conditioned place preference (CPP). In this experiment, we coupled optogenetic stimulation or suppression of NAc somatostatin interneurons with cocaine exposure, as previously performed in D1 and D2 MSNs21 (Fig. 1d). To study the effect of stimulating NAc somatostatin interneurons, we used a low dose of cocaine (5 mg/kg), as we hypothesized that this manipulation would increase the rewarding effects of the drug. Consistent with this hypothesis, AAV-DIO-CHR2-EYFP-injected mice developed a stronger CPP to cocaine than those injected NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunication 2 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 ARTICLE found that neither optogenetic stimulation nor suppression alters locomotor activity compared to EYFP-expressing control mice. We repeated the experiment the next day and again found no difference between the groups (Supplementary Fig. 3A,C). Like- wise, in separate groups of mice, neither optogenetic stimulation nor suppression of NAc somatostatin interneurons produced a CPP or aversion in the absence of cocaine (Supplementary Fig. 3B,D). We next tried to induce CPP using optogenetic sti- mulation or suppression of the neurons with a sub-threshold dose of cocaine (2.5 mg/kg) that did not produce CPP in control EYFP mice. Neither stimulation nor suppression of NAc somatostatin interneurons affected cocaine CPP at this lower drug dose (Supplementary Fig. 4). Together, these results show that soma- tostatin interneuron activity alone does not mimic the effect of cocaine on mouse behavior. Rather, these cells modulate behavior elicited by cocaine exposure. with AAV-DIO-EYFP (Fig. 1e). Conversely, we used a higher dose of cocaine (7.5 mg/kg) to test the effect of suppressing NAc somatostatin interneuron activity and found that, while control mice formed a strong preference to this dose of cocaine, this effect was blocked upon suppression of the somatostatin cells (Fig. 1f). Together, these results establish a critical role for NAc somatostatin interneuron activity in controlling behavioral responses to cocaine. NAc somatostatin interneurons and baseline behavior. Given the ﬁnding that NAc somatostatin interneuron activity can modulate behavioral responses to cocaine, we determined if the same optogenetic manipulations alter locomotor or place con- ditioning behavior in the absence of cocaine. To do this, we sti- mulated or suppressed NAc somatostatin interneuron activity using optogenetics during 30 min of open ﬁeld exploration. Results We Burst/10 seconds Stimulation 20 Hz Suppression AAV-DIO-EYFP or AAT-DIO-hChR2-EYFP SST-IRES-CRE a 20 Hz 10 cycle Morning Afternoon d Saline Laser OFF Laser ON Cocaine 800 600 ** 400 CPP score 200 –200 Pre Post Pre Post 0 5 mg/kg e EYFP Stimulation Habituation laser off 80 EYFP Stimulation Suppression 60 40 Total beambreaks 20 0 1 2 3 Day 4 5 b 5000 4000 3000 2000 1000 0 Total beambreaks 1 2 3 Day 4 5 Cocaine (7.5 mg/kg) laser on c 600 400 200 –200 0 * CPP score Pre Post Pre Post f 7.5 mg/kg EYFP Suppression Fig. 1 NAc somatostatin interneuron activity regulates cocaine-induced locomotor activity and reward. a AAV-DIO-EYFP or AAV-DIO-ChR2-EYFP were injected bilaterally into NAc of Sst-Cre mice 3 weeks prior to optogenetic stimulation or suppression, respectively, with the following parameters: Stimulation (473 nm, 20 Hz frequency, 1 burst of 10 pulses, 4 ms pulse width, every 10 s) or Suppression (473 nm, 20 Hz frequency, 1 burst of 20 pulses, 49 ms pulse width, every sec). b There is no difference between the locomotor activity of the three groups during habituation while the lasers remain off (repeated measures two-way ANOVA: no effect of activity F (2,104) = 1.953. p = 0.147; n = 8,8,8) or day F(4,104) = 0.3617. p = 0.9383; n = 8,8,8). c The activity of NAc somatostatin interneurons (EYFP control, Stimulation, or Suppression) controls the locomotor activity of mice after repeated cocaine injections. There is a signiﬁcant effect of activity (repeated measures two-way ANOVA: signiﬁcant effect of activity F(2,21) = 5.647. p < 0.05; n = 8,8,8) and Day (Signiﬁcant effect of Day F (4,84) = 8.856. p < 0.001; n = 8,8,8). d Experimental design for unbiased CPP with optogenetics. Groups were counterbalanced to ensure that cocaine was paired evenly in both the gray and striped sides of the CPP chambers. e Stimulation of NAc somatostatin interneurons enhances cocaine CPP (Student's t-test: EYFP Pre-test vs EYFP Post-test, p < 0.01; EYFP Pre-test mean = −16.50 ± 91.33 SEM vs EYFP Post- test mean = 376.4 ± 91.33 SEM (n = 8); CHR2-Stimulation Pre-test vs CHR2-Stimulation Post-Test, *p < 0.0001 CHR2-Stimulation Pre-test mean = 51.00 ± 77.37 SEM vs CHR2-Stimulation Post-Test mean = 655.1 ± 53.93 SEM (n = 7); EYFP Post-test vs. CHR2-Stimulation Post-test, p < 0.05 n = 8,7). Results f Cocaine CPP is blocked when NAc somatostatin interneurons are suppressed during training with cocaine. (Student’s t-test; AAV-DIO-EYFP Pre vs Post: p < 0.01, EYFP Pre-test mean = −7.11 ± 66.2 SEM vs EYFP Post-test mean = 348.3 ± 90.41 SEM (n = 9)); AAV-DIO-CHR2 Pre vs Post: P > 0.05, n = 14). Data are represented as ± SEM Habituation laser off 80 EYFP Stimulation Suppression 60 40 Total beambreaks 20 0 1 2 3 Day 4 5 b 5000 4000 3000 2000 1000 0 Total beambreaks 1 2 3 Day 4 5 Cocaine (7.5 mg/kg) laser on c Burst/10 seconds Stimulation 20 Hz Suppression AAV-DIO-EYFP or AAT-DIO-hChR2-EYFP SST-IRES-CRE a 20 Hz 10 cycle b c a AAV-DIO-EYFP or AAT-DIO-hChR2-EYFP 800 600 **** 400 CPP score 200 –200 Pre Post Pre Post 0 5 mg/kg e EYFP Stimulation 600 400 200 –200 0 * CPP score Pre Post Pre Post f 7.5 mg/kg EYFP Suppression Morning d Saline Laser OFF f f d e Afternoon Laser ON Cocaine Cocaine Fig. 1 NAc somatostatin interneuron activity regulates cocaine-induced locomotor activity and reward. a AAV-DIO-EYFP or AAV-DIO-ChR2-EYFP were injected bilaterally into NAc of Sst-Cre mice 3 weeks prior to optogenetic stimulation or suppression, respectively, with the following parameters: Stimulation (473 nm, 20 Hz frequency, 1 burst of 10 pulses, 4 ms pulse width, every 10 s) or Suppression (473 nm, 20 Hz frequency, 1 burst of 20 pulses, 49 ms pulse width, every sec). b There is no difference between the locomotor activity of the three groups during habituation while the lasers remain off (repeated measures two-way ANOVA: no effect of activity F (2,104) = 1.953. p = 0.147; n = 8,8,8) or day F(4,104) = 0.3617. p = 0.9383; n = 8,8,8). c The activity of NAc somatostatin interneurons (EYFP control, Stimulation, or Suppression) controls the locomotor activity of mice after repeated cocaine injections. There is a signiﬁcant effect of activity (repeated measures two-way ANOVA: signiﬁcant effect of activity F(2,21) = 5.647. p < 0.05; n = 8,8,8) and Day (Signiﬁcant effect of Day F (4,84) = 8.856. p < 0.001; n = 8,8,8). d Experimental design for unbiased CPP with optogenetics. Groups were counterbalanced to ensure that cocaine was paired evenly in both the gray and striped sides of the CPP chambers. Results e Stimulation of NAc somatostatin interneurons enhances cocaine CPP (Student's t-test: EYFP Pre-test vs EYFP Post-test, p < 0.01; EYFP Pre-test mean = −16.50 ± 91.33 SEM vs EYFP Post- test mean = 376.4 ± 91.33 SEM (n = 8); CHR2-Stimulation Pre-test vs CHR2-Stimulation Post-Test, *p < 0.0001 CHR2-Stimulation Pre-test mean = 51.00 ± 77.37 SEM vs CHR2-Stimulation Post-Test mean = 655.1 ± 53.93 SEM (n = 7); EYFP Post-test vs. CHR2-Stimulation Post-test, p < 0.05 n = 8,7). f Cocaine CPP is blocked when NAc somatostatin interneurons are suppressed during training with cocaine. (Student’s t-test; AAV-DIO-EYFP Pre vs Post: *p < 0.01, EYFP Pre-test mean = −7.11 ± 66.2 SEM vs EYFP Post-test mean = 348.3 ± 90.41 SEM (n = 9)); AAV-DIO-CHR2 Pre vs Post: P > 0.05, n = 14). Data are represented as ± SEM NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 Cell type-speciﬁc RNA-sequencing of NAc somatostatin interneurons. We next sought to understand the molecular adaptations induced in NAc somatostatin interneurons by use of RNA-sequencing. These cells offer a particular challenge to pro- ﬁle transcriptionally because of their minute numbers. To proﬁle the entire (non poly-A selected) transcriptome of NAc soma- tostatin interneurons, we generated a transgenic reporter line (SST-TLG498 mice) to label the nuclei of these cells with a modiﬁed form of EGFP that is retained in the nuclear membrane (EGFP-F)22, enabling their isolation from NAc dissections using FACS (Fig. 2a). We succeeded in FACS-isolating nuclei suitable for RNA-sequencing from individual SST-TLG498 mice (Fig. 2b), based on previously published methods (Supplementary Fig. 5)23–25. We extracted RNA from sorted EGFP(+) and EGFP (−) nuclei and performed qPCR for Sst transcripts, which were strongly enriched in the EGFP(+) fraction and barely detectable in the EGFP(−) fraction (Student’s t-test: EGFP(+) vs. EGFP(−), n = 3, P < 0.0001). that are speciﬁc to neurons (Bonferoni p < 0.05) (Fig. 2d). Underlying these GO terms are changes in transcripts encoding proteins that regulate cytoskeletal architecture and cellular adhesion: ankryn (Ankr), neuronal cell adhesion molecule (NrCam), and microtubule-associated protein 2 (Map2), which have well-established roles in structural and functional plasti- city27–29. Results Cocaine = 4.04 logCPM, logFC = 2.58, p < 0.05). We used the ENRICHR plat- form to determine transcription factors that may be upstream of highly expressed cocaine-altered transcripts, based on known transcription factor protein–protein interactions34,35. JUND was also predicted as upstream of the DETs in our dataset (Expression cutoff logCPM > 1; JUND: p < 0.05). Both lines of evidence sup- port the hypothesis that JUND may be a critical factor in med- iating cocaine-induced alterations in somatostatin interneurons in the NAc. Consistent with this hypothesis, we found that JunD expression levels in NAc somatostatin interneurons from individual mice were correlated with locomotor activity on the ﬁnal day of testing regardless of cocaine or saline exposure (Fig. 3a). To study cocaine regulation of JUND protein expression in NAc somatostatin interneurons, we performed immunohistochemistry for JUND in saline- and cocaine-treated SST-TLG498 mice. We determined ﬁrst that cocaine did not alter the percentage of EGFP (+) cells that were immunoreactive for JUND. Next, we measured the total corrected cell ﬂuorescence of JUND in individual NAc somatostatin interneurons and found that JUND levels in individual cells were increased in cocaine-treated mice compared to saline controls (Fig. 3b, c). In contrast, JUND levels are not altered by cocaine exposure in non-somatostatin cells—i.e., EGFP (−) cells—in NAc despite being expressed broadly in these cells at baseline (Supplementary Fig. 8). This result conﬁrms prior interpretations that while JUND functions in MSNs as a binding partner for ΔFOSB, it itself is not regulated by cocaine in that cell type30,34. Together, these ﬁndings support the RNA-sequencing ﬁnding that JunD expression is induced selectively in NAc somatostatin interneurons in response to repeated cocaine injections. We proceeded with differential expression analysis of the RNA-sequencing data to identify differentially expressed tran- scripts (DETs) in NAc somatostatin interneurons in response to repeated cocaine exposure: 778 transcripts were upregulated by cocaine and 322 were downregulated (Fig. 2c). These included many differentially expressed non-coding RNAs of each tran- script type, with the frequency of transcript types among DETs being similar to those in the whole transcriptome. We succeeded in identifying a DET signature for cocaine action: all mice in the cocaine arm are differentiated from all mice in the saline arm solely by unsupervised clustering of the expression values from the 75 most robust DETs (logFC, p < 0.05) (Supplementary Fig. 7). Results Our analysis also identiﬁed several additional differen- tially expressed protein-coding genes, e.g., calcium/calmodulin- dependent protein kinase II inhibitor 1 (Camk2n1) and several transcription factors—JunD, forkhead box O3 (FoxO3), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), all implicated previously in cocaine-induced plasticity in NAc30–33. However, it is important to note that these earlier studies relied on methods that manipulated and studied all NAc neurons using whole tissue lysates, which we avoid here by selectively studying somatostatin interneurons. The ﬁnding of JunD transcriptional regulation in particular drew our interest, as it is an AP-1 family transcription factor that has been implicated as a binding partner for ΔFOSB in principal MSNs of the NAc30,34. Our new results therefore raise the important question as to what role JUND may have in somatostatin interneurons, where ΔFOSB is not induced by cocaine, as these cells constitute a miniscule fraction of all cells contained in whole NAc homogenates. After validating our sorting method, we performed RNA- sequencing on EGFP(+) nuclei from a separate group of 20 individual SST-TLG498 mice that received 7 daily IP injections of either saline or cocaine (7.5 mg/kg). We observed increased locomotor activity in cocaine-treated SST-TLG498 mice (Supple- mentary Fig. 6). The resulting RNA-sequencing data were of high quality (Fig. 2c, d). This is evidenced by the wide distribution of transcript types that we identiﬁed and the high mapping rates within each sample. We also observed a small percentage of rRNA reads in our samples compared with other non-coding RNA transcript types, indicating that nuclear sorting can be used to sequence total RNA without bias for rRNA. Therefore, our data better represent dynamic transcriptional regulation that is partly lost in whole tissue or cell preparations where nuclear RNA is mixed with cytoplasmic steady-state mRNAs. We used the nuclear RNA-sequencing data to further conﬁrm the cell type- speciﬁcity of our sorted cell population (Fig. 2e). Sst transcripts, along with transcripts for several other molecules that are known to be expressed by somatostatin interneurons—Nos1, neuropep- tide Y (NPY), and the canonical MGE-derived post-mitotic GABAergic interneuron marker Lhx64,26, were all highly expressed. In contrast, markers of other GABAergic neuron subtypes or of NAc cholinergic interneurons were barely detectable (Fig. 2e). Cocaine alters JUND in NAc somatostatin interneurons. Our RNA-sequencing data revealed that JunD transcription was upregulated in NAc somatostatin interneurons after repeated cocaine exposure (JunD: Saline = 1.75 logCPM vs. NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunications Results C EGFP-F (Sst-TLG) Sst a c 0.53% snoRNA 1.07% snRNA 8.57% Antisense 5.63% lincRNA 2.94% miRNA 9.65% Pseudogene 3.48% Processed transcript 68.09% Protein coding 6 4 –5 10 15 logCPM Δ logCPM –4 –2 d e Neuron projection Synapse Synapse part Cell soma Cell projection Postsynaptic density Cell junction Axon 0 2 4 6 8 –log10 of Bonferoni p-value 0 2 4 6 8 10 Average logCPM SST NPY NOS1 LHX6 CCK PVALB CHAT VIP SSC-A (×1000) b 50 150 250 50 150 250 50 150 250 FSC-A (×1000) GFP 0 100 1000 10,000 100,000 0 100 P5 PE 50 150 250 SSC-A (×1000) SSC-W (×1000) EGFP-F (Sst-TLG) Sst a SSC-A (×1000) b 50 150 250 50 150 250 50 150 250 FSC-A (×1000) GFP 0 100 1000 10,000 100,000 0 100 P5 PE 50 150 250 SSC-A (×1000) SSC-W (×1000) b a a truncated form of JunD, lacking the N-terminus of the wild- type protein, we were able to use qPCR targeting the C- and N- termini of the JunD transcript to validate viral-mediated overexpression of JunD versus ΔJunD in NAc of Sst-Cre mice (Fig. 3d, e). We found that NAc of mice treated with HSV- Using this HSV system we found that JunD overexpression in NAc somatostatin interneurons increased baseline locomotor activity of mice in an open ﬁeld (Fig. 3f). However, we did not observe an increase in locomotor activity in response to cocaine after JunD overexpression or an effect on cocaine CPP c 0.53% snoRNA 1.07% snRNA 8.57% Antisense 5.63% lincRNA 2.94% miRNA 9.65% Pseudogene 3.48% Processed transcript 68.09% Protein coding 6 4 –5 10 15 logCPM Δ logCPM –4 –2 d e Neuron projection Synapse Synapse part Cell soma Cell projection Postsynaptic density Cell junction Axon 0 2 4 6 8 –log10 of Bonferoni p-value 0 2 4 6 8 10 Average logCPM SST NPY NOS1 LHX6 CCK PVALB CHAT VIP SSC 50 150 250 50 50 FSC-A (×1000) GFP 0 100 1000 10,000 100,000 0 100 P5 PE 50 150 250 SSC-A (×1000) SSC- a truncated form of JunD, lacking the N-terminus of the wild- type protein, we were able to use qPCR targeting the C- and N- termini of the JunD transcript to validate viral-mediated overexpression of JunD versus ΔJunD in NAc of Sst-Cre mice (Fig. 3d, e). Results We found that NAc of mice treated with HSV- JUND exhibit an equal proportion of C- and N-termini consistent with full-length JunD overexpression, whereas mice treated with HSV-ΔJUND exhibit greater expression of C- terminus JunD consistent with ΔJunD lacking the N-terminus of the wild-type transcript. Using this HSV system we found that JunD overexpression in NAc somatostatin interneurons increased baseline locomotor activity of mice in an open ﬁeld (Fig. 3f). However, we did not observe an increase in locomotor activity in response to cocaine after JunD overexpression or an effect on cocaine CPP (Supplementary Fig. 9). This could be due to high levels of JUND protein expressed by these neurons especially after cocaine exposure or a lack of temporal control over JunD transcription that may occur endogenously (see Discussion). Conversely, we found that overexpression of ΔJunD in NAc somatostatin c 0.53% snoRNA 1.07% snRNA 8.57% Antisense 5.63% lincRNA 2.94% miRNA 9.65% Pseudogene 3.48% Processed transcript 68.09% Protein coding 6 4 –5 10 15 logCPM Δ logCPM –4 –2 d e Neuron projection Synapse Synapse part Cell soma Cell projection Postsynaptic density Cell junction Axon 0 2 4 6 8 –log10 of Bonferoni p-value 0 2 4 6 8 10 Average logCPM SST NPY NOS1 LHX6 CCK PVALB CHAT VIP GFP 0 100 1000 10,000 100,000 0 100 P5 PE NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunications 5 c 0.53% snoRNA 1.07% snRNA 8.57% Antisense 5.63% lincRNA 2.94% miRNA 9.65% Pseudogene 3.48% Processed transcript 68.09% Protein coding 6 4 –5 10 15 logCPM Δ logCPM –2 GFP c –4 d Neuron projection Synapse Synapse part Cell soma Cell projection Postsynaptic density Cell junction Axon 0 2 4 6 8 –log10 of Bonferoni p-value e 0 2 4 6 8 10 Average logCPM SST NPY NOS1 LHX6 CCK PVALB CHAT VIP d Using this HSV system we found that JunD overexpression in NAc somatostatin interneurons increased baseline locomotor activity of mice in an open ﬁeld (Fig. 3f). However, we did not observe an increase in locomotor activity in response to cocaine after JunD overexpression or an effect on cocaine CPP (Supplementary Fig. 9). This could be due to high levels of JUND protein expressed by these neurons especially after cocaine exposure or a lack of temporal control over JunD transcription that may occur endogenously (see Discussion). Results Thus, we have high conﬁdence that the DETs we identiﬁed are genuinely regulated by cocaine, despite individual mouse variability in speciﬁc transcript expression levels. Database for annotation, visualization and integrated discovery (DAVID) was used to identify the functional gene ontology (GO) of the 1100 DETs in NAc somatostatin interneurons. We used this strategy to determine if a speciﬁc cellular sub-compartment was the target of cocaine-induced transcriptional alterations in somatostatin interneurons—for example, whether the differen- tially expressed genes are only enriched for proteins that function within synapses. On the contrary, we found that 8 cellular sub- compartments were signiﬁcantly enriched in our DET dataset j To test the behavioral relevance of such JunD regulation, we developed a conditional herpes simplex virus (HSV) vector to overexpress JunD, or its truncated, dominant-negative mutant termed ΔJunD—previously validated in vitro and in vivo36, selectively in NAc somatostatin interneurons. Because ΔJunD is NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 ed form of JunD, lacking the N-terminus of the wild- ein, we were able to use qPCR targeting the C- and N- of the JunD transcript to validate viral-mediated ssion of JunD versus ΔJunD in NAc of Sst-Cre mice e). We found that NAc of mice treated with HSV- xhibit an equal proportion of C- and N-termini t with full-length JunD overexpression, whereas mice ith HSV-ΔJUND exhibit greater expression of C- JunD consistent with ΔJunD lacking the N-terminus Using this HSV system we found that JunD over NAc somatostatin interneurons increased baselin activity of mice in an open ﬁeld (Fig. 3f). Howeve observe an increase in locomotor activity in respon after JunD overexpression or an effect on c (Supplementary Fig. 9). This could be due to h JUND protein expressed by these neurons especially exposure or a lack of temporal control over JunD that may occur endogenously (see Discussion). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 Nevertheless, it is likely that this population of cells utilizes multiple neuro- transmitter systems each of which alters the physiology of NAc microcircuits3. Future studies using optogenetics in combination with microﬂuidic infusion of speciﬁc receptor antagonists will eventually be able to systematically address this question. Cocaine alters NAc somatostatin interneuron excitability. Along with changes in transcripts related to transcriptional and structural plasticity, we observed cocaine-induced alterations in the expression levels of many types of ion channels in NAc somatostatin interneurons (Fig. 4a). Changes in the expression of these transcripts contributed to several identiﬁed GO terms (Fig. 2d). We therefore investigated whether the cellular excit- ability of NAc somatostatin neurons was altered by repeated cocaine exposure. First, we injected Sst-Cre mice with AAV-DIO- EYFP and performed recordings from EYFP(+) cells in brain slices of the NAc. The cells from which we recorded exhibited similar baseline properties previously seen in striatal somatostatin interneurons20,37. We found that cocaine does not alter the resting membrane potential of these cells (Cocaine: −59.19±0.64 mV; Control: −59.94±0.59 mV; Mann–Whitney test, p = 0.38; n = 31–36 cells and 5 mice per group). However, cocaine decreased the number of evoked action potentials in response to current injections compared to saline treatment (Fig. 4b, c). Consistent with this decreased neuronal excitability, we also observed an increased threshold to induce the ﬁrst spike (rheobase) in response to current injections in the cocaine-treated group (Fig. 4d). These results show that repeated cocaine exposure decreases the excitability of NAc somatostatin interneurons. y y y q We used a nuclear sorting method by which cells in frozen tissue are lysed rapidly, enabling the effective isolation of nuclei from other cellular organelles in a manner that avoids the use of enzymes to dissociate the tissue. As transcription of new genes requires ATP, it is unlikely that isolated nuclei possess the energetic requirements to induce transcription during FACS. Importantly, our data showing minimal expression levels of c-Fos transcripts in NAc somatostatin interneuron nuclei isolated from saline-treated mice are in line with recent reports showing little or no c-Fos activation in sorted nuclei from naive mice in contrast to sorted whole cells which display high levels of c-Fos25. This is a critical consideration for the ﬁeld moving forward, as transcrip- tional changes induced by FACS isolation of live whole cells may confound cell type-speciﬁc RNA-sequencing data. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 Fig. 2 Cocaine induces 1100 DETs in NAc somatostatin interneurons. a Coronal section of anterior forebrain showing EGFP-F/SST overlap in NAc. Scale in a = 200 µm; Scale in inset = 25 µm. b Representative FACS gating used to isolate EGFP(+) nuclei from NAc of SST-TLG498 mice. We isolated ~5000 nuclei per mouse. c Differential expression analysis identiﬁes 1100 cocaine-regulated transcripts in NAc somatostatin interneurons (p < 0.05). 10 mice were injected with saline and 10 with cocaine every day for 7 days. Mice were analyzed 1 h after the ﬁnal injection and tissue was ﬂash frozen and stored before nuclear FACS. On average, ~1–2 ng of RNA were collected per mouse as determined by analysis on RNA Pico Bioanaylzer chips (Agilent). 0.5–2 ng were then used to generate indexed libraries for sequencing using Clontech SMARTer Stranded Total RNA-seq library preparation kits. All libraries were analyzed for mean insert size and molar concentration prior to sequencing with V4 chemistry (Illumina) at Beckman Coulter Genomics (Now: GeneWiz). Samples were pooled in groups of 8 and all pools were sequenced across multiple lanes. We performed multiplexed Illumina HiSeq to obtain > 2 × 107 paired reads per sample with read length of 125 × 2 bp. Individual transcripts are depicted as individual squares. Transcripts above the x axis are upregulated, while transcripts below the x axis are downregulated. Transcripts are color coded by transcript type and transcript types are quantiﬁed for relative abundance in top right inset pie chart. d DETs are enriched for gene ontology terms related to multiple neuronal compartments demonstrating different ways in which genes can regulate cellular function (Bonferoni p < 0.05). Several of the genes implicated in regulating these cellular compartments (Ankr, Map2, NrCam) are implicated in regulating neuronal structural and functional plasticity. e Cell type-speciﬁc expression of GABAergic neuron and other interneuron subtype transcriptional markers. Npy neuropeptide Y, Nos1 nitrous oxide synthase 1, Lhx6 Lim-containing homeobox 6, Cck cholecystekinin, Pv parvalbumin, Chat choline acetyltransferase, Vip vasoactive intestinal peptide interneurons decreased locomotor responses to repeated doses of cocaine, as would be expected from our RNA-sequencing experiment (Fig. 3g). principal MSNs via GABAergic signaling7,20. Previous studies have shown that infusion of somatostatin peptide alters dopamine release in striatum and that somatostatin infusion alters amphetamine-induced locomotor activity in rodents, suggesting that somatostatin signaling is also involved10,13. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 Nuclear sequencing thus offers an advantage with the understanding that it captures a distinct population of transcripts. p p p p We identiﬁed several differentially expressed protein-coding genes that warrant future study in somatostatin interneurons (Supplementary Data 1). In particular, our sequencing results pointed toward JunD expression in NAc somatostatin inter- neurons as inﬂuencing behavioral responses to cocaine. Using immunohistochemistry, we found that cocaine increases JUND expression in somatostatin interneurons, but not in principal neurons, in the NAc. We used viral gene transfer to bidir- ectionally manipulate JunD activity selectively in NAc somatos- tatin interneurons and found that JunD overexpression increased baseline locomotor activity, while the dominant-negative ΔJunD mutant blunted locomotor responses to cocaine. The relatively high baseline expression of JUND in these neurons, and the further induction of JUND by cocaine, may contribute to the inability to modify behavioral responses to cocaine with JunD overexpression. Another possible explanation for the lack of effect of JunD overexpression on locomotor or CPP responses to cocaine relates to the temporal features of JunD regulation. Our RNA-sequencing and immunohistochemistry data show induc- tion of JUND expression in NAc somatostatin interneurons 1 h after the last dose of cocaine, while our HSV overexpression Results Conversely, we found that overexpression of ΔJunD in NAc somatostatin a truncated form of JunD, lacking the N-terminus of the wild- type protein, we were able to use qPCR targeting the C- and N- termini of the JunD transcript to validate viral-mediated overexpression of JunD versus ΔJunD in NAc of Sst-Cre mice (Fig. 3d, e). We found that NAc of mice treated with HSV- JUND exhibit an equal proportion of C- and N-termini consistent with full-length JunD overexpression, whereas mice treated with HSV-ΔJUND exhibit greater expression of C- terminus JunD consistent with ΔJunD lacking the N-terminus of the wild-type transcript. a truncated form of JunD, lacking the N-terminus of the wild- type protein, we were able to use qPCR targeting the C- and N- termini of the JunD transcript to validate viral-mediated overexpression of JunD versus ΔJunD in NAc of Sst-Cre mice (Fig. 3d, e). We found that NAc of mice treated with HSV- JUND exhibit an equal proportion of C- and N-termini consistent with full-length JunD overexpression, whereas mice treated with HSV-ΔJUND exhibit greater expression of C- terminus JunD consistent with ΔJunD lacking the N-terminus of the wild-type transcript. a truncated form of JunD, lacking the N-terminus of the wild- type protein, we were able to use qPCR targeting the C- and N- termini of the JunD transcript to validate viral-mediated overexpression of JunD versus ΔJunD in NAc of Sst-Cre mice (Fig. 3d, e). We found that NAc of mice treated with HSV- JUND exhibit an equal proportion of C- and N-termini consistent with full-length JunD overexpression, whereas mice treated with HSV-ΔJUND exhibit greater expression of C- terminus JunD consistent with ΔJunD lacking the N-terminus of the wild-type transcript. 5 NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 Saline Cocaine EGFP-F (Sst-TLG) JUND DAPI a b c r = 0.46 p < 0.05 JunD expression (logCPM) Locomotor activity: day 7 6000 *** 4000 JUND corrected fluorescence intensity 2000 0 Saline Cocaine 8 6 4 2 0 2000 4000 –2 –4 HSV-LSL-mCherry +/– HSV-LSL-JUND or HSV-LSL-ΔJUND SST-Cre d HSV-LSL-JUND HSV-LSL-ΔJunD 0.04 * 0.03 0.02 mRNA expression relative to β-actin 0.01 0.00 C-terminus N-terminus e * mCherry + JunD f Locomotor activity Total beambreaks 500 400 300 200 100 0 g Cocaine (7.5 mg/kg) Beambreaks 5000 4000 3000 2000 1000 0 0 1 2 Day 3 HSV-LSL-mCherry HSV-LSL-ΔJUND g. 3 JunD expression in NAc somatostatin interneurons regulates locomotor behavior. a There is a positive correlation in our RNA-sequencing data etween JunD expression levels and locomotor activity across saline- and cocaine-treated mice on the last day of testing (Pearson Correlation: r = 0.46, n 20, p = 0.043). Green circles represent JunD expression levels from individual mice. b Representative images used for quantiﬁcation of JUND corrected tal cell ﬂuorescence (CTCF) in EGFP(+) cells. Each image is a single plane in a z-stack to ensure true ﬂuorescence signal (not the sum of multiple anes). Scale bar = 25 µm. c JUND protein expression is increased in EGFP(+) cells of cocaine-treated mice (1 h after last dose) compared to saline ontrols (Student's t-test with Welch’s Correction: **p < 0.0001; Saline mean CTCF = 1807 ± 138.9 SEM (n = 20 cells/3 mice) vs Cocaine mean CTCF = 920 ± 207.7 SEM (n = 23 cells/3 mice). Welch’s correction: t = 4.54, df = 37.77). d Stereotaxic targeting of HSV-LSL-JUND + HSV-LSL-mCHERRY or SV-LSL-ΔJUND + HSV-LSL-mCHERRY in NAc of Sst-Cre mice. e qPCR quantiﬁcation of transgene expression in NAc of Sst-Cre mice infected with HSV- SL-JUND or HSV-LSL-mCHERRY. ΔJunD is a truncated form of JunD that lacks the N-terminus, making it a dominant-negative protein. (Student’s t-test ith Welch’s correction: p < 0.05; HSV-LSL-JUND mean = 0.01859 ± 0.008903 SEM (n = 8) vs HSV-LSL-ΔJUND mean = 0.0007039 ± 0.0004589 SEM n = 10) Welch’s Correction: t = 2.259, df = 16). f JunD overexpression in NAc somatostatin interneurons increases locomotor activity in an open ﬁeld Students t-test: p = 0.051; mCherry mean = 303.4 ± 26.25 SEM vs + JunD mean = 395.5 ± 35.62 SEM (n = 11,11)). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 g Dominant-negativeΔJunD expression NAc somatostatin interneurons decreases locomotor responses to cocaine (Repeated Measures Two-way ANOVA: Signiﬁcant effect of Virus F (1,20) = 937. p < 0.04; n = 10,10). Data are represented as ± SEM Saline Cocaine EGFP-F (Sst-TLG) JUND DAPI b c 600 400 JUND corrected fluorescence intensity 200 c 6000 ** 4000 JUND corrected fluorescence intensity 2000 0 Saline Cocaine a r = 0.46 p < 0.05 JunD expression (logCPM) Locomotor activity: day 7 8 6 4 2 0 2000 4000 –2 –4 a c JUND HSV-LSL-mCherry +/– HSV-LSL-JUND or HSV-LSL-ΔJUND SST-Cre d HSV-LSL-mCherry +/– HSV-LSL-JUND or HSV-LSL-ΔJUND SST-Cre d HSV-LSL-JUND HSV-LSL-ΔJunD 0.04 0.03 0.02 mRNA expression relative to β-actin 0.01 0.00 C-terminus N-terminus e * mCherry + JunD f Locomotor activity Total beambreaks 500 400 300 200 100 0 g Cocaine (7.5 mg/kg) Beambreaks 5000 4000 3000 2000 1000 0 0 1 2 Day 3 HSV-LSL-mCherry HSV-LSL-ΔJUND d f * mCherry + JunD f Locomotor activity Total beambreaks 500 400 300 200 100 0 g Cocaine (7.5 mg/kg) Beambreaks 5000 4000 3000 2000 1000 0 0 1 2 Day 3 HSV-LSL-mCherry HSV-LSL-ΔJUND e e g Cocaine (7.5 mg/kg) Fig. 3 JunD expression in NAc somatostatin interneurons regulates locomotor behavior. a There is a positive correlation in our RNA-sequencing data between JunD expression levels and locomotor activity across saline- and cocaine-treated mice on the last day of testing (Pearson Correlation: r = 0.46, n = 20, p = 0.043). Green circles represent JunD expression levels from individual mice. b Representative images used for quantiﬁcation of JUND corrected total cell ﬂuorescence (CTCF) in EGFP(+) cells. Each image is a single plane in a z-stack to ensure true ﬂuorescence signal (not the sum of multiple planes). Scale bar = 25 µm. c JUND protein expression is increased in EGFP(+) cells of cocaine-treated mice (1 h after last dose) compared to saline controls (Student's t-test with Welch’s Correction: **p < 0.0001; Saline mean CTCF = 1807 ± 138.9 SEM (n = 20 cells/3 mice) vs Cocaine mean CTCF = 2920 ± 207.7 SEM (n = 23 cells/3 mice). Welch’s correction: t = 4.54, df = 37.77). d Stereotaxic targeting of HSV-LSL-JUND + HSV-LSL-mCHERRY or HSV-LSL-ΔJUND + HSV-LSL-mCHERRY in NAc of Sst-Cre mice. e qPCR quantiﬁcation of transgene expression in NAc of Sst-Cre mice infected with HSV- LSL-JUND or HSV-LSL-mCHERRY. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 ΔJunD is a truncated form of JunD that lacks the N-terminus, making it a dominant-negative protein. (Student’s t-test with Welch’s correction: p < 0.05; HSV-LSL-JUND mean = 0.01859 ± 0.008903 SEM (n = 8) vs HSV-LSL-ΔJUND mean = 0.0007039 ± 0.0004589 SEM (n = 10) Welch’s Correction: t = 2.259, df = 16). f JunD overexpression in NAc somatostatin interneurons increases locomotor activity in an open ﬁeld (Students t-test: p = 0.051; mCherry mean = 303.4 ± 26.25 SEM vs + JunD mean = 395.5 ± 35.62 SEM (n = 11,11)). g Dominant-negativeΔJunD expression in NAc somatostatin interneurons decreases locomotor responses to cocaine (Repeated Measures Two-way ANOVA: Signiﬁcant effect of Virus F (1,20) = 4.937. p < 0.04; n = 10,10). Data are represented as ± SEM adaptations to repeated cocaine exposure have been previously shown to mediate such homeostatic functions.42,43 The results of this study thereby provide new insight into designing treatments for cocaine addiction that target this small but critical population of NAc interneurons. system induces sustained induction of the protein over several days. It is possible that pulsatile regulation of JunD activity would exert more potent behavioral effects. In any event, we show a positive correlation between JunD transcription and locomotor activity, which together with our behavioral data highlight JunD as a novel cell type-speciﬁc target for future study. Based on the regulation of large numbers of ion channels in NAc somatostatin interneurons following repeated cocaine administration, we studied whether cocaine alters the excitability of these cells and indeed established a cocaine-induced decrease in their intrinsic excitability. Our observation that optogenetic suppression of these cells blunts behavioral responses to cocaine, while activation of the neurons exerts the opposite effect, suggests that cocaine-induced adaptations in NAc somatostatin inter- neurons are homeostatic, namely, that they have the net effect of dampening cocaine responses. Such tolerance to the behavioral effects of cocaine have been observed both in human addicts and in animal models39–41, and several molecular and cellular Discussion h d In this study, we used complementary methods to characterize the role played by NAc somatostatin interneurons in molecular and behavioral responses to cocaine. Using RNA-sequencing, we obtained a total transcriptome map of gene expression changes elicited in NAc somatostatin interneurons by repeated cocaine administration, and used these novel cell type-speciﬁc data to identify a previously unappreciated role for JUND acting in this cell population in cocaine action. We also found that repeated cocaine reduces the intrinsic excitability of NAc somatostatin interneurons and, using optogenetics, that cocaine-induced locomotor activity and reward are dependent on the activity of these neurons, despite their sparse numbers in NAc. Our ﬁnding that optogenetic activation of NAc somatostatin interneurons promotes cocaine responses raises the question as to which neurotransmitter is responsible, given that somatostatin interneurons synthesize and release GABA, SST, and NPY, among other signaling molecules such as NO5,38. Somatostatin interneurons are known to directly inﬂuence distal dendrites of NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunications Methods A i l c Whole-cell quantiﬁcation of current-induced spike numbers in EYFP(+) NAc somatostatin neurons showed that repeated cocaine administration resulted in decreased cellular excitability with 25 and 50 pA current injections as compared to saline-treated controls (Two-way ANOVA, Bonferroni posthoc-test: cocaine treatment F(1, 65) = 7.02, p = 0.0101; current injection F(1, 65) = 269.89, p < 0.0001; interaction F(1, 65) = 0.10, p = 0.7561; n = 31–36 cells per group, 5 mice per group). d Whole-cell quantiﬁcation of current injection-induced ﬁrst spike numbers in EYFP(+) NAc somatostatin neurons showed that increased current was needed to induce the ﬁrst spike (rheobase) in repeated cocaine-treated mice compared to saline controls (Gaussian ﬁt (Shapiro–Wilk) normality test p < 0.05, two-tailed Mann–Whitney test p = 0.0415; n = 31–36 cells per group, 5 mice per group). Data are represented as ± SEM EGFP-F + nuclei were sorted through 100 µm nozzle at 20 PSI and collected in 750 µl of Trizol LS. Immediately after sorting, collection tubes were vortexed and ﬂash frozen on dry ice. Samples were stored frozen in Trizol LS at −80 °C until RNA extraction and library preparation. Immunohistochemistry. Immunohistochemical experiments were performed at room temperature in 2 ml microcentrifuge tubes on a rocking plate. All experi- ments used the same blocking buffer that consists of 0.1% Triton in phosphate- buffered saline and 3% bovine serum albumin (BSA). All sections were mounted using Prolong Gold Antifade (Life Technologies) and kept at 4 °C for short-term storage and −20 °C long-term storage. For SST staining, Rabbit Anti-Somatostatin from Santa Cruz (sc-55565) was used. For JUND staining, Rabbit Anti-JUND from abcam (ab134067) was used. For C-FOS staining, Rabbit Anti-C-FOS from Santa Cruz (sc-52) was used. RNA isolation and library preparation for total RNA-sequencing. RNA was extracted directly from Trizol LS using Directzol RNA-Mini prep (Zymo). Samples were exposed to DNAse for 15 min as per kit protocol. RNA was eluted into 12 µl H2O to ensure viable concentration of RNA for 8 µl library prep input volume limit. Libraries were prepared for paired-end sequencing on HiSeq 2000 using Takara (Formerly Clontech) Smarter Stranded Total RNA-seq Kit (Cat #: 634838) and validated using Agilent Bioanalyzer, Agilent Tapestation, and PCR prior to sequencing. Cruz (sc-52) was used. Quantitative polymerase chain reaction (qPCR). cDNA was generated using iScript cDNA synthesis kit. qPCR was then performed using the 7900HT qPCR system (Thermo Fisher). Methods A i l c Whole-cell quantiﬁcation o current-induced spike numbers in EYFP(+) NAc somatostatin neurons showed that repeated cocaine administration resulted in decreased cellular excitability with 25 and 50 pA current injections as compared to saline-treated controls (Two-way ANOVA, Bonferroni posthoc-test: cocaine treatmen F(1, 65) = 7.02, p = 0.0101; current injection F(1, 65) = 269.89, p < 0.0001; interaction F(1, 65) = 0.10, p = 0.7561; n = 31–36 cells per group, 5 mice per group). d Whole-cell quantiﬁcation of current injection-induced ﬁrst spike numbers in EYFP(+) NAc somatostatin neurons showed that increased curren was needed to induce the ﬁrst spike (rheobase) in repeated cocaine-treated mice compared to saline controls (Gaussian ﬁt (Shapiro–Wilk) normality tes p < 0.05, two-tailed Mann–Whitney test p = 0.0415; n = 31–36 cells per group, 5 mice per group). Data are represented as ± SEM a Avg logCPM 10 ΔlogCPM –2.5 2.5 KCN SCN AMPA GABA A GABA B CaCN NMDA Cacna1e Gabrb2 Gabra2 Gria2 Kcna2 Kcnq2 Kcnh1 Scn8a Scn3b Kcng3 Scn2a1 Cocaine Saline 0 a Excitability Current (pA) 0 5 10 15 20 25 50 25 * s c Spike number d 0 20 40 60 80 * Rheobase Current (pA) Saline Cocaine 20 mV 25 pA b d c c b Current (pA) Fig. 4 The excitability and rheobase of NAc somatostatin interneurons are altered by repeated cocaine exposure. a Expression proﬁling of neuronal ion channels in NAc somatostatin interneurons. Average expression of each transcript is shown as a vertical line for each treatment group with blue intensity representing level of expression. White colored transcripts are lowly or not expressed whereas dark blue transcripts are more highly expressed. Signiﬁcant changes in the transcription of speciﬁc channels after cocaine are shown under the heat map with the change in expression depicted by the font color of the gene name, such that yellow represents increased expression, while blue indicates decreased expression. b Representative traces of spike number obtained by 25 pA current injection in EYFP(+) NAc somatostatin neurons, as a measure of intrinsic neuronal excitability. Methods A i l Methods Animals and drugs. Sst-IRES-Cre(+/+) mice (Sst-Cre; Jackson Labs: Ssttm2.1 (CRE)Zjh/J Stock –013044) were crossed with ROSA-TLG498 (TLG498) condi- tional reporter mice22 which express EGFP-F, a hydrophobic variant of GFP that intercalates in the nuclear and other cellular membranes, in Sst expressing cells. Male 8–12-week-old Sst-Cre mice or Sst-Cre x TLG498 (SST-TLG498) mice, all on a C57BL/6 J background, were fed ad libitum and housed at 22–25 °C on a 12 h light/dark cycle. All experiments were performed in accordance with guidelines from the Society for Neuroscience and the institutional animal care and use committee at Mount Sinai. All mice were acclimated to vivarium conditions for at least 1 week prior to experimental manipulations. Sample sizes were constrained by the availability of transgenic mice but were ultimately determined based on our prior studies. For all experiments, cocaine-HCl (Sigma) was dissolved in sterile saline and administered via intraperitoneal injection. 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 a Avg logCPM 10 ΔlogCPM –2.5 2.5 KCN SCN AMPA GABA A GABA B CaCN NMDA Cacna1e Gabrb2 Gabra2 Gria2 Kcna2 Kcnq2 Kcnh1 Scn8a Scn3b Kcng3 Scn2a1 Cocaine Saline 0 Excitability Current (pA) 0 5 10 15 20 25 50 25 * * Saline Cocaine 20 mV 0.1 s 25 pA b c d 0 20 40 60 80 * Rheobase Current (pA) Spike number Fig. 4 The excitability and rheobase of NAc somatostatin interneurons are altered by repeated cocaine exposure. a Expression proﬁling of neuronal io channels in NAc somatostatin interneurons. Average expression of each transcript is shown as a vertical line for each treatment group with blue intensit representing level of expression. White colored transcripts are lowly or not expressed whereas dark blue transcripts are more highly expressed. Signiﬁcan changes in the transcription of speciﬁc channels after cocaine are shown under the heat map with the change in expression depicted by the font color o the gene name, such that yellow represents increased expression, while blue indicates decreased expression. b Representative traces of spike number obtained by 25 pA current injection in EYFP(+) NAc somatostatin neurons, as a measure of intrinsic neuronal excitability. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 following a stop codon surrounded by loxP sites driven by the IE 4/5 promoter allowing expression of GFP in all transduced cells and expression of our constructs only in cells also expressing Cre recombinase (i.e., somatostatin interneurons). Expression of HSV transgenes is maximal by 1–5 days after infusion. qPCR and immunohistochemistry were used to validate targeting, speciﬁcity, and expression of the transgenes. qPCR primers were made targeting either the C-Terminus or N- terminus of endogenous JunD protein. Since ΔJunD lacks the N-terminus of endogenous JunD, we expect to see more qPCR product with the N-terminus primers in NAc of mice injected with HSV-LSL-JUND compared to HSV-LSL- ΔJUND. transcardially with ice-cold oxygenated artiﬁcial cerebrospinal ﬂuid (ACSF) which contained 128 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 10 mM D-glucose, 24 mM NaHCO3, 2 mM CaCl2 and 2 mM MgCl2 (oxygenated with 95% O2 and 5% CO2, pH 7.35, 295–305 mOsm). Acute brain slices containing the NAc were sec- tioned at 250 µm in cold sucrose ACSF, which was derived by replacing NaCl with 254 mM sucrose. Slices recovered at 37 °C for 1 h in oxygenated ACSF. Somatos- tatin cells infected in vivo with the CHR2-expressing AAV were identiﬁed via ﬂuorescence visualization. Whole-cell current-clamp recordings were performed at −60 mV, the average resting membrane potential of somatostatin cells in NAc. Glass electrodes (3–5 MΩ) were ﬁlled with an internal solution containing 115 mM potassium gluconate, 20 mM KCl, 1.5 mM MgCl2, 10 mM phosphocreatine, 10 mM HEPES, 2 mM magnesium ATP, and 0.5 mM GTP (pH 7.2, 285 mOsm). Optic ﬁbers were connected using an FC/PC adapter to a 473-nm blue laser diode (Crystal Laser) and a stimulator (Agilent Technologies, no. 33220A) was used to generate blue light pulses. For ex vivo slice electrophysiological validation of CHR2 activation, we tested a series of burst stimulations to mimic the plateau low threshold spiking patterns observed physiologically in these neurons. Neurons were exposed to 10 spikes (4 ms width) over each 10 s epoch with increasing frequencies. Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) in sterile saline and administered by intraperitoneal injection. Thirty-three gauge needles were used to bilaterally infuse AAV or HSV vectors into NAc (AP = 1.5, ML = ± 1.5, and DV = −4.4; 10° angle). References 1. Lin, L.-C. & Sibille, E. Reduced brain somatostatin in mood disorders: a common pathophysiological substrate and drug target? Front. Pharmacol. 4, 110 (2013). 2. Lin, L. C. & Sibille, E. Somatostatin, neuronal vulnerability and behavioral emotionality. Mol. Psychiatry 20, 377–387 (2015). 3. Tepper, J. M., Tecuapetla, F., Koos, T. & Ibanez-Sandoval, O. Heterogeneity and diversity of striatal GABAergic interneurons. Front. Neuroanat. 4, 150 (2010). 4. Marin, O., Anderson, S. A. & Rubenstein, J. L. Origin and molecular speciﬁcation of striatal interneurons. J. Neurosci. 20, 6063–6076 (2000). 5. Kepecs, A. & Fishell, G. Interneuron cell types are ﬁt to function. Nature 505, 318–326 (2014). CPP with optogenetic manipulation of NAc somatostatin interneuron. An unbiased CPP paradigm utilizing Med Associates software and 3-chamber CPP boxes. The boxes consist of a small middle chamber, and two larger chambers on eat side. The larger conditioning chambers include different contextual cues (i.e., different spacing on wire grid ﬂooring, different colored striped walls). Sst-Cre mice infected with AAV-DIO-EYFP or AAV-DIO-CHR2-EYFP were placed in a three- chamber CPP box for a 20 min “pretest” to ensure that groups had no chamber bias; <10% of all mice show a bias deﬁned as 100 s. Groups were then balanced to adjust for any chamber bias that remained between the groups. In total, a small number of mice with pre-existing bias >600 s were excluded from the studies prior to training. Mice were then trained for 2 days to cocaine (2.5, 5 or 7.5 mg/kg) without or with optogenetic stimulation or suppression or to optogenetic manip- ulations alone. For optogenetics experiments, optical ﬁbers were not attached during the pretest or test days. During training ﬁber optic patch cords were secured to the cannulas immediately following injection of either saline or cocaine. In the ﬁrst training session, mice were injected with saline and conﬁned to one side of the apparatus during which time they were connected to ﬁber optics but the lasers were not turned on. In the second session, lasers were turned on after cocaine injections and mice were conﬁned to the opposite chamber. Cocaine was only given in the second session to ensure that no cocaine was present in the mice during saline pairing. The experiments were counterbalanced to ensure that cocaine was paired evenly in both sides of the chambers. After two training days, mice were tested and CPP scores determined. References CPP scores represent: (time spent in the Cocaine/Opto- genetics paired chamber) −(time spent in the unpaired chamber). 6. Stefanelli, T., Bertollini, C., Lüscher, C., Muller, D. & Mendez, P. Hippocampal somatostatin interneurons control the size of neuronal memory ensembles. Neuron 89, 1074–1085 (2016). 7. Chiu, C. Q. et al. Compartmentalization of GABAergic inhibition by dendritic spines. Science 340, 759–762 (2013). 8. Hathway, G. J., Humphrey, P. P. A. & Kendrick, K. M. Somatostatin induces striatal dopamine release and contralateral turning behaviour in the mouse. Neurosci. Lett. 358, 127–131 (2004). 9. Kouvidi, E., Papadopoulou-Daifoti, Z. & Thermos, K. Somatostatin modulates dopamine release in rat retina. Neurosci. Lett. 391, 82–86 (2006). 10. Ikeda, H., Kotani, A., Koshikawa, N. & Cools, A. R. Somatostatin receptors in the nucleus accumbens modulate dopamine-dependent but not acetylcholine- dependent turning behaviour of rats. Neuroscience 159, 974–981 (2009). p g pp p g p ulations alone. For optogenetics experiments, optical ﬁbers were not attached during the pretest or test days. During training ﬁber optic patch cords were secured to the cannulas immediately following injection of either saline or cocaine. In the ﬁrst training session, mice were injected with saline and conﬁned to one side of the apparatus during which time they were connected to ﬁber optics but the lasers were not turned on. In the second session, lasers were turned on after cocaine injections and mice were conﬁned to the opposite chamber. Cocaine was only given in the second session to ensure that no cocaine was present in the mice during saline pairing. The experiments were counterbalanced to ensure that cocaine was paired evenly in both sides of the chambers. After two training days, mice were tested and CPP scores determined. CPP scores represent: (time spent in the Cocaine/Opto- genetics paired chamber) −(time spent in the unpaired chamber). 11. Lopez-Huerta, V. G., Tecuapetla, F., Guzman, J. N., Bargas, J. & Galarraga, E. Presynaptic modulation by somatostatin in the neostriatum. Neurochem. Res. 33, 1452–1458 (2008). 12. Ikeda, H., Kamei, J., Koshikawa, N. & Cools, A. R. Nucleus accumbens and dopamine-mediated turning behavior of the rat: role of accumbal non- dopaminergic receptors. J. Pharmacol. Sci. 120, 152–164 (2012). 13. Raynor, K., Lucki, I. & Reisine, T. Somatostatin receptors in the nucleus accumbens selectively mediate the stimulatory effect of somatostatin on locomotor activity in rats. J. Pharmacol. Exp. Ther. 265, 67–73 (1993). 14. Santis, S. et al. Cocaine-induced locomotor activity with optogenetic manipulation of NAc Cocaine-induced locomotor activity with optogenetic manipulation of NAc somatostatin interneuron activity. Locomotor activity was performed in the following manner using rat-sized cages set up in locomotor apparati such that mice were assessed for movement on an x–y plane as measured by laser-grid beambreaks with Med Associates software. On day 1–5, pre-made optical ﬁbers (Doric Lenses) were secured bilaterally to the ﬁber optic cannula (Doric Lenses) and the mice were allowed to habituate to the test environment for 5 min prior to drug administration and light manipulation. Then, either saline or cocaine (7.5 mg/kg IP) was admi- nistered and the light function generator was activated to induce “Stimulation” (473 nm, 20 Hz frequency, 1 burst of 10 pulses, 4 ms pulse width, every 10 s) or “Suppression/Depolarization Block” (473 nm, 20 Hz frequency, 1 burst of 20 pul- ses, 49 ms pulse width, every sec) in somatostatin interneurons adapted from previous studies17. Both stimulation and suppression were validated in brain slices (Supplementary Fig. 2). Control EYFP mice were also exposed to laser manip- ulations. Locomotor activity was assessed for 30 min on each day while being paired with optogenetic manipulation. Mice were perfused 1 h after the ﬁnal injection of cocaine on day 7. Received: 2 September 2017 Accepted: 12 July 2018 ARTICLE An infusion volume of 0.5 μl was delivered using a 5 μl Hamilton syringe over the course of 5 min (at a rate of 0.1 μl/min). The infusion needle remained in place for at least 5 min after the infusions before removal to prevent backﬂow of the viruses. For optogenetics experiments, custom- made optical ferrules (Doric Lenses) with ﬁbers 3.9 mm in length from the base of the cannula were implanted up to 3 weeks after viral injection targeting the NAc (AP = 1.5; ML = 1.3; DV = –3.9; 0° angle) and mice were allowed to recover for at least 1 day before behavioral experiments. Electrophysiological recording of NAc somatostatin interneurons after chronic cocaine. Sst-Cre mice were injected with AAV-DIO-EYFP and injected with either saline or cocaine for 7 days in the home cage. Mice were sacriﬁced 1 h following the ﬁnal injection of cocaine/saline, as described above. All recordings were carried out blind to the experimental conditions. The anatomical location of the EYFP(+) neurons was validated within the NAc in each recording. Whole-cell recordings were obtained from 250 µm NAc brain slices. Spike number was obtained by 25 pA and 50 pA current injections in 0.5 s increments in current- clamp mode, as a measure of intrinsic neuronal excitability. Whole-cell quantiﬁ- cation of current injection-induced ﬁrst spike numbers was used to determine the rheobase. Cocaine-induced locomotor activity in SST-TLG498 mice. A population of 20 individual SST-TLG498 mice were exposed to either daily saline or cocaine injections (7.5 mg/kg) for 7 days and locomotor activity was assessed21. Brieﬂy, activity was assessed in rat-sized cages within a locomotor apparatus and mice were evaluated for horizontal ambulation in the x and y planes by quantifying laser-grid beambreaks with Med Associates software. An hour after the ﬁnal injection of saline or cocaine, the mice were euthanized by decapitation and individual brains were extracted. Brains were immediately sectioned and a 200 µm thick section containing NAc was obtained. 14 gauge blunted needles were used to collect bilateral tissue punches of NAc from individual mice, which were immediately frozen on dry ice and stored at −80 °C until the day of FACS. Data availability statement. All of the individual raw RNA-sequencing ﬁles are available to the public through the NIH GEO data repository via ascension number: GSE116484 Data availability statement. Methods A i l CT values were normalized to GAPDH, which was not affected by cocaine exposure. Primers used include: Sst – FWD: AAGGAA GATGCTGTCCTGCC, REV: TTGGCCAGTTCCTGTTTCCC; JunD C-Terminus – FWD: TTCTATGGCGAGGAGGCTCT, REV: GAGCTCCAGGGAAAGC TCTG; JunD N-Terminus – FWD: CTGGTGACCACCACACCTAC, REV: CGGCAAATTCCTGCTCTTCG. Bioinformatics analyses. Raw 100 bp cDNA reads were aligned to the Ensembl GRCm38.75 mouse reference genome via STAR. Raw read count values were deduced from quantitation against the Ensembl GRCm38.75 annotation features using featureCounts from the Subread package44. These raw count values were normalized by the weighted trimmed mean of M-values, which scales the samples by the raw library sizes. This normalized gene expression matrix used was used to study differences in gene expression between cocaine- and saline-treated groups. Differential expression was estimated using the Voom method and genes were considered differentially regulated at a nominal p-value threshold of 0.05. Nuclear FACS isolation from individual SST-TLG498 mice. Nuclei were isolated from SST-TLG498 NAc punches of individual mice and prepared for FACS with modiﬁcations to established methods23,24. Triton X-100 was omitted from all buffers to maintain EGFP-F in the nuclear membrane. Lysed cellular extracts were spun on a sucrose gradient in an ultracentrifuge at 24.4 K RPM at 4 °C for 1 h with a swing bucket rotor. Nuclei were triturated and vortexed into a suspension and ﬁltered through a 35 µm mesh. Wild-type mice were used to establish gating strategy on a FACS machine before transgenic samples were analyzed. On average, EGFP-F + nuclei accounted for a small but replicable percentage (1–2%) of the nuclei in each set of punches, consistent with previous histological estimates3. Viral vectors and stereotaxic surgery. AAV-Ef1a-DIO-CHR2(H134R)-EYFP or AAV-EF1A-DIO-EYFP were obtained from the UNC viral vector core facility. JunD and ΔJunD were mouse codon optimized, de novo synthesized and sequence-validated. They were individually sub-cloned into bi-cistronic HSV vectors that express GFP under the CMV promoter. The transgenes were inserted AAV-EF1A-DIO-EYFP were obtained from the UNC viral vector core facility. JunD and ΔJunD were mouse codon optimized, de novo synthesized and sequence-validated. They were individually sub-cloned into bi-cistronic HSV vectors that express GFP under the CMV promoter. The transgenes were inserted NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunications 8 ARTICLE All of the individual raw RNA-sequencing ﬁles are available to the public through the NIH GEO data repository via ascension number: GSE116484 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-05657-9 15. Heiman, M. et al. A translational proﬁling approach for the molecular characterization of CNS cell types. Cell 135, 738–748 (2008). 40. Hammer, R. P., Egilmez, Y. & Emmett-Oglesby, M. W. Neural mechanisms of tolerance to the effects of cocaine. Behav. Brain. Res. 84, 225–239 (1997). yp 16. Zhang, F. et al. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nat. Protoc. 5, 439–456 (2010). 41. Schenk, S. & Partridge, B. Sensitization and tolerance in psychostimulant self- administration. Pharmacol. Biochem. Behav. 57, 543–550 (1997). 17. Herman, A. M., Huang, L., Murphey, D. K., Garcia, I. & Arenkiel, B. R. Cell type-speciﬁc and time-dependent light exposure contribute to silencing in neurons expressing Channelrhodopsin-2. eLife 3, e01481 (2014). 42. Carlezon, W. A., Duman, R. S. & Nestler, E. J. The many faces of CREB. Trends Neurosci. 28, 436–445 (2005). 43. Dong, Y. et al. CREB modulates excitability of nucleus accumbens neurons. Nat. Neurosci. 9, 475–477 (2006). 18. Veit, J., Hakim, R., Jadi, M. P., Sejnowski, T. J. & Adesnik, H. Cortical gamma band synchronization through somatostatin interneurons. Nat. Neurosci. 20, 951–959 (2017). 44. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efﬁcient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014). 19. Hamm, J. P. & Yuste, R. Somatostatin interneurons control a key component of mismatch negativity in mouse visual cortex. Cell Rep. 16, 597–604 (2016). 20. Straub, C. et al. Principles of synaptic organization of GABAergic interneurons in the striatum. Neuron 92, 84–92 (2016). Author Contributions 25. Lacar, B. et al. Nuclear RNA-seq of single neurons reveals molecular signatures of activation. Nat. Commun. 7, 11022 (2016). E.A.R. and E.J.N. wrote the manuscript; E.A.R., J.W.K., R.C.B., E.S.C., P.J.H., S.M.K., M.-H.H. and E.J.N. designed research; E.A.R., H.K., E.S.C., R.C.B., J.W.K., M.D., C.L., B.J., S.M.K. and M.S. performed experiments. E.A.R., J.R.S., A.K., I.P., J.W.K., B.J., E.A.R. and E.J.N. wrote the manuscript; E.A.R., J.W.K., R.C.B., E.S.C., P.J.H., S.M.K., M.-H.H. and E.J.N. designed research; E.A.R., H.K., E.S.C., R.C.B., J.W.K., M.D., C.L., B.J., S.M.K. and M.S. performed experiments. E.A.R., J.R.S., A.K., I.P., J.W.K., B.J., S.M.K. and M.S. analyzed data; E.A.R., E.S.C., and E.J.N made the ﬁgures; E.S.C. made the ﬁgure schematics; P.J.H., R.L.N., D.B., S.P., M.-H.H, A.S. and E.M. provided and prepared reagents and materials necessary for the experiments. 26. Petilla Interneuron Nomenclature Group. et al. Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex. Nat. Neurosci. 9, 557–568 (2008). B.J., S.M.K. and M.S. performed experiments. E.A.R., J.R.S., A.K., I.P., J.W.K., B.J., S.M.K. and M.S. analyzed data; E.A.R., E.S.C., and E.J.N made the ﬁgures; E.S.C. made the ﬁgure schematics; P.J.H., R.L.N., D.B., S.P., M.-H.H, A.S. and E.M. provided and prepared reagents and materials necessary for the experiments. 27. Fanara, P. et al. Changes in microtubule turnover accompany synaptic plasticity and memory formation in response to contextual fear conditioning in mice. Neuroscience 168, 167–178 (2010). Additional information 28. 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Neurosci. 29, 3529–3537 (2009). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 32. Robison, A. J. et al. Behavioral and structural responses to chronic cocaine require a feedforward loop involving ΔFosB and calcium/calmodulin- dependent protein kinase II in the nucleus accumbens shell. J. Neurosci. 33, 4295–4307 (2013). 33. Ferguson, D. et al. SIRT1-FOXO3a regulate cocaine actions in the nucleus accumbens. J. Neurosci. 35, 3100–3111 (2015). 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BMC Bioinform. 14, 128 (2013). enrichment analysis tool. BMC Bioinform. 14, 128 (2013) 35. Kuleshov, M. V. et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Acknowledgements interneurons in the striatum. Neuron 92, 84–92 (2016). We would like to acknowledge the Mount Sinai Flow Cytometry Core Facility for their help and expertise in the cell sorting experiments performed. This work was supported by grants from the National Institute on Drug Abuse (P01DA008227 and R01DA007359 to EJN and K99DA024111 to E.S.C.), the Brain and Behavior Research Foundation (to E.S.C.), and Korea Brain Research Institute basic research program Grant No. 18-BR-02- 03 (to J.W.K.). 21. Lobo, M. K. et al. Cell type-speciﬁc loss of BDNF signaling mimics optogenetic control of cocaine reward. Science 330, 385–390 (2010). 22. Chakravarthy, S. et al. Cre-dependent expression of multiple transgenes in isolated neurons of the adult forebrain. PLoS ONE 3, e3059 (2008). 23. Matevossian, A., ., & Akbarian, S. Neuronal nuclei isolation from human postmortem brain tissue. J. Vis. Exp. 1, 914 (2008). 24. Jiang, Y., Matevossian, A., Huang, H.-S., Straubhaar, J. & Akbarian, S. Isolation of neuronal chromatin from brain tissue. BMC Neurosci. 9, 42 (2008). References Somatostatin increases rat locomotor activity by activating sst (2) and sst (4) receptors in the striatum and via glutamatergic involvement. N. S. Arch. Pharmacol. 379, 181–189 (2009). Ex vivo slice validation of optogenetic stimulation protocol in NAc soma- tostatin interneurons. Three weeks after viral injections, the mice were perfused 9 NATURE COMMUNICATIONS \| (2018) 9:3149 \| DOI: 10.1038/s41467-018-05657-9 \| www.nature.com/naturecommunications © The Author(s) 2018 Additional information Nucleic Acids Res. 44, W90–W97 (2016). 36. Winstanley, C. A. et al. DeltaFosB induction in orbitofrontal cortex mediates tolerance to cocaine-induced cognitive dysfunction. J. 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https://openalex.org/W2908223980	https://science.swsu.ru/jour/article/download/347/340	Russian	null	PENSION AGE IN THE RUSSIAN FEDERATION: ASSESSMENT OF THE EFFECTIVENESS OF THE PREVENTIVE INCREASE	Izvestiâ Ûgo-Zapadnogo gosudarstvennogo universiteta	2,018	cc-by	3,986	Т. А. Бочкова, канд. экон. наук, доцент, Кубанский государственный аграрный университет (Краснодар, Россия) (e-mail: bochkova-tatiana@mail.ru) Т. А. Бочкова, канд. экон. наук, доцент, Кубанский государственный аграрный университет (Краснодар, Россия) (e-mail: bochkova-tatiana@mail.ru) 111 111 УДК 331 УДК 331 Т.А. Световцева, канд. экон. наук, профессор, ФГБОУ ВО «Юго-Западный государственный университет» (Курск, Россия) (e-mail:svetovtseva2018@yandex.ru) С.А. Мамий, канд. экон. наук, доцент, Кубанский государственный аграрный университет (Краснодар, Россия) (e-mail:aslambechevna-sima@mail.ru) С.А. Мамий, канд. экон. наук, доцент, Кубанский государственный аграрный универс (Краснодар, Россия) (e-mail:aslambechevna-sima@mail.ru) Т. А. Бочкова, канд. экон. наук, доцент, Кубанский государственный аграрный университет (Краснодар, Россия) (e-mail: bochkova-tatiana@mail.ru) ПЕНСИОННЫЙ ВОЗРАСТ В РОССИЙСКОЙ ФЕДЕРАЦИИ: ОЦЕНКА ЭФФЕКТИВНОСТИ ПРЕДПОЛАГАЕМОГО ПОВЫШЕНИЯ Время выхода на пенсию является рычагом, регулирующим соотношение численности трудоспособ- ных россиян и пенсионеров - чем оно выше, тем большее число граждан становятся плательщиками, а не получателями пенсионных выплат. В данной работе изучена проблематика увеличения пенсионного возраста как фактор макроэкономи- ческого развития Российской Федерации, а также проанализированы возможные последствия данного увеличения для возрастной категории граждан и молодежи. Цель настоящей работы – произвести оценку эффективности предполагаемого повышения пенсионного возраста в нашей стране и спрогнозировать влияние данного экономического рычага на занятость населения. Выявлено, что показатель пенсионного возраста, являясь своеобразным рычагом, может и должен применяться не как инструмент экономии бюджетных средств, а прежде всего, как механизм социального регулирования. Рассмотрено влияние данной меры на государство, экономику и благосостояние различных групп населения. В конце работы сделаны выводы о выгодах и издержках от предполагаемого повышения. Ключевые слова: пенсионный возраст, безработица, пенсионная реформа, дефицит бюджета. DOI: 10.21869/2223-1560-2018-22-2-111-118 Ссылка для цитирования: Световцева Т.А., Мамий С.А., Бочкова Т. А. Пенсионный возраст в Российской Федерации: оценка эффективности предполагаемого повышения // Известия Юго-Западного государственного университета. 2018. Т. 22, № 2(77). С. 111-118. Пенсионный возраст – установлен- ный государством возраст гражданина, по достижении которого он имеет право претендовать на получение пенсии по старости. Достигнув пенсионного возрас- та, гражданин превращается из плательщи- ка пенсионных взносов в получателя пен- сионных выплат из пенсионного фонда. жащих с тяжёлыми условиями труда воз- раст выхода на пенсию еще ниже. Однако в правительстве России в по- следние годы активно обсуждается воз- можность повышения пенсионного воз- раста граждан как залог устойчивости пенсионной системы в долгосрочной пер- спективе, а также один из инструментов преодоления кризиса в экономике. Экс- пертное сообщество не приняло оконча- тельного решения по данному вопросу, но сходится в понимании того, что уве- личение пенсионного возраста неизбеж- но. Расхождения во мнениях встречаются лишь относительно сроков и механизмов реализации данной реформы. На сегодняшний день пенсионный возраст в большинстве стран мира со- ставляет порядка 60 - 65 лет. В Россий- ской Федерации возрастная планка выхо- да на пенсию значительно ниже. Дей- ствующее в нашей стране пенсионное обеспечение по старости ввели в СССР в 1932 году. Был утвержден следующий пенсионный возраст: мужчины – 60 лет, женщины – 55 лет. Для рабочих и слу- Проблематика усугубляется тем, что в работоспособный возраст начинают вступать представители поколения, ро- ISSN 2223-1560. Известия Юго-Западного государственного университета. 2018. Т. 22, № 2 Т.А. Световцева, С.А. Мамий, Т. А. Бочкова 112 дившиеся в 90-е годы прошлого столетия, когда отмечался рекордно низкий уро- вень рождаемости. ПЕНСИОННЫЙ ВОЗРАСТ В РОССИЙСКОЙ ФЕДЕРАЦИИ: ОЦЕНКА ЭФФЕКТИВНОСТИ ПРЕДПОЛАГАЕМОГО ПОВЫШЕНИЯ Это означает, что чис- ленность пенсионеров будет неуклонно расти, а число работающих граждан сни- жаться. Все это потребует решительных мер со стороны государства, так как сба- лансировать бюджет пенсионного фонда в данных условиях без дополнительных вливаний не получится. Цель настоящей работы – произвести оценку эффективности предполагаемого повышения пенсионного возраста в нашей стране и спрогнозировать влияние данного экономического рычага на заня- тость населения. дившиеся в 90-е годы прошлого столетия, когда отмечался рекордно низкий уро- вень рождаемости. Это означает, что чис- ленность пенсионеров будет неуклонно расти, а число работающих граждан сни- жаться. Все это потребует решительных мер со стороны государства, так как сба- лансировать бюджет пенсионного фонда в данных условиях без дополнительных вливаний не получится. Исходя из вышеуказанной цели, формируем следующие задачи: 1. Рассчитать влияние предполагаемо- го увеличения пенсионного возраста на пенсионную систему РФ, занятость насе- ления, а также бюджет и экономику РФ. На тринадцатой по счету пресс- конференции президента России, органи- зованной 14 декабря 2017 года, журнали- сты выяснили точку зрения Владимира Путина касаемо данной непопулярной меры: «Возможное повышение пенсион- ного возраста не должно быть шоковым, оно не будет приниматься кулуарно». Глава государства заявил, что резолюция об увеличении не утверждена, но развер- нутый ответ президента сигнализировал, что это вопрос времени. Владимир Путин апеллировал к опыту стран ближнего за- рубежья – Белоруссии, Казахстана и Украины, где уже принято решение о по- вышении пенсионного возраста. 2. Оценить обновленную расстановку сил различных возрастных групп на рын- ке труда, определить возможные послед- ствия изменений для каждой возрастной группы. На стыке XIX–XX веков в мировом сообществе начали формироваться пен- сионные системы, основной целью кото- рых являлось оказание помощи людям, достигшим возраста нетрудоспособности и утратившим возможность обеспечивать собственное существование. Германия стала первым государством, внедрившим данную систему. Системы каждой страны формировались в соответствии с их финан- совыми возможностями, что в большин- стве случаев прямым образом повлияло на установление высокого возрастного преде- ла выхода на пенсию (табл. 1). В данной работе спрогнозированы вероятные последствия увеличения пен- сионного возраста как фактора макроэко- номического развития Российской Феде- рации, что крайне актуально в настоящее время. Таблица 1 Таблица 1 Нормативный возраст выхода на пенсию по странам Страна Пенсионный возраст (лет) Год Мужчины Женщины Россия 60 55 2017 Белоруссия 60,5 55,5 2017 Казахстан 63 58 2017 Украина 60 58 2017 Германия 67 65 2017 Великобритания 65 65 2017 США 67 65 2017 Япония 65 65 2017 И В е й б Е Р ISSN 2223-1560. Известия Юго-Западного государственного университета. 2018. Т. Источники: Всемирный банк, Евростат, Росстат. ISSN 2223-1560. Известия Юго-Западного государственного университета. 2018. Т. 22, № 1(76) В СССР низкий пенсионный возраст преподносился как завоевание трудящих- ся [1]. инструментом решения назревшей про- блемы. Однако в условиях экономическо- го спада повышение налогообложения окажет дестимулирующий эффект на средний и малый бизнесы, а также про- чих экономических агентов, что может стать дополнительным препятствием для выхода из кризиса. Повышение же пен- сионного возраста, по мнению бывшего министра финансов Алексея Кудрина, позволит проводить более высокую ин- дексацию пенсий, что станет дополни- тельным социальным плюсом. Выгоды для государства достаточно очевидны [3]. В СССР низкий пенсионный возраст преподносился как завоевание трудящих- ся [1]. Время выхода на пенсию является рычагом, регулирующим соотношение численности трудоспособных россиян и пенсионеров - чем оно выше, тем боль- шее число граждан становятся платель- щиками, а не получателями пенсионных выплат. Пенсионный возраст – один из ос- новных инструментов макроэкономиче- ской системы страны. Сокращение числен- ности пенсионеров оказывает положитель- ное влияние на страну в целом: система становится финансово устойчивой [2]. С экономической точки зрения мож- но обозначить следующие плюсы: повы- шение пенсионного возраста будет спо- собствовать росту ВВП, так как увели- чившаяся численность трудовых ресур- сов спровоцирует рост занятости в пери- од экономического подъема [13, 14]. Но сможет ли отечественный рынок полно- стью принять предложение труда работ- ников в возрасте? Существует вероятность того, то эти работники могут остаться без работы и без пенсии, что, безусловно, спровоцирует социальную напряженность, так как размер пособия по безработице в России практически вдвое меньше средней пенсии (табл. 2 и 3) [4]. В начале 2017 года Минтруд Россий- ской Федерации осуществил публикацию проектов нормативных актов на Феде- ральном портале, где, в том числе, пред- ставил проект бюджета Пенсионного фонда России на 2017-2019 годы. Со- гласно документу, к концу текущего года ожидается дефицит бюджета в размере 181,7 миллиарда рублей, в 2018 году - 177,3 миллиарда рублей, в 2019 году - 167,6 миллиарда рублей. На покрытие дефицита бюджета Правительством РФ выделяются колос- сальные средства, и повышение пенсион- ного возраста, наряду с повышением налогов, представляется эффективным Таблица 2 Таблица 2 Средний размер пенсионных выплат Год 2013 2014 2015 2016 2017 Средний размер пенсии, руб. 9153,6 10029,7 10888,7 12406,0 13700,0 Источники: Росстат, Пенсионный фонд. программы для лиц старшего возраста, чтобы увеличить продолжительность их активной жизни, а также быть готовыми к выплатам пособий по безработице. Со- ответственно, повышение пенсионного возраста для экономики нашей страны может иметь двоякие последствия [5]. программы для лиц старшего возраста, чтобы увеличить продолжительность их активной жизни, а также быть готовыми к выплатам пособий по безработице. Со- ответственно, повышение пенсионного возраста для экономики нашей страны может иметь двоякие последствия [5]. ПЕНСИОННЫЙ ВОЗРАСТ В РОССИЙСКОЙ ФЕДЕРАЦИИ: ОЦЕНКА ЭФФЕКТИВНОСТИ ПРЕДПОЛАГАЕМОГО ПОВЫШЕНИЯ 22, № 1(76) Пенсионный возраст в Российской Федерации: оценка эффективности предполагаемого повышения 113 В СССР низкий пенсионный возраст преподносился как завоевание трудящих- ся [1]. В СССР низкий пенсионный возраст преподносился как завоевание трудящих- ся [1]. В любом случае государство будет вынуждено нести определенные издерж- ки, так как необходимо повышать квали- фикацию и обучать лиц старшего возрас- та с целью повышения их конкуренто- способности на рынке труда, в чем не за- интересованы работодатели. Помимо прочего, нужно разработать медицинские ISSN 2223-1560. Известия Юго-Западного государственного университета. 2018. Т. 22, № 2(77) Т.А. Световцева, С.А. Мамий, Т. А. Бочкова 114 Таблица 3 Таблица 3 Величина пособия по безработице Год Величина пособия Основание (постановление) Минимум Максимум 2017 850 рублей 4900 рублей Постановление Пра- вительства РФ от 8.12.2016 г. № 1326 2016 850 рублей 4900 рублей Постановление Пра- вительства РФ от 12.11.2015 № 1223 2015 850 рублей 4900 рублей Постановление Пра- вительства РФ от 17.12.2014 № 1382 Источники: КонсультантПлюс, Гарант.ру Источники: КонсультантПлюс, Гарант.ру С социальной точки зрения ситуация такова: с 2015 года и по настоящее время в нашей стране действует добровольная система, разработанная для того, чтобы не принимать болезненных для каждого будущего пенсионера решений. По дан- ной системе пособие формируется из стажа работы и официальной заработной платы. Величина упомянутых элементов прямо пропорционально влияет на вели- чину пособия. В формуле имеются при- бавочные коэффициенты: если гражданин отложил выход на пенсию на пять лет и не получал в это время пособие по старо- сти, размер его пенсии будет в полтора раза выше и т.д. [6]. чтобы иметь возможность больше полу- чать в недалеком будущем. По факту вы- яснилось, что граждане имеют недоверие к государственной системе и предпочи- тают безотлагательно воспользоваться даже небольшой прибавкой. Другими словами, добровольная схема в России не сработала. Откладывать прибыль на бу- дущее россияне не готовы и сопротивля- ются перспективе повышения пенсионно- го возраста. На вопрос исследовательского цен- тра портала Superjob.ru: «Согласны ли Вы с тем, что возраст выхода на пенсию нужно повысить (сегодня он составляет 55 лет для женщин и 60 лет — для муж- чин)?» в 2015 году жители России отве- тили следующим образом (табл. 4). Каждый третий россиянин, по пред- варительным прогнозам, должен был воспользоваться предложенной схемой, Таблица 4 ц Результаты опроса населения РФ Ответ Все Пол Возраст, лет Мужчины Женщины До 24 25-34 35-44 45 Да, мужчинам, и женщинам 8% 7% 8% 9% 7% 8% 7% Да, мужчинам 2% 2% 2% 2% 2% 1% 2% Да, женщинам 2% 4% 1% 2% 2% 2% 4% Нет 81% 79% 82% 76% 4% 2% 79% Затрудняюсь 7% 8% 7% 11% 5% 7% 8% Источник: Superjob.ru. 2015. – 24 сентября. ISSN 2223-1560. Известия Юго-Западного государственного университета. 2018. Т. В СССР низкий пенсионный возраст преподносился как завоевание трудящих- ся [1]. 22, № 1(76) 115 Пенсионный возраст в Российской Федерации: оценка эффективности предполагаемого повышения Опрошенные считают: Опрошенные считают: Это позволит гражданам России лучше адаптироваться к новым условиям. – Средняя продолжительность жизни в России ниже, чем в развитых странах мира (66 лет), повышение пенсионного возраста приведет к тому, что многие не смогут дожить до собственной пенсии. Граждане в этом возрасте будут вы- нуждены обеспечивать себя, поэтому, как следствие, можно ожидать рост предло- жения труда на рынке. В 2008 году рос- сийским экономистом Е. Т. Гурвичем были озвучены следующие прогнозы: ес- ли повысить пенсионный возраст до 62,5 лет и мужчинам, и женщинам, то общая численность работников увеличилась бы на 9,9 %, численность пенсионеров со- кратилась бы на 35,9 %, что привело бы к росту ставки замещения до 71 % [8]. – Выплата пенсий – социальное обя- зательство государства, от которых оно, таким образом, косвенно уклоняется. – Отсутствие пенсионных выплат снизит уровень жизни населения. – Возможностей трудоустройства для возрастной категории граждан крайне мало. Возрастная категория граждан на- чнет конкурировать с более молодыми работниками, что также может повлечь рост безработицы среди молодежи. Рабо- тодателю выгодно продолжить сотрудни- чество с опытным работником, обладаю- щим профессиональным багажом знаний, нежели нести издержки на адаптацию и обучение нового сотрудника. Таким образом, население нашей страны не одобряет повышение пенсион- ного возраста, в связи с чем, правитель- ство РФ пока не решается применить данный экономический рычаг [7]. В настоящий момент предлагаются различные схемы повышения пенсионно- го возраста, какое-либо единое мнение по этому счету отсутствует. Опираясь на опыт соседних стран, некоторые экспер- ты считают, что наиболее рационально - повышать пенсионный возраст ежегодно по три-шесть месяцев до 63-х лет. По данным Росстата, в июне 2017 года среди безработных доля молодежи до 25 лет составила 18,7%, данный про- цент, при увеличении пенсионного воз- раста, будет неуклонно расти [9]. Рис. 1. Безработица по социальным группам в июне 2017 г. сточник: Росстат 0 10 20 30 40 50 60 70 01.07.2017 Женщины Городские жители Молодежь до 25 лет Лица, не имеющие опыта трудовой деятельности Женщины Городские жители Молодежь до 25 лет Лица, не имеющие опыта трудовой деятельности Рис. 1. Безработица по социальным группам в июне 2017 г. сточник: Росстат ISSN 2223-1560. Известия Юго-Западного государственного университета. 2018. Т. 22, № 2(77) Т.А. Световцева, С.А. Мамий, Т. А. Бочкова 116 сии неуклонно стареет, поэтому выше- указанное повышение будет являться действенным рычагом в регулировании экономического баланса потребителей национального дохода и трудоспособно- го населения. Обратная сторона медали: усугубляющее проблемы бедности сни- жение ставки заработной платы в связи с повышением предложения рабочей силы. Опрошенные считают: В периоды кризиса имеется вероятность массовой безработицы. Помимо прочего, государство понесет расходы на образо- вательные и медицинские программы для возрастных работников, стабилизирую- щую поддержку россиян, утративших трудоспособность до наступления пенси- онного возраста. Если плюсы от увеличе- ния пенсионного возраста превысят ми- нусы, меру можно считать экономически эффективной. Безработица ожидает и лиц от 55 лет, которым особенно тяжело будет трудо- устроиться, оказавшись среди безработ- ных в немолодом возрасте. Проблемы со здоровьем также будут отрицательно влиять на конкурентоспособность. Не ис- ключена возможность массовой безрабо- тицы, как социальной катастрофы. Некоторые эксперты имеют и прямо противоположное мнение: молодежь и люди в возрасте крайне редко пересека- ются на рынке труда. Молодежь, как пра- вило, устраивается в частные организа- ции, тогда как возрастная категория граждан работает в государственных учреждениях и бюджетных организаци- ях. Среди людей в возрасте многие явля- ются инженерными специалистами, а мо- лодые работники наполняют IT и фи- нансовый сектор [10]. Появление на рынке труда опытных работников негативно отразится на заня- тости и карьере молодежи, так как пожи- лые работники будут продолжать зани- мать выгодные рабочие места. Так как увеличение предложения ра- бочей силы обратно пропорционально уве- личению цены труда, повышение пенсион- ного возраста также спровоцирует сниже- ние темпов роста заработной платы. Оптимальный вариант: поэтапное повышение возраста выхода на пенсию для определенных категорий граждан России. Подводя итоги, можно заметить сле- дующее: повышение пенсионного возрас- та в стране эффективно для пенсионной системы: пенсионеров в стране меньше – объем пенсионных выплат уменьшается. Соответственно, увеличивается финансо- вая устойчивость пенсионной системы. Высвободившиеся средства позволяют индексировать пенсии, что повышает размер ежемесячных пенсионных выплат, а, как следствие, и качество жизни самих пенсионеров. ISSN 2223-1560. Известия Юго-Западного государственного университета. 2018. Т. 22, № 2(77) T.A. Svetovtseva, Candidate of Economic Sciences, Professor, Southwest State University (Kursk, Russia) (e-mail:svetovtseva2018@yandex.ru) S. A. Mamiy, Candidate of Economic Sciences, Associate Professor, Kuban State Agrarian University (Krasnodar, Russia) (e-mail:aslambechevna-sima@mail.ru) T. A. Bochkova, Candidate of Economic Sciences, Associate Professor, Kuban State Agrarian University (Krasnodar, Russia) (e-mail: bochkova-tatiana@mail.ru) PENSION AGE IN THE RUSSIAN FEDERATION: ASSESSMENT OF THE EFFECTIVENESS OF THE PREVENTIVE INCREASE Time of retirement is the lever regulating a ratio of number of able-bodied Russians and pensioners - the it is higher, the bigger number of citizens become payers, but not recipients of pension payments. In this work the perspective of increase in a retirement age as a factor of macroeconomic development of the Russian Federation is studied and also possible consequences of this increase for age category of citizens and youth T.A. Svetovtseva, Candidate of Economic Sciences, Professor, Southwest State University (Kursk, Russia) (e-mail:svetovtseva2018@yandex.ru) S. A. Mamiy, Candidate of Economic Sciences, Associate Professor, Kuban State Agrarian University (Krasnodar, Russia) (e-mail:aslambechevna-sima@mail.ru) T. A. Bochkova, Candidate of Economic Sciences, Associate Professor, Kuban State Agrarian University (Krasnodar, Russia) (e-mail: bochkova-tatiana@mail.ru) PENSION AGE IN THE RUSSIAN FEDERATION: ASSESSMENT OF THE EFFECTIVENESS OF THE PREVENTIVE INCREASE Список литературы Поступила в редакцию 19.01.18 Поступила в редакцию 19.01.18 Поступила в редакцию 19.01.18 10. Нужно ли повышение пенсионно- го возраста в России? [Электронный ре- 10. Нужно ли повышение пенсионно- го возраста в России? [Электронный ре- Список литературы 1. Киященко Е. А., Бочкова Т. А. Управленческие решения в органах соци- альной защиты населения // Современные технологии в мировом научном про- странстве: сб. статей конф. Уфа, 2016. С. 26-29. С экономической точки зрения по- добное средство способно привести к различным исходам. Плюсы в том, что число граждан, пригодных к труду, уве- личится, соответственно, ВВП возрастет. Благодаря возрастной категории граждан исчезнет проблема нехватки квалифици- рованной рабочей силы. Население Рос- 2. Бурковский П. В., Петрова К. В. Проблемы управления инвестиционной деятельностью на региональном уровне // Инновационная экономика: перспективы и совершенствования. 2015. №2 (7). С. 56-59. 3. Вишневский А., Васин С., Рамонов А. Возраст выхода на пенсию и продол- 3. Вишневский А., Васин С., Рамонов А. Возраст выхода на пенсию и продол- ISSN 2223-1560. Известия Юго-Западного государственного университета. 2018. Т. 22, № 1(76) 117 Пенсионный возраст в Российской Федерации: оценка эффективности предполагаемого повышения сурс] // Versia.ru. URL: https: // versia.ru/ nuzhno-li-povyshenie-pensionnogo- vozrasta-v-rossii жительность жизни // Вопросы экономи- ки. 2012. № 9. С. 88–109. жительность жизни // Вопросы экономи- ки. 2012. № 9. С. 88–109. 4. Гурвич Е.Т. Приоритеты нового этапа пенсионной реформы // SPERO – Социальная политика: экспертиза, реко- мендации, обзоры. 2008. № 8. 11. Мамий С.А., Петров А.А. Эконо- мический потенциал возобновляемых ис- точников энергии // Политематический электронный журнал КубГАУ. 2017. №127,. С.164-175. 5. Коровкин А.Г., Долгова И.Н., Ко- ролев И.Б. Последствия инерционного развития трудового потенциала РФ // Проблемы прогнозирования. 2011. №6. 12. Мамий С.А., Бондаренко Л.Л. Развитие малого бизнеса в России как перспектива экономико-политической са- мостоятельности // Политематический сетевой электронный журнал КубГАУ. 2015. №113. С.1243-1257. 6. Коровкин А.Г. Последствия по- вышения пенсионного возраста для рын- ка труда [Электронный ресурс] // Ведо- мости. 2015. 27 мая. URL: http:// www.vedomosti.ru/management/blogs/2015 /05/27/593847-ugrozi-povisheniya 13. Вертакова Ю.В., Харченко Е.В., Железняков С.С.Интеграция подходов к управлению современной организацией / монография / под ред. Ю. В. Вертаковой; Юго-Западный государственный универ- ситет. Курск, 2010. 7. Малева Т.М., Синявская О.В. По- вышение пенсионного возраста: pro et сontra // Журнал новой экономической ассоциации. 2010. № 8. С. 117-137. 8. Колосницына М.Г. Экономика труда: учебное пособие. М.: ГУ ВШЭ, 2013. 14. Ершова И.Г., Вертакова Ю.В. Выбор приоритетов эффективного управ- ления интеллектуальными ресурсами в экономике знаний // Известия Юго-Запад- ного государственного университета. Се- рия: Экономика. Социология. Менедж- мент. 2012. № 2. С. 260-270. 9. Ниворожкина Л.И., Арженовский С.В. Эффективность программ сокраще- ния бедности: декомпозиция динамики и структуры российской бедности. М.: Консорциум экономических исследова- ний и образования, 2008. UDC 331 ISSN 2223-1560. Известия Юго-Западного государственного университета. 2018. Т. 22, № 2(77) UDC 331 T.A. Svetovtseva, Candidate of Economic Sciences, Professor, Southwest State University (Kursk, Russia) (e-mail:svetovtseva2018@yandex.ru) S. A. Mamiy, Candidate of Economic Sciences, Associate Professor, Kuban State Agrarian University (Krasnodar, Russia) (e-mail:aslambechevna-sima@mail.ru) T. A. Bochkova, Candidate of Economic Sciences, Associate Professor, Kuban State Agrarian University (Krasnodar, Russia) (e-mail: bochkova-tatiana@mail.ru) PENSION AGE IN THE RUSSIAN FEDERATION: ASSESSMENT OF THE EFFECTIVENESS OF THE PREVENTIVE INCREASE Time of retirement is the lever regulating a ratio of number of able-bodied Russians and pensioners - the it is higher, the bigger number of citizens become payers, but not recipients of pension payments. In this work the perspective of increase in a retirement age as a factor of macroeconomic development of the Russian Federation is studied and also possible consequences of this increase for age category of citizens and youth Key words: pension age, unemployment, pension reform, budget defic Key words: pension age, unemployment, pension reform, budget deficit. DOI: 10.21869/2223-1560-2018-22-2-111-118 For citation: Svetovtseva T.A., Mamiy S. A., Bochkova T. A. Pension Age in the Russian Federation: Assessment of the Effectiveness of the Preventive Increase. Proceedings of the Southwest State University, 2018, vol. 22, no. 2(77), pp. 111-118 (in Russ.). 8. Kolosnitsyina M.G. Ekonomika tru- da. Moscow, 2013. PENSION AGE IN THE RUSSIAN FEDERATION: ASSESSMENT OF THE EFFECTIVENESS OF THE PREVENTIVE INCREASE Time of retirement is the lever regulating a ratio of number of able-bodied Russians and pensioners - the it is higher, the bigger number of citizens become payers, but not recipients of pension payments. In this work the perspective of increase in a retirement age as a factor of macroeconomic development of the Russian Federation is studied and also possible consequences of this increase for age category of citizens and youth ISSN 2223-1560. Известия Юго-Западного государственного университета. 2018. Т. 22, № 2(77) Т.А. Световцева, С.А. Мамий, Т. А. Бочкова 118 are analysed. The purpose of the real work – to make assessment of efficiency of alleged raising the retirement age in our country and to predict influence of this economic lever on employment of the population. It is revealed that the indicator of a retirement age, being a peculiar lever, can and has to be applied not as the instrument of economy of budgetary funds, and first of all as the mechanism of social regulation. Influence of this measure on the state, economy and welfare of various groups of the population is considered. At the end of work conclusions are drawn on benefits and costs of alleged increase. are analysed. The purpose of the real work – to make assessment of efficiency of alleged raising the retirement age in our country and to predict influence of this economic lever on employment of the population. It is revealed that the indicator of a retirement age, being a peculiar lever, can and has to be applied not as the instrument of economy of budgetary funds, and first of all as the mechanism of social regulation. Influence of this measure on the state, economy and welfare of various groups of the population is considered. At the end of work conclusions are drawn on benefits and costs of alleged increase. Reference 1. Kiyaschenko E. A., Bochkova T. A. Upravlencheskie resheniya v organah sotsi- alnoy zaschityi naseleniya. Sovremennyie tehnologii v mirovom nauchnom prostranstve. Sb. statey konf. Ufa, 2016, pp. 26-29. 1. Kiyaschenko E. A., Bochkova T. A. Upravlencheskie resheniya v organah sotsi- alnoy zaschityi naseleniya. Sovremennyie tehnologii v mirovom nauchnom prostranstve. Sb. statey konf. Ufa, 2016, pp. 26-29. 9. Nivorozhkina L.I., Arzhenovskiy S.V. Effektivnost programm sokrascheniya bednosti: dekompozitsiya dinamiki i struk- turyi rossiyskoy bednosti. Moscow, 2008. 10. Nuzhno li povyishenie pensionnogo vozrasta v Rossii? [Elektronnyiy resurs]. Versia.ru. URL: https://versia.ru/nuzhno-li- povyshenie-pensionnogo-vozrasta-v-rossii 2. Burkovskiy P. V., Petrova K. V. Prob- lemyi upravleniya investitsionnoy deyatel- nostyu na regionalnom urovne. Innovatsion- naya ekonomika: perspektiv i sovershenstvo- vaniya, 2015, no.2 (7), pp. 56-59. 11. Mamiy S.A., Petrov A.A. Ekonomicheskiy potentsial vozobnovlyae- myih istochnikov energii. Politematicheskiy elektronnyiy zhurnal KubGAU, 2017, no. 127, pp.164-175. 3. Vishnevskiy A., Vasin S., Ramonov A. Vozrast vyihoda na pensiyu i prodolzhite- lnost zhizni. Voprosyi ekonomiki, 2012, no. 9, pp. 88–109. 3. Vishnevskiy A., Vasin S., Ramonov A. Vozrast vyihoda na pensiyu i prodolzhite- lnost zhizni. Voprosyi ekonomiki, 2012, no. 9, pp. 88–109. 12. Mamiy S.A., Bondarenko L.L. Raz- vitie malogo biznesa v Rossii, kak perspek- tiva ekonomiko-politicheskoy samostoya- telnosti. Politematicheskiy setevoy elektron- nyiy zhurnal KubGAU, 2015, no.113, pp.1243-1257. 4. Gurvich E.T. Prioritetyi novogo eta- pa pensionnoy reformyi. SPERO – Sotsi- alnaya politika: ekspertiza, rekomendatsii, obzoryi, 2008, no. 8. 4. Gurvich E.T. Prioritetyi novogo eta- pa pensionnoy reformyi. SPERO – Sotsi- alnaya politika: ekspertiza, rekomendatsii, obzoryi, 2008, no. 8. 5. Korovkin A.G., Dolgova I.N., Korolev I.B. Posledstviya inertsionnogo razvitiya trudovogo potentsiala RF. Prob- lemyi prognozirovaniya, 2011, no.6. 13. Vertakova Yu.V. Harchenko E.V., Zheleznyakov S.S. Integratsiya podhodov k upravleniyu sovremennoy organizatsiey. Kursk, 2010. 6. Korovkin A.G. Posledstviya povy- isheniya pensionnogo vozrasta dlya ryinka truda [Elektronnyiy resurs]. Vedomosti. 2015, 27 maya. URL: http:// www.vedomosti.ru/ management/blogs/2015/05/27/593847- ugrozi-povisheniya 14. Ershova I.G., Vertakova Yu.V. Vyibor prioritetov effektivnogo upravleniya intellektualnyimi resursami v ekonomike znaniy. 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https://openalex.org/W4392755582	https://holrev.uho.ac.id/index.php/journal/article/download/105/28	Indonesian	null	Penggunaan Regulatory Impact Analysis: Studi Penyusunan Peraturan Daerah Jawa Timur Tentang Desa Wisata	Halu oleo law review	2,024	cc-by	8,148	ABSTRACT Keywords: Legislation Drafting; Regulatory Impact Analysis; Tourism Villages This scientific article discusses the analysis of the use of Regulatory Impact Analysis (RIA) in the formation of East Java Regional Regulations concerning Tourism Villages. The research reveals that in the development of the Academic Text of these regulations, regulatory impact assessment methods such as RIA and ROCCIPI are not utilized. Instead, socio-legal methods are employed to compare the rules with the academic texts that have been produced. The main finding is that the omission of RIA in the Academic Text may lead to formal defects in the regional regulations, given the requirements of Law No. 13/2022 emphasizing the importance of this method. In the socio- legal method analysis, this article observes that a focus on legality and philosophical aspects in the Academic Text without using RIA may result in a lack of understanding of the regulation's impact on society, the economy, and the environment. The significant social and economic impacts of empowering tourism villages may not be adequately considered. The implications of these findings are crucial. The absence of RIA in the Academic Text necessitates urgent revisions or replacements of regional regulations in accordance with the guidelines of Law No. 13/2022. Remedial steps are required in the regional regulation formation process, including the application of RIA to ensure the desired positive impacts and a balance between empowering tourism villages and environmental preservation. Thus, the regulations can meet legal requirements, provide maximum benefits, and achieve the holistic goals of empowering tourism villages in East Java. Agatha Retnosari1, Radian Salman2, Humaidi Rizqi Alfath Syaif3 1. Universitas Airlangga, Indonesia. E-mail: agatha.retnosari73@gmail.com. 2. Universitas Airlangga, Indonesia. E-mail: radian.salman@fh.unair.ac.id. 3. Universitas Airlangga, Indonesia. E-mail: humaidirizqi96@gmail.com. ARTICLE INFO Keywords: Legislation Drafting; Regulatory Impact Analysis; Tourism Villages Volume 8 Issue 1, March 2024: pp. 29-48. Copyright © 2024 Halu Oleo Law Review. Faculty of Law, Halu Oleo University, Kendari, Southeast Sulawesi, Indonesia. P-ISSN: 2548-1762 \| E-ISSN: 2548-1754. Open Access at: https://holrev.uho.ac.id Halu Oleo Law Review is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Penggunaan Regulatory Impact Analysis: Studi Penyusunan Peraturan Daerah Jawa Timur Tentang Desa Wisata Usage of Regulatory Impact Analysis: Study on Drafting East Java Regional Regulation Regarding Tourism Village Agatha Retnosari1, Radian Salman2, Humaidi Rizqi Alfath Syaif3 1. Universitas Airlangga, Indonesia. E-mail: agatha.retnosari73@gmail.com. 2. Universitas Airlangga, Indonesia. E-mail: radian.salman@fh.unair.ac.id. 3. Universitas Airlangga, Indonesia. E-mail: humaidirizqi96@gmail.com. INFO ARTIKEL Kata kunci: Desa Wisata; Perancangan Perundang-undangan; Regulatory Impact Analysis Kata kunci: Desa Wisata; Perancangan Perundang-undangan; Regulatory Impact Analysis Artikel ilmiah ini membahas analisis penggunaan Regulatory Impact Analysis (RIA) dalam pembentukan Peraturan Daerah Jawa Timur terkait Desa Wisata. Penelitian menemukan bahwa dalam pembentukan Naskah Akademik peraturan tersebut, metode evaluasi dampak regulasi seperti RIA dan ROCCIPI tidak digunakan. Sebagai gantinya, metode sosiolegal digunakan untuk membandingkan aturan dengan naskah akademik yang telah dibuat. Temuan utama adalah ketidakgunakan RIA dalam Naskah Akademik dapat menyebabkan cacat formil dalam peraturan daerah, 29 P-ISSN: 2548-1762 \| E-ISSN: 2548-1754 mengingat persyaratan UU No. 13/2022 menekankan pentingnya metode ini. Dalam analisis metode sosiolegal, artikel ini mencermati bahwa fokus pada legalitas dan aspek filosofis dalam Naskah Akademik tanpa menggunakan RIA dapat menyebabkan kurangnya pemahaman dampak peraturan terhadap masyarakat, ekonomi, dan lingkungan. Dampak sosial dan ekonomi yang signifikan dari pemberdayaan desa wisata mungkin tidak diperhitungkan secara memadai. Implikasi dari temuan ini sangat penting. Tidak adanya penggunaan RIA dalam Naskah Akademik memunculkan kebutuhan mendesak untuk merevisi atau mengganti peraturan daerah sesuai dengan pedoman UU No. 13/2022. Diperlukan langkah-langkah perbaikan dalam proses pembentukan peraturan daerah, termasuk penerapan RIA untuk memastikan dampak positif yang diinginkan dan keseimbangan antara pemberdayaan desa wisata dengan pelestarian lingkungan. Dengan cara ini, peraturan dapat memenuhi persyaratan hukum, memberikan manfaat maksimal, dan mencapai tujuan pemberdayaan desa wisata di Jawa Timur secara holistik. mengingat persyaratan UU No. 13/2022 menekankan pentingnya metode ini. Dalam analisis metode sosiolegal, artikel ini mencermati bahwa fokus pada legalitas dan aspek filosofis dalam Naskah Akademik tanpa menggunakan RIA dapat menyebabkan kurangnya pemahaman dampak peraturan terhadap masyarakat, ekonomi, dan lingkungan. Dampak sosial dan ekonomi yang signifikan dari pemberdayaan desa wisata mungkin tidak diperhitungkan secara memadai. Implikasi dari temuan ini sangat penting. Tidak adanya penggunaan RIA dalam Naskah Akademik memunculkan kebutuhan mendesak untuk merevisi atau mengganti peraturan daerah sesuai dengan pedoman UU No. 13/2022. Diperlukan langkah-langkah perbaikan dalam proses pembentukan peraturan daerah, termasuk penerapan RIA untuk memastikan dampak positif yang diinginkan dan keseimbangan antara pemberdayaan desa wisata dengan pelestarian lingkungan. Dengan cara ini, peraturan dapat memenuhi persyaratan hukum, memberikan manfaat maksimal, dan mencapai tujuan pemberdayaan desa wisata di Jawa Timur secara holistik. 1 Viva Budy Kusnandar, “Berapa Jumlah Desa/Kelurahan Di Indonesia?,” Katadata Databoks, last modified 2021, accessed September 19, 2023, https://databoks.katadata.co.id/datapublish/2021/11/22/berapa- jumlah-desakelurahan-di-indonesia. 2 Peni Widarti, “6 Desa Wisata Jatim Terpilih Jadi Desa Wisata Terbaik 2021,” Bisnis.Com, last modified 2021, accessed September 19, 2023, https://surabaya.bisnis.com/read/20210826/531/1434482/6- desa-wisata-jatim-terpilih-jadi-desa-wisata-terbaik-2021. 1 Viva Budy Kusnandar, “Berapa Jumlah Desa/Kelurahan Di Indonesia?,” Katadata Databoks, last modified 2021, accessed September 19, 2023, https://databoks.katadata.co.id/datapublish/2021/11/22/berapa- jumlah-desakelurahan-di-indonesia. j 2 Peni Widarti, “6 Desa Wisata Jatim Terpilih Jadi Desa Wisata Terbaik 2021,” Bisnis.Com, last modified 2021, accessed September 19, 2023, https://surabaya.bisnis.com/read/20210826/531/1434482/6- desa-wisata-jatim-terpilih-jadi-desa-wisata-terbaik-2021. 1. Pendahuluan Provinsi Jawa Timur ialah salah satu provinsi dengan jumlah desa yang cukup masif di Indonesia. Jawa Timur memiliki potensi pariwisata yang dapat meningkatkan Pendapatan Daerah Regional Bruto (selanjutnya disebut sebagai “PDRB”) dan pertumbuhan ekonomi daerahnya. Pulau Jawa menempati urutan teratas dalam jumlah kelurahan atau desa di Indonesia. Provinsi Jawa Tengah menempati urutan pertama dengan jumlah sebanyak 8.562 desa dan diikuti oleh Provinsi Jawa Timur dengan jumlah sebanyak 8.501 desa.1 Berdasarkan data Asosiasi Desa Wisata Indonesia atau ASIDEWI pada 2020, tercatat jumlah desa wisata di Indonesia mencapai 1.302 desa dengan 6 peringkat teratas yakni 138 desa wisata di Jawa Barat, 132 desa wisata di Jawa Tengah, 114 desa wisata di Jawa Timur, 92 di NTT, 87 di Sumatera Utara, dan 57 Yogyakarta.2 Besarnya angka tersebut khususnya bagi Provinsi Jawa Timur, menunjukkan bahwa perkembangan desa teruslah meningkat. Pengaturan mengenai desa dapat ditemui pada Undang-Undang Nomor 6 Tahun 2014 tentang Desa (selanjutnya disebut sebagai “UU Desa”). UU Desa mengakomodasi hal tersebut dengan memberikan kewenangan yang lebih besar bagi masyarakat di desa untuk membangun dan mengembangkan wilayahnya. Hal tersebut dapat dicapai salah satunya dengan memanfaatkan potensi sumber daya alam yang ada di desa untuk kegiatan pariwisata. Hal ini nantinya akan disebut sebagai Desa Wisata. 30 Halu Oleo Law Review \| Volume 8 Issue 1, March 2024 Pariwisata dianggap sebagai sektor yang cukup potensial untuk dikembangkan karena dianggap mampu mengakselerasi pertumbuhan ekonomi dan cukup strategis bagi suatu negara atau daerah karena pariwisata dipandang sebagai penunjang sektor lainnya. Pariwisata dianggap sebagai sektor yang cukup potensial untuk dikembangkan karena dianggap mampu mengakselerasi pertumbuhan ekonomi dan cukup strategis bagi suatu negara atau daerah karena pariwisata dipandang sebagai penunjang sektor lainnya. Tabel 1.1 Persentase Pertumbuhan PDRB Pariwisata Tabel 1.1 Persentase Pertumbuhan PDRB Pariwisata Indikator 2016 2017 2018 2019 2020 Kontribusi Pariwisata terhadap PDRB Jawa Timur (%) 5,72 5,83 5,86 6,00 5,64 Pertumbuhan PDRB Pariwisata - - 6,83 6,92 -9,6 Sumber: Dinas Kebudayaan dan Pariwisata Provinsi Jawa Timur. Provinsi Jawa Tengah saat ini menjadi satu-satunya wilayah di Indonesia yang memiliki Peraturan Daerah (selanjutnya disebut sebagai “Perda”) khusus untuk Desa Wisata, yaitu Perda Provinsi Jawa Tengah No. 2 Tahun 2019 tentang Pemberdayaan Desa Wisata di wilayah tersebut. Rincian implementasi Perda ini dijabarkan dalam Pergub No. 53 Tahun 2019, memberikan panduan pelaksanaan Perda No. 2 Tahun 2019. Begitu juga, Bali telah mengesahkan Perda Provinsi Bali No. 5 Tahun 2020, yang berfokus pada Standar Penyelenggaraan Kepariwisataan Budaya Bali. / p j p p g j 4 Abraham Ferry Rosando et al., “Legalisasi Pembentukan Desa Wisata Budaya Plunturan Dari Pemerintah Kabupaten Ponorogo,” in Seminar Nasional Konsorsium Universitas 17 Agustus 1945 Surabaya Ke-2 (Surabaya: Universitas 17 Agustus 1945, 2020), 22. 3 Dadi Haryadi, “DPRD Jabar: Perda Desa Wisata Untuk Pembangunan Jawa Barat,” AyoPurwakarta.Com, last modified 2021, accessed September 19, 2023, https://purwakarta.ayoindonesia.com/umum/pr- 321274975/dprd-jabar-perda-desa-wisata-untuk-pembangunan-jawa-barat. 1. Pendahuluan Peraturan ini menekankan hak desa adat/lembaga tradisional/kelompok masyarakat untuk mengembangkan pariwisata pedesaan sesuai potensi setempat dan ketentuan perundang-undangan. Desa Wisata ditetapkan melalui Keputusan Bupati/Walikota, dengan kewajiban pengelola untuk mengutamakan investasi dan sumber daya lokal. Ketentuan lebih lanjut diatur dalam Peraturan Gubernur. Di Jawa Barat, pembahasan Perda Desa Wisata sedang berlangsung di Pansus V DPRD Provinsi Jawa Barat. Di Jawa Timur, beberapa kota atau kabupaten akhirnya membentuk perda khusus terkait desa wisata, seperti yang terjadi di Kota Batu yang mengesahkan Perda Kota Batu No. 1 Tahun 2021 tentang Desa Wisata pada tahun 2021.3 Namun, tantangan masih ada dalam implementasi peraturan-peraturan tersebut. Sebagai contoh, di Ponorogo, ketidaksiapan pemasaran Desa Plunturan, yang diakui sebagai desa terbaik dalam melestarikan budaya, disebabkan oleh ketidakadaan legalitas terkait pembentukan Desa Wisata Plunturan.4 Baru pada tahun 2021 disahkan Perda Kabupaten Ponorogo No. 4 Tahun 2021 tentang Desa Wisata. Sumenep menghadapi situasi serupa, di mana ketidakjelasan regulasi hukum di tingkat daerah menyebabkan ketidakpastian hukum dalam upaya pelestarian budaya keris dan menjadikan Sumenep sebagai Kota Keris dengan pembuat keris terbanyak di Indonesia. Kekosongan hukum ini berdampak pada keputusan lewat Surat Keputusan Bupati Sumenep, tanpa pengaturan terkait pengelolaan Desa Aeng Tong-Tong sebagai desa 31 P-ISSN: 2548-1762 \| E-ISSN: 2548-1754 wisata. Di Banyuwangi, meskipun memiliki Perda Desa Wisata sejak 2017, ketidakadaan peraturan di tingkat provinsi mengakibatkan kekosongan hukum, karena hanya mengacu pada Perda Provinsi Jawa Timur No. 6 Tahun 2017 tentang Rencana Induk Pembangunan Pariwisata Provinsi Jawa Timur Tahun 2017-2032. Meski demikian, keberhasilan Desa Kemiren dalam pengembangan ekonomi melalui pariwisata budaya dan edukasi, sebagaimana diatur dalam Perda Kabupaten Banyuwangi No. 1 Tahun 2017, menunjukkan dampak positif dari intervensi kebijakan lokal dan keterlibatan masyarakat.5 Menurut Satria, agar suatu kebijakan diterapkan dengan tepat sasaran dan efisien maka harus disusun dengan suatu metode yang ketat. Salah satu metode itu adalah Regulatory Impact Assessment (selanjutnya disebut sebagai “RIA”).6 RIA adalah suatu alat analisis untuk membantu pembuat kebijakan menilai dampak suatu regulasi. RIA digunakan untuk mengukur dan menguji kemungkinan manfaat, biaya dan dampak peraturan baik usulan peraturan maupun peraturan yang sudah ada. Fitur utama dalam RIA adalah pertimbangan dampak ekonomi dari sebuah peraturan, dimana komponen utamanya adalah pelibatan para stakeholder dalam setiap tahapan pembentukan peraturan perundang-undangan.7 Provinsi Jawa Timur telah memiliki Perencanaan Pariwisata yang dituangkan dalam dokumen Rencana Induk Pengembangan Pariwisata Provinsi Jawa Timur 2017-2023 (selanjutnya disebut sebagai “Ripparprov”). Namun di dalam Ripparprov ini tidak ada penjelasan lebih rinci terkait Desa Wisata. 5 Siti Ngaisah, Bagus Ananda Kurniawan, and Chusnul Abadi, “Implementasi Program Desa Wisata Dalam Menunjang Pelestarian Dan Pengembangan Budaya Keris,” Kaganga: Jurnal Pendidikan Sejarah dan Riset Sosial Humaniora 4, No. 1 (March 10, 2021): 51, https://journal.ipm2kpe.or.id/index.php/KAGANGA/ article/view/1863. / / 6 Rahmad Satria, “Penerapan Metode Regulatory Impact Assessment (RIA) Dalam Penyusunan Regulasi Daerah,” Masalah-Masalah Hukum 2, No. 2 (April 24, 2015): 179, http://ejournal.undip.ac.id/index.php/ mmh/article/view/11432. 1. Pendahuluan Menilik adanya beberapa contoh kasus di atas terkait problematik yang muncul di beberapa daerah berkaitan dengan keberadaan Desa Wisata, maka dipandang perlu adanya usulan kebijakan terkait dengan Usaha Desa Wisata. Oleh karena ada hubungan erat terkait dengan peningkatan ekonomi, maka menjadi penting jika dalam proses pembentukan Raperda Usaha Desa Wisata ini dikaji dengan menggunakan RIA. Agar tujuan untuk mencapai keadilan sosial dapat tercapai, di mana penduduk setempat ikut merasakan manfaat ekonomi dengan adanya Usaha Desa Wisata. Bentuk pengejawantahan dari hal ini ialah dengan dibentuknya Peraturan Daerah Provinsi Jawa Timur Nomor 4 Tahun 2022 tentang Pemberdayaan Desa Wisata (selanjutnya disebut sebagai “Perda No./2022”) yang telah digodok sejak 2017, namun baru diundangkan pada tahun 2022. Setelah UU Nomor 13 Tahun 2022 tentang Perubahan Kedua Atas UU Nomor 12 Tahun 2011 tentang Pembentukan Peraturan Perundang-undangan (selanjutnya disebut sebagai “UU 13/2022”) diberlakukan, terdapat beberapa perubahan menarik yang perlu 32 Halu Oleo Law Review \| Volume 8 Issue 1, March 2024 diperhatikan lebih lanjut. Penjelasan umum UU tersebut mencantumkan bahwa ada beberapa hal yang perlu ditingkatkan, antara lain: diperhatikan lebih lanjut. Penjelasan umum UU tersebut mencantumkan bahwa ada beberapa hal yang perlu ditingkatkan, antara lain: Penambahan metode omnibus. 1. Penambahan metode omnibus. 2. Perbaikan kesalahan teknis setelah mendapatkan persetujuan bersama antara DPR dan Presiden dalam rapat paripurna sebelum pengesahan, serta peningkatan keterlibatan dan partisipasi masyarakat yang bermakna (meaningful participation). 2. Perbaikan kesalahan teknis setelah mendapatkan persetujuan bersama antara DPR dan Presiden dalam rapat paripurna sebelum pengesahan, serta peningkatan keterlibatan dan partisipasi masyarakat yang bermakna (meaningful participation). Pembentukan Peraturan Perundang-undangan secara elektronik. 3. Pembentukan Peraturan Perundang-undangan secara elektronik. 4. Perubahan sistem pendukung dari peneliti menjadi pejabat fungsional lain yang tugasnya terkait dengan Pembentukan Peraturan Perundang-undangan. 4. Perubahan sistem pendukung dari peneliti menjadi pejabat fungsional lain yang tugasnya terkait dengan Pembentukan Peraturan Perundang-undangan. 5. Perubahan teknik penyusunan Naskah Akademik. 5. Perubahan teknik penyusunan Naskah Akademik. 5. Perubahan teknik penyusunan Naskah Akademik. 6. Perubahan teknik penyusunan Peraturan Perundang-undangan. Spesifik terkait perubahan teknik penyusunan Naskah Akademik, dijelaskan lebih lanjut bahwa: Spesifik terkait perubahan teknik penyusunan Naskah Akademik, dijelaskan lebih lanjut bahwa: “Penambahan teknik penyusunan Naskah Akademik Rancangan Undang-Undang, Rancangan Peraturan Daerah Provinsi, dan Rancangan Peraturan Daerah Kabupaten/Kota dimuat dalam Lampiran I. 1. Penambahan metode omnibus. 1. Pendahuluan Perubahan terhadap teknik penyusunan Naskah Akademik Rancangan Undang-Undang, Rancangan Peraturan Daerah Provinsi, dan Rancangan Peraturan Daerah Kabupaten/Kota dilakukan terhadap ketentuan Bab II huruf D tentang kajian terhadap implikasi penerapan sistem baru yang akan diatur dalam Undang-Undang atau Peraturan Daerah terhadap aspek kehidupan masyarakat dan dampaknya terhadap aspek beban keuangan negara. Kajian tersebut didukung dengan analisis yang menggunakan metode tertentu, antara lain metode Regulatory Impact Analysis (RIA) dan metode Rule, Opportunity, Capacity, Communication, Interest, Process, and ldeology (ROCCIPI).” “Penambahan teknik penyusunan Naskah Akademik Rancangan Undang-Undang, Rancangan Peraturan Daerah Provinsi, dan Rancangan Peraturan Daerah Kabupaten/Kota dimuat dalam Lampiran I. Perubahan terhadap teknik penyusunan Naskah Akademik Rancangan Undang-Undang, Rancangan Peraturan Daerah Provinsi, dan Rancangan Peraturan Daerah Kabupaten/Kota dilakukan terhadap ketentuan Bab II huruf D tentang kajian terhadap implikasi penerapan sistem baru yang akan diatur dalam Undang-Undang atau Peraturan Daerah terhadap aspek kehidupan masyarakat dan dampaknya terhadap aspek beban keuangan negara. Kajian tersebut didukung dengan analisis yang menggunakan metode tertentu, antara lain metode Regulatory Impact Analysis (RIA) dan metode Rule, Opportunity, Capacity, Communication, Interest, Process, and ldeology (ROCCIPI).” Penyebutan yang eksplisit mengenai metode RIA dalam Penjelasan Umum UU yang kemudian diuraikan lebih lanjut dalam Lampiran I UU yang berlaku saat ini merupakan perkembangan penting yang memerlukan tinjauan lebih mendalam. Hal ini menjadi signifikan karena penyebutan metode RIA dan ROCCIPI sebagai alternatif metode analisis membuka peluang bagi penerapan metode analisis lain selama mampu menganalisis implikasi dari sistem baru yang akan diatur dalam Undang-Undang (UU) atau Peraturan Daerah (Perda) terhadap berbagai aspek kehidupan masyarakat dan dampaknya terhadap aspek keuangan negara atau daerah. Penyebutan metode RIA dalam UU yang berlaku saat ini juga menandai tahap baru dalam penggunaan metode RIA di Indonesia karena telah diintegrasikan dalam peraturan perundang-undangan nasional. Sebelumnya, metode RIA digunakan secara tidak teratur dalam proses pembentukan peraturan perundang-undangan maupun dalam perumusan kebijakan di Indonesia. Meskipun beberapa kementerian/lembaga telah mencoba menerapkan metode RIA dalam ruang lingkup mereka, penggunaannya sering kali tidak sesuai dengan kerangka kerja yang telah ditetapkan oleh Organization for Economic Co- operation and Development (selanjutnya disebut sebagai “OECD”). 33 P-ISSN: 2548-1762 \| E-ISSN: 2548-1754 OECD adalah sebuah organisasi internasional yang terdiri dari 38 negara anggota. Tujuan utamanya adalah untuk mendorong pertumbuhan ekonomi, meningkatkan standar hidup, dan berkontribusi pada pembangunan berkelanjutan. OECD menyediakan platform bagi negara-negara anggotanya untuk mendiskusikan dan mengkoordinasikan kebijakan, berbagi pengalaman, melakukan penelitian, dan mengumpulkan data di berbagai bidang seperti ekonomi, pendidikan, lingkungan, dan perpajakan. 1. Pendahuluan Organisasi ini memainkan peran penting dalam mempromosikan kerja sama internasional dan membantu negara- negara mencapai tujuan ekonomi dan sosial mereka. Oleh karenanya, penelitian ini akan mengkaji bagaimana penerapan RIA yang sangat dianjurkan oleh UU 13/2022 pada pembentukan Perda No. 4/2022 agar diketahui apakah penyusunannya sudah sesuai dengan ketentuan atau tidak. 2. Metode Penelitian ini menggunakan jenis penelitian socio-legal dalam pengkajian bahan penelitian. Penelitian socio-legal menggunakan metode penelitian dimana ilmu sosial mempengaruhi kinerja ilmu hukum dalam berbagai aspek yang berhubungan keberlakuan hukum di masyarakat. Penelitian dengan metode socio-legal mempunyai kacamata dalam memandang hukum dengan detail, karena mengkaji norma-norma hukum dan menggunakan beberapa disiplin ilmu secara interdisipliner. Metode penelitian selanjutnya ialah menggunakan metode Yuridis Normatif dan Yuridis Empiris. Penelitian yuridis normatif dilakukan dengan mengkaji aturan perundang- undangan terkait dengan teori dan proses pembentukan perundang-undangan. Sedangkan Metode empiris digunakan untuk mengetahui diperlukan RIA dalam proses pembentukan Raperda Usaha Desa Wisata Provinsi Jawa Timur sebagai bagian dari kajian yang harus dilakukan sebelum masuk ke dalam norma-norma hukumnya. Ini juga untuk mengetahui dari sekian perda yang ada di Jawa Timur, apakah ada yang menggunakan metode RIA sebagai alat analisis melakukan kajian. 8 Edward A Suchman, Evaluative Research: Principles and Practice in Public Service and Social Action Progr (New York: Russell Sage Foundation, 1967), 243, http://www.jstor.org/stable/10.7758/ 9781610445177. b d 11 Rahman Hasima, “Penerapan Metode Regulatory Impact Assessment Dalam Penyusunan Peraturan Daerah Di Kota Kendari,” Halu Oleo Law Review 4, No. 1 (March 20, 2020): 53, http://ojs.uho.ac.id/index.php/holrev/article/view/9258. p // j / p p/ / / / 12 Kai Hauerstein and Peter Bissenger, Training Manual Regulatory Impact Assessment (Jakarta: GTZ-Red dan Bappenas RI, 2009), 7. 13 Agus Ediawan et al., Arti Penting Regulatory Impact Assessment (RIA) (Jakarta: The Asia Foundation, 2008), 8. 14 Scott H. Jacobs, “An Overview of Regulatory Impact Analysis in OECD Countries,” in Regulatory Impact Analysis: Best Practices in OECD Countries (Paris: OECD Publication, 1997), 20. 9 Randall B. Ripley and Grace A. Franklin, Policy Implementation and Bureaucracy (Illinois: The Dorsey Press, 1982), 30. 10 Ibid 3. Penerapan RIA Sebagai Alat Analisis Penyusunan Perda Desa Wisata Di Jawa Timur Evaluasi ialah suatu istilah yang sering kali digunakan pada setiap sektor kehidupan berbangsa, terlebih pada sektor publik. Dalam mengevaluasi sektor publik setidaknya ada tiga (3) hal yang harus diingat, yaitu:8 1. Objek evaluasi ialah suatu tindakan yang sengaja dibuat atau diprogram oleh pemerintah; 1. Objek evaluasi ialah suatu tindakan yang sengaja dibuat atau diprogram oleh pemerintah; 2. Objek evaluasi diciptakan oleh pemerintah dengan tujuan tertentu yang dapat diperkirakan; dan 34 Halu Oleo Law Review \| Volume 8 Issue 1, March 2024 3. Objek evaluasi dapat diukur keberhasilannya berdasarkan tujuan dibuatnya. Pentingnya evaluasi ini merupakan bentuk dari adanya intervensi pemerintah dalam melihat dampak yang terjadi atas tindakan pemerintah kepada masyarakat. Suatu kejadian yang terjadi di masyarakat harus dapat dievaluasi karena berdampak dengan skala besar pada khalayak umum. Dalam segi peraturan perundang-undangan, evaluasi dapat dilakukan dengan menggunakan RIA. Regulatory Impact Assessment atau Regulatory Impact Assessment (selanjutnya disebut sebagai “RIA”) ialah suatu metode terencana yang digunakan oleh pemerintah dan instansi pengatur untuk mengevaluasi potensi efek dari regulasi atau kebijakan yang diajukan sebelum diterapkan. Asal usul RIA bisa dilacak hingga pertengahan abad ke-20 ketika pemerintah mulai menyadari pentingnya pendekatan terstruktur dalam menilai dampak regulasi terhadap berbagai pihak terkait, termasuk bisnis, individu, dan ekonomi secara umum.9 RIA dapat berbentuk suatu dokumen yang dibuat sebelum suatu peraturan perundang- undangan yang akan diundangkan oleh pemerintah dibuat.10 RIA diharapkan dapat menyediakan penilaian serta potensi dampak dari suatu peraturan baru dengan sistematis dan terperinci. RIA dapat membantu pembuat peraturan perundang-undangan dalam menganalisis banyaknya dampak serta sulitnya meramalkan dampak suatu peraturan perundang-undangan tanpa melibatkan pihak-pihak yang terdampak oleh hal tersebut. 11 RIA juga dapat digunakan untuk memperbaiki kualitas suatu peraturan perundang- undangan baik yang sedang diusulkan (proposed regulation) maupun yang sedang berjalan (existing regulation).12 Sehingga terkadang RIA juga disebut sebagai alat evaluasi suatu kebijakan yang diambil pemerintah. Oleh karenanya, penggunaan RIA dapat membantu pemangku kepentingan dalam melakukan pengambilan keputusan.13 Secara sistematis serta konsisten, RIA dapat digunakan untuk mengkaji pengaruh apa saja yang akan muncul sebagai konsekuensi tindakan pemerintah. Jacobs berpendapat sebagai berikut:14 “RIA is a decision tool, a method of (i) systematically and consistently examining selected potential impacts arising from government action and of (ii) communicating the information to decision makers.” Jacobs menyimpulkan bahwa RIA ialah suatu alat penentu berupa metode untuk secara sistematis dan konsisten 35 P-ISSN: 2548-1762 \| E-ISSN: 2548-1754 memunculkan potensi dampak yang muncul dari tindakan pemerintah agar dapat dikomunikasikan pada penentu kebijakan. 3. Penerapan RIA Sebagai Alat Analisis Penyusunan Perda Desa Wisata Di Jawa Timur Aspek analisis serta komunikasi ialah aspek yang sangat krusial dalam penggunaan RIA. Pemilihan RIA sebagai indikator langkah-langkah dan perubahan dalam evaluasi didasarkan pada berbagai alasan yang terkait dengan karakteristik tertentu yang menjadikannya contoh yang baik untuk memahami bagaimana pemerintah bertindak selama bertahun-tahun. RIA adalah salah satu alat evaluasi yang paling banyak digunakan di seluruh dunia. RIA terdiri dari analisis ekonomi dan dengan demikian mudah dibandingkan. Selain itu, RIA terkait dengan regulasi pasar dan dalam konteks ini dianggap sangat penting. Asumsi penelitian terkait dengan elemen terakhir ini: gagasan utamanya adalah bahwa evaluasi awalnya diciptakan sebagai solusi untuk masalah pengeluaran publik dan dengan tujuan mendukung pasar bebas, tetapi sekarang telah berkembang menjadi lebih dari sekadar alat kontrol. Saat ini, evaluasi lebih berperan sebagai bentuk dukungan untuk pengambilan keputusan yang efektif dan memberikan manfaat bagi seluruh sistem. Sebagai kesimpulan, dapat disimpulkan bahwa penerapan RIA sebagai paradigma evaluasi memiliki keterkaitan yang erat dengan peran pemerintah dalam mengelola ekonomi, mengatur pasar, serta meningkatkan tingkat akuntabilitas. 15 Office of the Assistant Secretary for Planning and Evaluation, Guidelines for Regulatory Impact Analysis (Washington, D.C.: U.S. Department of Health and Human Services, 2016), 2. 16 Jacobs, “An Overview of Regulatory Impact Analysis in OECD Countries,” 20. (Washington, D.C.: U.S. Department of Health and Human Services, 2016), 2. 16 Jacobs, “An Overview of Regulatory Impact Analysis in OECD Countries,” 20. 3.1. Metodologi Penggunaan RIA Secara Umum Metodologi RIA adalah alat yang kuat dalam pengambilan keputusan pemerintah yang digunakan oleh banyak negara di seluruh dunia. Walaupun pendekatannya dapat bervariasi antara negara-negara, terdapat prinsip-prinsip dasar yang menjadi panduan dalam proses ini. Langkah-langkah yang telah dijelaskan di atas mencerminkan kerangka kerja umum yang digunakan dalam RIA.15 Namun, setiap negara mungkin mengadaptasi atau menyesuaikan pendekatan RIA mereka sesuai dengan konteks, tujuan, dan tantangan yang unik. Variasi dapat muncul dalam hal data yang tersedia, tingkat kompleksitas regulasi, atau tingkat partisipasi publik dalam proses. Misalnya, negara-negara dengan ekonomi yang berbeda atau tantangan lingkungan yang berbeda dapat menekankan aspek-aspek tertentu dari RIA. 16 Meskipun terdapat perbedaan dalam pelaksanaan RIA di berbagai negara, prinsip-prinsip dasarnya tetap sama: mengidentifikasi masalah, mengembangkan kebijakan yang memadai, melibatkan pemangku kepentingan, menganalisis dampak, dan memastikan bahwa manfaat kebijakan melebihi biayanya. Proses ini dirancang untuk memastikan bahwa regulasi yang diusulkan adalah yang paling efektif, efisien, dan sesuai dengan kebutuhan masyarakat. 36 Halu Oleo Law Review \| Volume 8 Issue 1, March 2024 Metode RIA melibatkan beberapa langkah kunci yang berkontribusi pada analisis yang rinci. Langkah-langkah ini mencakup:17 Metode RIA melibatkan beberapa langkah kunci yang berkontribusi pada analisis yang rinci. Langkah-langkah ini mencakup:17 Metode RIA melibatkan beberapa langkah kunci yang berkontribusi pada analisis yang rinci. Langkah-langkah ini mencakup:17 a. Identifikasi Permasalahan Tahap pertama dalam RIA adalah mengidentifikasi masalah atau isu yang memerlukan perhatian regulasi. Ini melibatkan analisis mendalam untuk memahami akar permasalahan dan dampaknya pada masyarakat. Misalnya, jika tingkat pengangguran di suatu daerah meningkat secara signifikan, identifikasi permasalahannya adalah masalah pengangguran yang perlu diatasi. b. Pengembangan Kebijakan Setelah masalah teridentifikasi, langkah berikutnya adalah mengembangkan berbagai opsi kebijakan yang mungkin untuk mengatasi masalah tersebut. Opsi-opsi ini bisa mencakup regulasi baru, perubahan dalam hukum yang ada, atau tindakan non- regulasi. Dalam hal peningkatan pengangguran, opsi kebijakan dapat mencakup pelatihan kerja bagi penduduk setempat, insentif bagi perusahaan untuk mempekerjakan lebih banyak pekerja, atau program dukungan bagi pengangguran. c. Konsultasi dengan Pihak Terkait RIA mengedepankan keterlibatan pihak-pihak terkait, seperti perusahaan, kelompok masyarakat, dan ahli di bidang terkait, untuk memberikan masukan dan perspektif yang beragam selama proses perencanaan kebijakan. Misalnya, saat merancang kebijakan pengurangan penggunaan plastik, konsultasi dengan produsen plastik, LSM lingkungan, dan konsumen dapat membantu mengidentifikasi dampak dan solusi yang lebih efektif. d. Analisis Dampak Pada tahap ini, dilakukan analisis mendalam tentang potensi dampak dari setiap opsi kebijakan yang diusulkan, termasuk dampak ekonomi, sosial, dan lingkungan. 17 Ibid. 17 Ibid. 3.1. Metodologi Penggunaan RIA Secara Umum Contohnya, dalam analisis dampak regulasi pengurangan penggunaan plastik, diperiksa bagaimana regulasi tersebut akan mempengaruhi biaya produksi, konsumsi plastik, dan dampaknya pada lingkungan. e. Cost-Benefit Analysis RIA mencakup analisis manfaat-biaya untuk menilai apakah manfaat dari regulasi diharapkan melebihi biayanya. Ini melibatkan perbandingan antara biaya dan manfaat yang diantisipasi. Misalnya, dalam analisis manfaat-biaya regulasi pengurangan penggunaan plastik, maka biaya pelaksanaan regulasi akan dibandingkan dengan manfaat seperti pengurangan polusi plastik di lingkungan. f. Penilaian Risiko RIA juga mencakup penilaian risiko untuk mengidentifikasi potensi risiko yang terkait dengan regulasi yang diusulkan, serta cara untuk mengelolanya. Misalnya, dalam regulasi pengurangan penggunaan plastik, penilaian risiko dapat mencakup potensi dampak ekonomi jika perusahaan plastik kehilangan pendapatan atau risiko pencemaran lingkungan yang lebih lanjut. 37 P-ISSN: 2548-1762 \| E-ISSN: 2548-1754 g. Penilaian Alternatif Pembuat kebijakan mempertimbangkan berbagai alternatif dalam mengembangkan regulasi, memastikan bahwa pilihan yang diusulkan adalah yang paling efektif dan efisien. Misalnya, dalam pengurangan penggunaan plastik, alternatif bisa mencakup larangan total, pajak plastik, atau promosi penggunaan bahan pengganti ramah lingkungan. h. Dokumentasi dan Transparansi Selama proses RIA, semua informasi, data, dan metodologi yang digunakan didokumentasikan secara rinci untuk memastikan transparansi dan akuntabilitas. Misalnya, semua hasil analisis dan laporan yang digunakan dalam RIA pengurangan penggunaan plastik harus didokumentasikan untuk mengizinkan pemeriksaan oleh pihak eksternal. i. Pembuatan Keputusan Tahap akhir melibatkan penyajian temuan RIA kepada pembuat kebijakan, yang kemudian menggunakan informasi ini untuk membuat keputusan tentang implementasi regulasi yang diusulkan. Setelah melihat hasil RIA, pembuat kebijakan dapat memutuskan untuk menerapkan regulasi pengurangan penggunaan plastik dengan mengadopsi salah satu dari alternatif yang telah diidentifikasi. Langkah-langkah tersebut membantu memastikan bahwa setiap regulasi yang diusulkan dipertimbangkan secara cermat, berdasarkan bukti dan keterlibatan pihak-pihak terkait, sehingga dapat menghasilkan kebijakan yang lebih baik dan berdampak positif pada masyarakat dan lingkungan. 3.2. RIA sebagai Metode Evaluasi dalam Penyusunan Peraturan Perundang- undangan di Indonesia Setelah diberlakukannya UU Nomor 13 Tahun 2022, terdapat perubahan menarik yang perlu diperhatikan. Beberapa perbaikan mencakup adopsi metode omnibus, koreksi kesalahan teknis dengan persetujuan bersama DPR dan Presiden, serta peningkatan partisipasi masyarakat. Selain itu, terdapat perubahan dalam pembentukan peraturan perundang-undangan secara elektronik, serta modifikasi dalam sistem pendukung dan teknik penyusunan Naskah Akademik. Secara khusus, perubahan teknik penyusunan Naskah Akademik menekankan penambahan metode seperti RIA atau Roccipi. Pentingnya penyebutan eksplisit metode RIA dalam UU ini menandai tahap baru dalam penggunaannya di Indonesia, membuka peluang untuk menerapkan metode analisis lain. Penyebutan ini juga merupakan langkah signifikan karena memperkenalkan metode RIA secara resmi dalam peraturan perundang-undangan nasional, menyiratkan evolusi dalam pendekatan analisis kebijakan di Indonesia Dalam konteks pembentukan Perda, metode RIA telah diperkenalkan sebagai alat untuk melibatkan masyarakat dalam proses legislasi di tingkat daerah. Namun, penerapannya 38 Halu Oleo Law Review \| Volume 8 Issue 1, March 2024 tidak selalu sesuai dengan pedoman RIA yang diusulkan oleh OECD, meskipun tidak ada instrumen hukum yang mengatur penggunaan RIA dalam proses pembentukan peraturan perundang-undangan. Dengan adanya penyebutan eksplisit mengenai metode RIA dalam UU 13/2022, penting untuk mengkaji kembali dasar konseptual dan kerangka kerja RIA. Bagaimana seharusnya metode RIA ditempatkan dalam konteks pembentukan peraturan perundang-undangan di Indonesia, khususnya dalam pembentukan UU dan Perda? Kapan metode RIA sebaiknya digunakan dalam proses pembentukan peraturan perundang-undangan di Indonesia? Semua pertanyaan ini memerlukan analisis lebih lanjut untuk memahami implikasi dan peran metode RIA dalam konteks regulasi di Indonesia. Dengan pemahaman ini, penting untuk menegaskan bahwa metode RIA seharusnya dianggap sebagai alat analisis dalam pemecahan masalah, bukan hanya sebagai metode dalam penyusunan Naskah Akademik (selanjutnya disebut sebagai “NA”). NA adalah dasar akademis dalam perancangan peraturan perundang-undangan. Pemahaman yang keliru sering kali menyebabkan RIA dianggap hanya sebagai langkah dalam penyusunan NA. Namun, jika RIA dianggap sebagai bagian integral dalam penyusunan NA, maka akan sulit untuk muncul opsi "tidak melakukan apa-apa" dalam RIA, karena NA selalu akan mengarah pada pembentukan peraturan, bahkan dalam struktur NA sendiri harus melibatkan rancangan peraturan perundang-undangan. Penting untuk dicatat bahwa UU 13/2022 mengakui metode RIA sebagai salah satu metode analisis yang sah. Namun, perlu dipahami bahwa perubahan yang diakui dalam UU 13/2022 terkait dengan Lampiran I UU 12/2011 lebih berfokus pada teknik penyusunan Naskah Akademik (NA) daripada metode penyusunan NA itu sendiri. 3.2. RIA sebagai Metode Evaluasi dalam Penyusunan Peraturan Perundang- undangan di Indonesia Lebih rinci lagi, perubahan yang terjadi pada Lampiran I UU 13/2022 berfokus pada Bab II Sub D yang berkaitan dengan Kajian terhadap Implikasi Penerapan Sistem Baru, bukan pada Bab I Sub D yang terkait dengan Metode. Oleh karena itu, alternatif metode analisis seperti RIA dan/atau ROCCIPI dalam Lampiran I UU 13/2022 lebih ditujukan untuk menyusun Kajian terhadap Implikasi Penerapan Sistem Baru daripada mengubah metode penyusunan NA itu sendiri. Dalam konteks UU 13/2022, metode penyusunan Naskah Akademik (NA) tidak mengalami perubahan seperti yang dielaborasikan di bawah ini: “Penyusunan Naskah Akademik pada dasarnya merupakan suatu kegiatan penelitian sehingga digunakan metode penyusunan Naskah Akademik yang berbasiskan metode penelitian hukum atau penelitian lain. Penelitian hukum dapat dilakukan melalui metode yuridis normatif dan metode yuridis empiris. Metode yuridis empiris dikenal juga dengan penelitian sosiolegal. Metode yuridis normatif dilakukan melalui studi pustaka yang menelaah (terutama) data sekunder yang berupa Peraturan Perundang-undangan, putusan pengadilan, perjanjian, kontrak, atau dokumen hukum lainnya, serta hasil penelitian, hasil pengkajian, dan referensi lainnya. Metode yuridis normatif dapat dilengkapi dengan wawancara, diskusi (seperti pada focus group discussion), dan rapat dengar pendapat. Metode yuridis empiris atau sosiolegal adalah penelitian yang diawali dengan penelitian normatif atau penelaahan terhadap Peraturan Perundang-undangan (normatif) yang 39 P-ISSN: 2548-1762 \| E-ISSN: 2548-1754 dilanjutkan dengan observasi yang mendalam serta penyebarluasan kuesioner untuk mendapatkan data faktor non hukum yang terkait dan yang berpengaruh terhadap Peraturan Perundang-undangan yang diteliti.” Dalam konteks UU 13/2022, metode RIA sebaiknya tidak diterapkan secara penuh untuk menyusun Bagian II Sub D terkait Kajian terhadap Implikasi Penerapan Sistem Baru. RIA memiliki opsi "do nothing" yang dapat menyiratkan bahwa tidak perlu membentuk peraturan atau kebijakan non-peraturan, yang bertentangan dengan keberadaan NA yang digunakan sebagai dasar akademis dalam pembentukan peraturan. Oleh karena itu, implementasi RIA dalam menyusun Bagian II Sub D terkait Kajian terhadap Implikasi Penerapan Sistem Baru harus mempertimbangkan aspek CBA (Cost-Benefit Analysis) yang relevan. Namun, dalam konteks ini, CBA hanya relevan untuk mengkaji tindakan satu-satunya yang dapat dianalisis, yaitu tindakan untuk membentuk peraturan. Penekanan harus diberikan pada pemahaman bahwa penyusunan NA merupakan langkah lanjutan dari keputusan politik untuk membentuk peraturan. Dengan demikian, meskipun metode RIA dapat digunakan untuk menganalisis implikasi penerapan sistem baru, implementasinya harus disesuaikan dengan konteks ini. Penting untuk memahami bahwa penyebutan metode RIA secara eksplisit dalam UU 13/2022 seharusnya diterjemahkan dengan cermat. 18 Erwinsyah Agus, Muhammad Jufri Dewa, and Muhammad Sabaruddin Sinapoy, “Analisis Hukum Peraturan Daerah Tanpa Melalui Tahapan Perencanaan Pembentukan Peraturan Perundang-Undangan,” Halu Oleo Law Review (2020): 177. 3.2. RIA sebagai Metode Evaluasi dalam Penyusunan Peraturan Perundang- undangan di Indonesia Ini hanya berlaku dalam konteks menyusun Bagian II Sub D terkait Kajian terhadap Implikasi Penerapan Sistem Baru, dan bahkan dalam konteks ini, penggunaan metode RIA dibatasi hanya pada analisis biaya dan manfaat yang relevan (CBA). Tidak boleh salah mengartikan bahwa metode RIA yang disebutkan dalam UU 13/2022 dimaksudkan untuk metode penyusunan NA. Hal ini dikarenakan metode penyusunan NA tidak mengalami perubahan dan masih menggunakan metode yang diperkenalkan dalam Lampiran I UU 12/2011. Namun, dalam tulisan ini, Penulis juga mengusulkan pemahaman baru tentang penggunaan metode RIA dalam pembentukan peraturan di Indonesia. Dengan dasar konseptual bahwa metode RIA adalah bagian dari Good Regulatory Practices (GRP), metode RIA dapat dianggap sebagai alat dalam penyusunan rencana daftar peraturan yang akan dibentuk, atau dalam konteks Indonesia, Program Legislasi Nasional untuk UU atau program pembentukan peraturan untuk peraturan di bawah UU. RIA dan GRP memiliki keterkaitan yang erat dalam konteks peraturan perundang-undangan. RIA, sebagai metode analisis, memberikan landasan untuk mengevaluasi dampak regulasi terhadap berbagai aspek, termasuk implikasi terhadap masyarakat dan beban keuangan negara. Sementara itu, GRP mencakup serangkaian prinsip dan prosedur yang ditujukan untuk memastikan regulasi yang efektif, efisien, dan memperhatikan kepentingan stakeholder. Integrasi RIA dalam GRP membantu memastikan bahwa setiap regulasi yang diusulkan telah melalui evaluasi dampak menyeluruh, memastikan bahwa keputusan regulasi didasarkan pada analisis yang solid dan memperhitungkan kebutuhan masyarakat serta meminimalkan risiko dampak yang tidak diinginkan. Dengan demikian, RIA dan GRP 40 Halu Oleo Law Review \| Volume 8 Issue 1, March 2024 bekerja bersama untuk menciptakan lingkungan regulasi yang lebih baik dan berkelanjutan. ( ) 19 Dian Agung Wicaksono, “Quo Vadis Pengaturan Regulatory Impact Analysis (RIA) Dalam Pembentukan Peraturan Perundang-Undangan,” Jurnal Legislasi Indonesia 20, No. 2 (2023): 45. 3.3. Analisis Penggunaan RIA dalam Penyusunan Peraturan Daerah Provinsi Jawa Timur Nomor 4 Tahun 2022 tentang Pemberdayaan Desa Wisata Perlu diketahui bahwa Naskah Akademik dari Peraturan Daerah Provinsi Jawa Timur Nomor 4 Tahun 2022 tentang Pemberdayaan Desa Wisata (selanjutnya disebut sebagai “Perda No. 4/2022”) ditulis dan diselesaikan pada tahun 2018, namun baru disahkan pada tahun 2022 setelah diundangkannya UU 13/2022. Tepatnya Perda No. 4/2022 disahkan pada tanggal 2 September 2022 sedangkan UU 13/2022 diundangkan pada 16 Juni 2022. Peraturan daerah ialah peraturan yang memiliki sifat terendah dari hierarki perundang- undangan. Peraturan daerah dibuat agar otonomi daerah secara strategis dapat dilaksanakan baik di tingkat provinsi maupun daerah.18 Penulisan peraturan perundang-undangan, terutama peraturan daerah provinsi, merupakan suatu tugas yang sangat penting dan rumit dalam konteks pembentukan hukum yang efektif. Untuk memastikan bahwa peraturan ini memiliki kualitas dan dampak yang positif, perlu diterapkan metode analisis yang tepat. Dua metode yang sering digunakan dalam pembuatan peraturan adalah Regulatory Impact Analysis (RIA) dan Regulatory Oversight for Compliance, Competitiveness, and Impact (ROCCIPI).19 Telah dijelaskan sebelumnya bahwa pada Lampiran I UU 13/2022 mensyaratkan bahwa: “Penambahan teknik penyusunan Naskah Akademik Rancangan Undang-Undang, Rancangan Peraturan Daerah Provinsi, dan Rancangan Peraturan Daerah Kabupaten/Kota dimuat dalam Lampiran I. Perubahan terhadap teknik penyusunan Naskah Akademik Rancangan Undang-Undang, Rancangan Peraturan Daerah Provinsi, dan Rancangan Peraturan Daerah Kabupaten/ Kota dilakukan terhadap ketentuan Bab II huruf D tentang kajian terhadap implikasi penerapan sistem baru yang akan diatur dalam Undang-Undang atau Peraturan Daerah terhadap aspek kehidupan masyarakat dan dampaknya terhadap aspek beban keuangan negara. Kajian tersebut didukung dengan analisis yang menggunakan metode tertentu, antara lain metode Regulatory Impact Analysis (RIA) dan metode Rule, Opportunity, Capacity, Communication, Interest, Process, and ldeology (ROCCIPI).” Namun, dalam analisis terhadap naskah akademik peraturan daerah provinsi Jawa Timur yang berkaitan dengan pemberdayaan desa wisata, terdapat ketidaksesuaian yang mencolok. Sebagai sebuah perangkat penting dalam proses pembentukan peraturan, 41 P-ISSN: 2548-1762 \| E-ISSN: 2548-1754 naskah akademik tersebut seharusnya mencakup penerapan metode seperti ROCCIPI dan RIA untuk memastikan bahwa dampak peraturan terhadap masyarakat dan ekonomi setempat diperhitungkan dengan baik.20 Namun, dalam naskah akademik yang telah diajukan, terdapat kekurangan yang signifikan. Naskah tersebut hanya berfokus pada legalitas desa wisata dan pembahasan filosofis, tanpa mengintegrasikan metode analisis yang sangat dibutuhkan dalam pembuatan peraturan yang efektif. Hal ini menunjukkan bahwa perlu adanya perbaikan dalam proses penyusunan peraturan daerah provinsi tersebut, dengan memasukkan metode analisis seperti RIA dan ROCCIPI guna memastikan peraturan yang dihasilkan memiliki dampak positif yang diinginkan dan sesuai dengan tujuan pemberdayaan desa wisata di Jawa Timur. 20 R.D.H. Koesoemahatmadja, “Pengantar Ke Arah Sistem Pemerintahan Daerah Di Indonesia” (Bandung: Binacipta, 1979), 4. 3.3. Analisis Penggunaan RIA dalam Penyusunan Peraturan Daerah Provinsi Jawa Timur Nomor 4 Tahun 2022 tentang Pemberdayaan Desa Wisata Dalam proses pembuatan peraturan perundang-undangan, termasuk peraturan daerah provinsi, penting untuk memahami bahwa peraturan tersebut akan memiliki dampak langsung pada masyarakat, ekonomi, dan lingkungan. Oleh karena itu, metode seperti Regulatory Oversight for Compliance, Competitiveness, and Impact (ROCCIPI) dan Regulatory Impact Analysis (RIA) sangat relevan. ROCCIPI adalah sebuah kerangka kerja yang bertujuan untuk memastikan peraturan- peraturan yang ada benar-benar ditaati dan efektif dalam mencapai tujuannya. Hal ini mencakup evaluasi secara menyeluruh terhadap peraturan yang ada, pemantauan implementasi, dan penyesuaian jika diperlukan. Dalam konteks peraturan daerah provinsi yang berkaitan dengan pemberdayaan desa wisata, penggunaan ROCCIPI akan memastikan bahwa peraturan tersebut benar-benar memberdayakan desa-desa wisata secara efektif, dan tidak hanya menjadi dokumen hukum yang tidak digunakan. Di sisi lain, RIA adalah suatu metode yang digunakan untuk menganalisis dampak sosial, ekonomi, dan lingkungan dari peraturan yang akan diterapkan. Dalam kasus peraturan daerah provinsi tentang pemberdayaan desa wisata, RIA akan membantu dalam mengidentifikasi dampak positif dan negatif dari peraturan tersebut. Ini termasuk dampak ekonomi terhadap industri pariwisata, dampak sosial terhadap masyarakat lokal, dan dampak lingkungan. Dengan memasukkan RIA dalam naskah akademik peraturan, pemerintah dapat membuat kebijakan yang lebih berkelanjutan dan sesuai dengan tujuan pemberdayaan desa wisata di Jawa Timur. Namun, perlu dicatat bahwa dalam naskah akademik yang telah diajukan, kedua metode ini tampaknya tidak digunakan. Fokus naskah hanya pada legalitas dan aspek filosofis, tanpa memberikan analisis yang komprehensif mengenai bagaimana peraturan tersebut akan memengaruhi masyarakat dan lingkungan sekitar. Inilah yang membuatnya kurang memadai dan perlu adanya perbaikan dalam proses penyusunan peraturan daerah provinsi tersebut. Dengan menggabungkan metode ROCCIPI dan RIA, naskah akademik dapat diperkaya dengan analisis yang lebih mendalam, yang akan memastikan bahwa 42 Halu Oleo Law Review \| Volume 8 Issue 1, March 2024 peraturan yang dihasilkan benar-benar bermanfaat dan sesuai dengan tujuan pemberdayaan desa wisata di Jawa Timur, serta memenuhi persyaratan hukum yang berlaku. Analisis lebih dalam mengenai penggunaan metode ROCCIPI dan RIA dalam pembuatan peraturan perundang-undangan yang berlaku memperlihatkan pentingnya pendekatan yang terstruktur dan holistik dalam perumusan kebijakan. Dalam konteks peraturan daerah provinsi yang menyangkut pemberdayaan desa wisata, perlu dijelaskan lebih rinci mengapa kedua metode ini sangat relevan.21 ROCCIPI, pertama-tama, berfokus pada peran kritis pengawasan dan penegakan hukum dalam peraturan yang ada. Dalam konteks peraturan perundang-undangan tentang pemberdayaan desa wisata, implementasi peraturan tersebut harus dipantau secara ketat untuk memastikan bahwa desa-desa wisata sebenarnya mendapatkan manfaat yang diharapkan. 21 Agussalim Andi Gadjong, Pemerintahan Daerah: Kajian Politik Dan Hukum (Jakarta: Ghalia Indonesia, 2005), 76. 3.3. Analisis Penggunaan RIA dalam Penyusunan Peraturan Daerah Provinsi Jawa Timur Nomor 4 Tahun 2022 tentang Pemberdayaan Desa Wisata Hal ini mencakup pengukuran efektivitas peraturan, pemantauan kompetitivitas desa wisata, serta evaluasi dampaknya terhadap tingkat kepatuhan dan kinerja ekonomi desa. Dengan menggunakan ROCCIPI, pemerintah dapat memastikan bahwa peraturan yang diterapkan bukan hanya "kertas dan pena", melainkan alat yang berfungsi dengan baik untuk mencapai tujuan pemberdayaan desa wisata. Selain itu, RIA memiliki peran penting dalam memahami implikasi jangka panjang dari peraturan. Peraturan perundang-undangan yang berkaitan dengan pemberdayaan desa wisata akan memiliki dampak ekonomi, sosial, dan lingkungan yang signifikan. Analisis RIA akan membantu mengidentifikasi risiko dan manfaat dari peraturan tersebut. Misalnya, dapat mengukur potensi pertumbuhan sektor pariwisata yang mungkin terjadi sebagai dampak positif, namun juga memperhatikan dampak lingkungan, seperti peningkatan limbah atau kerusakan lingkungan yang mungkin terjadi. Dengan pemahaman yang lebih mendalam tentang dampak ini, peraturan dapat disusun dengan lebih cermat, menciptakan keseimbangan antara pemberdayaan desa wisata dan pelestarian lingkungan. Penting untuk mencatat bahwa dalam naskah akademik yang telah diajukan, kekurangan dalam penggunaan kedua metode ini mengisyaratkan bahwa peraturan yang disusun mungkin belum cukup komprehensif. Dampak sosial, ekonomi, dan lingkungan tidak dibahas secara mendalam, dan ini dapat berpotensi menghasilkan peraturan yang kurang efektif dalam mencapai tujuan pemberdayaan desa wisata. Oleh karena itu, penulis naskah akademik perlu memahami bahwa metode analisis yang lebih mendalam seperti ROCCIPI dan RIA sangat penting dalam merumuskan peraturan yang efektif dan berkelanjutan, yang tidak hanya mematuhi hukum tetapi juga memberikan manfaat maksimal bagi masyarakat dan lingkungan yang terlibat. Analisis dampak regulasi atau RIA adalah komponen penting dalam penyusunan peraturan perundang-undangan yang efektif. Dalam konteks peraturan daerah provinsi 43 P-ISSN: 2548-1762 \| E-ISSN: 2548-1754 yang berkaitan dengan pemberdayaan desa wisata, penggunaan RIA memiliki implikasi yang mendalam.22 Pertama-tama, RIA membantu pemerintah memahami dampak sosial dari peraturan tersebut. Ini melibatkan analisis dampak terhadap masyarakat lokal dan komunitas desa yang akan terkena dampak langsung dari peraturan. Dengan menggunakan RIA, pemerintah dapat mengidentifikasi apakah peraturan tersebut akan memberdayakan masyarakat lokal atau malah membawa konsekuensi sosial yang tidak diinginkan. Misalnya, peraturan dapat menciptakan peluang pekerjaan baru dan meningkatkan pendapatan bagi warga desa, tetapi juga dapat memiliki dampak seperti perubahan sosial atau masalah ketidaksetaraan ekonomi. RIA membantu memahami kompleksitas dan risiko-risiko sosial ini. Selanjutnya, RIA melibatkan analisis dampak ekonomi dari peraturan tersebut. Dalam konteks pemberdayaan desa wisata, peraturan dapat memiliki implikasi ekonomi yang signifikan. RIA akan membantu mengukur pertumbuhan sektor pariwisata yang dapat terjadi sebagai akibat dari peraturan tersebut, serta dampaknya pada lapangan pekerjaan, pendapatan, dan perkembangan ekonomi lokal. 22 The Liang Gie, Pertumbuhan Pemerintah Daerah Di Negara Republik Indonesia (Jakarta: Gunung Agung, 1968), 35. 3.3. Analisis Penggunaan RIA dalam Penyusunan Peraturan Daerah Provinsi Jawa Timur Nomor 4 Tahun 2022 tentang Pemberdayaan Desa Wisata Dengan pemahaman yang lebih mendalam tentang dampak ekonomi, pemerintah dapat menyusun peraturan yang mendukung pertumbuhan ekonomi berkelanjutan. Terakhir, RIA juga memperhatikan dampak lingkungan dari peraturan tersebut. Di daerah dengan desa-desa wisata, pelestarian lingkungan sering kali menjadi fokus penting. Dengan RIA, pemerintah dapat mengukur dampak potensial peraturan terhadap lingkungan seperti polusi, kerusakan ekosistem, atau pemanfaatan sumber daya alam. Dengan demikian, peraturan dapat dirancang untuk memastikan perlindungan lingkungan yang adekuat. Dalam konteks naskah akademik yang telah diajukan, kurangnya analisis dampak sosial, ekonomi, dan lingkungan dari peraturan menunjukkan bahwa naskah tersebut kurang lengkap. Dalam rangka untuk memastikan bahwa peraturan pemberdayaan desa wisata di Jawa Timur berfungsi dengan baik dan sesuai dengan tujuannya, diperlukan analisis RIA yang lebih mendalam. Dengan demikian, peraturan dapat dirancang dengan lebih baik untuk memaksimalkan manfaat sosial dan ekonomi sambil meminimalkan dampak negatif pada lingkungan. Analisis Ex Post yang telah dilakukan mengungkapkan sejumlah kekurangan signifikan dalam implementasi Peraturan Daerah (Perda) No. 4/2022. Dalam konteks pemberdayaan desa wisata di Jawa Timur, sejumlah aspek yang sangat penting tampaknya tidak berhasil diatasi secara efektif. Terdapat ketidaksesuaian antara tujuan yang diharapkan dalam peraturan tersebut dan kenyataan lapangan. Dalam beberapa hal, Perda 44 Halu Oleo Law Review \| Volume 8 Issue 1, March 2024 ini mungkin belum mencapai potensi penuhnya dalam mencapai tujuan pemberdayaan desa wisata. Sebagai contoh, pada Pasal 2 yang bertujuan untuk menguraikan prinsip-prinsip yang harus diperhatikan dalam pemberdayaan Desa Wisata, termasuk keaslian, partisipasi masyarakat, nilai tambah, dan konservasi, guna memastikan pengembangan destinasi wisata yang berkelanjutan dan memperkaya nilai budaya lokal. Pasal ini menyediakan beberapa opsi prinsip-prinsip yang harus dipertimbangkan dalam pemberdayaan Desa Wisata, seperti keaslian, kolaborasi, dan konservasi. Dengan demikian, setiap Desa Wisata memiliki ruang untuk mengembangkan program yang sesuai dengan identitas dan kebutuhan lokal mereka. Pemberdayaan Desa Wisata yang memperhatikan prinsip- prinsip ini memungkinkan pengelolaan destinasi wisata yang berkelanjutan, mempertahankan warisan budaya, dan melibatkan masyarakat secara aktif. Dengan mengikuti prinsip-prinsip ini, Desa Wisata memiliki peluang untuk menjadi destinasi yang unik dan menarik bagi pengunjung sambil memperhatikan keberlangsungan lingkungan dan nilai-nilai budaya. Lalu, pada Pasal 5 bertujuan untuk menetapkan lembaga pengelola Desa Wisata agar dapat mengelola destinasi tersebut secara efektif. Hal ini untuk memastikan adanya entitas yang mampu merencanakan, mengkoordinasikan, dan mengembangkan potensi wisata setempat. Pasal ini memberikan beberapa opsi lembaga yang dapat mengelola Desa Wisata, seperti Badan Usaha Milik Desa, kelompok sadar wisata (Pokdarwis), atau kelompok masyarakat lainnya. 3.3. Analisis Penggunaan RIA dalam Penyusunan Peraturan Daerah Provinsi Jawa Timur Nomor 4 Tahun 2022 tentang Pemberdayaan Desa Wisata Dengan demikian, ada keragaman struktur organisasi yang dapat diadaptasi sesuai kebutuhan dan kondisi lokal. Dengan adanya lembaga pengelola yang bervariasi, Desa Wisata dapat mengadopsi struktur organisasi yang sesuai dengan kebutuhan mereka. Ini memungkinkan pengelolaan yang lebih efisien dan inklusif, melibatkan berbagai kelompok dalam mengelola dan mempromosikan destinasi wisata mereka. Terakhir pada Pasal 18 memiliki tujuan untuk memberikan pedoman dalam memberikan bantuan pembiayaan untuk pemberdayaan Desa Wisata dengan mempertimbangkan ketersediaan dana daerah. Pasal ini menegaskan bahwa bantuan pembiayaan harus disesuaikan dengan kemampuan keuangan daerah dan harus sesuai dengan ketentuan perundang-undangan yang berlaku. Dengan mempertimbangkan ketersediaan dana daerah dan mengikuti ketentuan hukum yang berlaku, bantuan pembiayaan untuk pemberdayaan Desa Wisata akan menjadi lebih terencana, efektif, dan sesuai dengan kapasitas keuangan daerah. Hal ini mencegah pengeluaran yang berlebihan dan memastikan penggunaan dana yang efisien untuk pengembangan Desa Wisata. Salah satu aspek penting yang perlu ditekankan adalah bahwa Naskah Akademik Perda No. 4/2022 tidak memasukkan analisis RIA. RIA adalah alat yang sangat berharga untuk memahami dampak sosial, ekonomi, dan lingkungan dari sebuah peraturan, terutama dalam konteks peraturan yang berkaitan dengan pembangunan desa wisata. Kehilangan analisis RIA dalam naskah akademik dapat mengakibatkan peraturan yang kurang 45 P-ISSN: 2548-1762 \| E-ISSN: 2548-1754 mendalam dan mungkin tidak memadai dalam mengatasi masalah yang dihadapi oleh desa wisata di Jawa Timur. Peraturan Daerah (Perda) No. 4/2022 yang mengatur pemberdayaan desa wisata di Jawa Timur telah menjadi fokus perhatian, terutama terkait ketiadaan ketentuan yang mengatur Kampung Wisata. Sementara banyak desa wisata sudah ada di wilayah Jawa Timur, tidak dapat diabaikan bahwa Kampung Wisata juga memiliki eksistensi yang signifikan dalam sektor pariwisata. Contohnya, Surabaya memiliki beragam Kampung Wisata yang menjadi destinasi wisata yang populer. Namun, Perda No. 4/2023 sepertinya belum mengakomodasi atau mengatur secara khusus tentang Kampung Wisata, meskipun mereka merupakan bagian penting dari lanskap pariwisata di provinsi ini. Ketidakinklusian ketentuan untuk Kampung Wisata dalam Perda ini menimbulkan pertanyaan penting tentang pengakuan dan dukungan yang diberikan kepada wilayah- wilayah tersebut dalam upaya pemberdayaan. Pengaturan yang jelas dan komprehensif tentang Kampung Wisata dapat membantu dalam mengintegrasikan upaya pemberdayaan desa dan kampung wisata, serta memastikan bahwa semua elemen pariwisata lokal mendapat perhatian yang layak. Oleh karena itu, penting untuk mempertimbangkan untuk merevisi atau melengkapi Perda No. 4/2022 dengan mengakomodasi peran dan keberadaan Kampung Wisata dalam pemberdayaan desa wisata di Jawa Timur. 3.3. Analisis Penggunaan RIA dalam Penyusunan Peraturan Daerah Provinsi Jawa Timur Nomor 4 Tahun 2022 tentang Pemberdayaan Desa Wisata Dengan demikian, peraturan akan lebih reflektif terhadap realitas lapangan dan dapat memberikan landasan hukum yang kuat untuk pengembangan sektor pariwisata yang lebih holistik di wilayah ini. Dalam rangka memperbaiki Perda No. 4/2022, salah satu pilihan adalah memasukkan analisis RIA yang mendalam dan komprehensif. Hal ini akan membantu dalam memahami dampak peraturan tersebut terhadap masyarakat, ekonomi, dan lingkungan, dan juga dalam mengidentifikasi potensi hambatan atau masalah yang mungkin timbul. Jika diperlukan, perubahan dalam naskah Perda atau pembentukan ulang peraturan dengan mempertimbangkan pedoman yang ada dalam UU No. 13/2022 dapat menjadi langkah yang tepat untuk memastikan bahwa peraturan ini efektif dalam mencapai tujuan pemberdayaan desa wisata di Jawa Timur. 4. Kesimpulan Setelah diberlakukannya UU Nomor 13 Tahun 2022, terdapat perubahan penting dalam proses pembentukan peraturan perundang-undangan di Indonesia. Beberapa aspek yang mencolok melibatkan penambahan metode omnibus, perbaikan kesalahan teknis, pembentukan peraturan perundang-undangan secara elektronik, dan perubahan teknik penyusunan naskah akademik. Paling mencolok adalah penyebutan metode Regulatory Impact Analysis (RIA) sebagai alternatif analisis, membuka peluang untuk metode analisis lain dalam mengevaluasi implikasi peraturan terhadap masyarakat dan keuangan negara. Seiring dengan perubahan ini, penting untuk melakukan tinjauan mendalam terhadap implementasi metode RIA dan peran serta masyarakat dalam proses perencanaan pembentukan peraturan. Evaluasi tersebut perlu mencakup pemahaman yang 46 Halu Oleo Law Review \| Volume 8 Issue 1, March 2024 komprehensif mengenai perintah pembentukan UU dari UUD NRI Tahun 1945, Tap MPR, dan UU lainnya, serta penerapan mekanisme yang memungkinkan validasi aspirasi dan kebutuhan hukum masyarakat dalam Prolegnas. Integrasi RIA sebagai alat analisis dalam tahap perencanaan dapat memperkuat partisipasi masyarakat dan memastikan keberlanjutan Good Regulatory Practices dalam pengambilan kebijakan. 5. Saran Saran untuk perbaikan melibatkan tiga aspek utama. Pertama, melakukan evaluasi lebih lanjut terhadap implementasi peraturan dengan fokus pada pemahaman sejauh mana peraturan menjadi landasan efektif bagi program pemberdayaan desa wisata. Kedua, merevisi Peraturan Daerah dengan memasukkan ketentuan yang mengakomodasi peran dan keberadaan Kampung Wisata dalam pemberdayaan desa wisata. Ketiga, memperkaya naskah akademik peraturan dengan analisis Regulatory Impact Analysis (RIA) guna memahami dampak sosial, ekonomi, dan lingkungan secara mendalam. Dengan langkah- langkah ini, diharapkan peraturan dapat lebih efektif dalam mencapai tujuan pemberdayaan desa wisata di Jawa Timur. Daftar Pustaka Agus, Erwinsyah, Muhammad Jufri Dewa, and Muhammad Sabaruddin Sinapoy. “Analisis Hukum Peraturan Daerah Tanpa Melalui Tahapan Perencanaan Pembentukan Peraturan Perundang-Undangan.” Halu Oleo Law Review (2020). Ediawan, Agus, Yuyu Qomariah, Frida Rustanti, Hari Kusdaryanto, Muhammad Mustafa, and Bayu Wijayanto. Arti Penting Regulatory Impact Assessment (RIA). Jakarta: The Asia Foundation, 2008. Gadjong, Agussalim Andi. Pemerintahan Daerah: Kajian Politik Dan Hukum. Jakarta: Ghalia Indonesia, 2005. Gie, The Liang. Pertumbuhan Pemerintah Daerah Di Negara Republik Indonesia. Jakarta: Gunung Agung, 1968. Haryadi, Dadi. “DPRD Jabar: Perda Desa Wisata Untuk Pembangunan Jawa Barat.” AyoPurwakarta.Com. Last modified 2021. Accessed September 19, 2023. https://purwakarta.ayoindonesia.com/umum/pr-321274975/dprd-jabar-perda- desa-wisata-untuk-pembangunan-jawa-barat. Hasima, Rahman. “Penerapan Metode Regulatory Impact Assessment Dalam Penyusunan Peraturan Daerah Di Kota Kendari.” Halu Oleo Law Review 4, No. 1 (March 20, 2020): 54. http://ojs.uho.ac.id/index.php/holrev/article/view/9258. Hauerstein, Kai, and Peter Bissenger. Training Manual Regulatory Impact Assessment. Jakarta: GTZ-Red dan Bappenas RI, 2009. Jacobs, Scott H. “An Overview of Regulatory Impact Analysis in OECD Countries.” In Regulatory Impact Analysis: Best Practices in OECD Countries. Paris: OECD Publication, 1997. Koesoemahatmadja, R.D.H. “Pengantar Ke Arah Sistem Pemerintahan Daerah Di 47 P-ISSN: 2548-1762 \| E-ISSN: 2548-1754 Indonesia.” Bandung: Binacipta, 1979. Kusnandar, Viva Budy. “Berapa Jumlah Desa/Kelurahan Di Indonesia?” Katadata Databoks. Last modified 2021. Accessed September 19, 2023. https://databoks.katadata.co.id/ datapublish/2021/11/22/berapa-jumlah-desakelurahan-di-indonesia. Ngaisah, Siti, Bagus Ananda Kurniawan, and Chusnul Abadi. “Implementasi Program Desa Wisata Dalam Menunjang Pelestarian Dan Pengembangan Budaya Keris.” Kaganga: Jurnal Pendidikan Sejarah dan Riset Sosial Humaniora 4, No. 1 (March 10, 2021): 1–6. https://journal.ipm2kpe.or.id/index.php/KAGANGA/article/view/1863. Office of the Assistant Secretary for Planning and Evaluation. Guidelines for Regulatory Impact Analysis. Washington, D.C.: U.S. Department of Health and Human Services, 2016. Ripley, Randall B., and Grace A. Franklin. Policy Implementation and Bureaucracy. Illinois: The Dorsey Press, 1982. Rosando, Abraham Ferry, Hilyatun Nuha, Paramitha Rachmawati, and Dimas Mahendra Putra. “Legalisasi Pembentukan Desa Wisata Budaya Plunturan Dari Pemerintah Kabupaten Ponorogo.” In Seminar Nasional Konsorsium Universitas 17 Agustus 1945 Surabaya Ke-2, 38–46. Surabaya: Universitas 17 Agustus 1945, 2020. Satria, Rahmad. “Penerapan Metode Regulatory Impact Assessment (RIA) Dalam Penyusunan Regulasi Daerah.” Masalah-Masalah Hukum 2, No. 2 (April 24, 2015): 178. http://ejournal.undip.ac.id/index.php/mmh/article/view/11432. Suchman, Edward A. Evaluative Research: Principles and Practice in Public Service and Social Action Progr. New York: Russell Sage Foundation, 1967. http://www.jstor.org/ stable/10.7758/9781610445177. Wicaksono, Dian Agung. “Quo Vadis Pengaturan Regulatory Impact Analysis (RIA) Dalam Pembentukan Peraturan Perundang-Undangan.” Jurnal Legislasi Indonesia 20, No. 2 (2023): 44–60. Widarti, Peni. “6 Desa Wisata Jatim Terpilih Jadi Desa Wisata Terbaik 2021.” Bisnis.Com. Last modified 2021. Accessed September 19, 2023. https://surabaya.bisnis.com/ read/20210826/531/1434482/6-desa-wisata-jatim-terpilih-jadi-desa-wisata- terbaik-2021. 48
https://openalex.org/W4385201181	https://lexrussica.msal.ru/jour/article/download/3191/1390	Russian	null	Modern Aspects of Violent Juvenile Delinquency	Lex Russica/Lex russica (Russkij zakon)	2,023	cc-by	7,655	* Корнакова Светлана Викторовна, кандидат юридических наук, доцент, доцент кафедры теории и истории государства и права Байкальского государственного университета (БГУ) Ленина ул., д. 11, г. Иркутск, Россия, 664003 svetlana-kornakova@yandex.ru ** Корягина Светлана Анатольевна, кандидат юридических наук, доцент, доцент кафедры уголовного права и криминологии Байкальского государственного университета (БГУ) Ленина ул., д. 11, г. Иркутск, Россия, 664003 koryagina.sveta@yandex.ru DOI: 10.17803/1729-5920.2023.200.7.076-086 С. В. Корнакова, С. А. Корягина* © Корнакова С. В., Корягина С. А., 2023 Науки криминального цикла JUS CRIMINALE Науки криминального цикла JUS CRIMINALE Современные аспекты насильственной преступности несовершеннолетних Аннотация. В статье проводится анализ современного состояния насильственной преступности несовер- шеннолетних в нашей стране. Приводятся данные официальной статистики. Результатом анализа таких данных явилась констатация того, что наряду с тенденцией снижения преступности несовершеннолетних отмечается вызывающий тревогу прирост совершения несовершеннолетними тяжких насильственных преступлений. Демонстрируются сводные показатели преступности несовершеннолетних, обосновыва- ющие такой вывод. Авторами рассматриваются точки зрения ученых на социальные факторы, которые являются детерми- нирующими для проявления насилия в подростковой среде. В этой связи делается обобщающий вывод о том, что к основным факторам относятся демонстрация насилия в СМИ и Интернете, его привычное проявление в ближайшем окружении, социальные девиации и психические аномалии у несовершенно- летних, а также существенные недостатки досуговой деятельности. Уделяется внимание ситуативному характеру преступлений, совершенных несовершеннолетними, а также влиянию на совершение неко- торых видов преступлений личности и поведения несовершеннолетних потерпевших. Делается вывод о насущной необходимости выработки комплексных превентивных мер, которые предполагают скоор- динированную деятельность различных учреждений (образовательных, просветительных, спортивных, культурно-досуговых учреждений, правоохранительных органов). При этом, по мнению авторов, основ- ным вектором политики нашего государства в этой сфере должно быть дальнейшее усиление мер по нравственному и идеологическому воспитанию несовершеннолетних, организации для них доступной досуговой деятельности, поскольку обусловленную возрастом избыточную поведенческую активность несовершеннолетних следует не подавлять, а переориентировать. Авторы приходят к выводу, что молодежная политика государства должна быть направлена на повыше- ние ценности здорового образа жизни, образования и культуры, а основным ее вектором в этой сфере должно быть усиление мер по нравственному и идеологическому воспитанию несовершеннолетних. Ключевые слова: преступность несовершеннолетних; насильственные преступления несовершеннолет- них; детерминация насилия; меры предупреждения преступности несовершеннолетних. Для цитирования: Корнакова С. В., Корягина С. А. Современные аспекты насильственной преступ- ности несовершеннолетних // Lex russica. — 2023. — Т. 76. — № 7. — С. 76–86. — DOI: 10.17803/1729- 5920 2023 200 7 076-086 Авторы приходят к выводу, что молодежная политика государства должна быть направлена на повыше- ние ценности здорового образа жизни, образования и культуры, а основным ее вектором в этой сфере должно быть усиление мер по нравственному и идеологическому воспитанию несовершеннолетних. Ключевые слова: преступность несовершеннолетних; насильственные преступления несовершеннолет- них; детерминация насилия; меры предупреждения преступности несовершеннолетних Авторы приходят к выводу, что молодежная политика государства должна быть направлена на повыше- ние ценности здорового образа жизни, образования и культуры, а основным ее вектором в этой сфере должно быть усиление мер по нравственному и идеологическому воспитанию несовершеннолетних. должно быть усиление мер по нравственному и идеологическому воспитанию несовершеннолетних. Ключевые слова: преступность несовершеннолетних; насильственные преступления несовершеннолет- них; детерминация насилия; меры предупреждения преступности несовершеннолетних. Современные аспекты насильственной преступности несовершеннолетних Для цитирования: Корнакова С В Корягина С А Современные аспекты насильственной преступ Ключевые слова: преступность несовершеннолетних; насильственные преступления несовершеннолет- них; детерминация насилия; меры предупреждения преступности несовершеннолетних. Для цитирования: Корнакова С. В., Корягина С. А. Современные аспекты насильственной преступ- ности несовершеннолетних // Lex russica. — 2023. — Т. 76. — № 7. — С. 76–86. — DOI: 10.17803/1729- 5920.2023.200.7.076-086. Том 76 № 7 (200) июль 2023 76 Корнакова С. В., Корягина С. А. Современные аспекты насильственной преступности несовершеннолетних 1 Указ Президента РФ от 02.07.2021 № 400 «О Стратегии национальной безопасности Российской Феде- рации» // СЗ РФ. 2021. № 27 (ч. II). Ст. 5351. Modern Aspects of Violent Juvenile Delinquency Svetlana V. Kornakova, Cand. Sci. (Law), Associate Professor, Associate Professor, Department of Theory and History of State and Law, Baikal State University (BSU) ul. Lenina, d. 11, Irkutsk, Russia, 664003 svetlana-kornakova@yandex.ru Svetlana V. Kornakova, Cand. Sci. (Law), Associate Professor, Associate Professor, Department of Theory and History of State and Law, Baikal State University (BSU) ul. Lenina, d. 11, Irkutsk, Russia, 664003 svetlana kornakova@yandex ru Svetlana A. Koryagina, Cand. Sci. (Law), Associate Professor, Associate Professor, Department of Criminal Law and Criminology, Baikal State University (BSU) ul. Lenina, d. 11, Irkutsk, Russia, 664003 koryagina.sveta@yandex.ru Svetlana A. Koryagina, Cand. Sci. (Law), Associate Professor, Associate Professor, Department of Criminal Law and Criminology, Baikal State University (BSU) ul. Lenina, d. 11, Irkutsk, Russia, 664003 koryagina.sveta@yandex.ru Abstract. The paper analyzes the current state of violent juvenile delinquency in our country and gives the data of official statistics. The analysis of the data shows that, along with the decreasing juvenile delinquency, there is an alarming increase in the commission of serious violent crimes by minors. Summary indicators of juvenile delinquency are demonstrated, justifying such a conclusion. The authors consider the point of view of scientists on social factors that are determinative for the manifestation of violence in the adolescent environment. In this regard, the authors conclude that in general the main factors include the demonstration of violence in the media and the Internet, its habitual manifestation in the immediate environment, social deviations and mental anomalies in minors, as well as significant disadvantages of leisure activities. Attention is given to the situational nature of crimes committed by minors, as well as the impact on the commission of certain types of crimes of the personality and behavior of juvenile victims. It is concluded that there is an urgent need to develop comprehensive preventive measures that involve coordinated activities of various institutions (educational, sports, cultural and leisure institutions, law enforcement agencies). At the same time, according to the authors, the main vector of our state’s policy in this area should be the further strengthening of measures for the moral and ideological education of minors, the organization of accessible leisure activities for them, since excessive behavioral activity of minors due to age should not be suppressed, but reoriented. The authors conclude that the state’s youth policy should aimed at increasing the value of a healthy lifestyle, education and culture, and its main vector in this area should be strengthening measures for the moral and ideological education of minors. Keywords: juvenile delinquency; violent crimes of minors; determination of violence; measures to prevent juvenile delinquency. Cite as: Kornakova SV, Koryagina SA. Sovremennye aspekty nasilstvennoy prestupnosti nesovershennoletnikh [Modern Aspects of Violent Juvenile Delinquency]. Lex russica. 2023;76(7):76-86. DOI: 10.17803/1729- 5920.2023.200.7.076-086. (In Russ., abstract in Eng.). всестороннему духовному, интеллектуальному, нравственному и физическому развитию. Svetlana V. Kornakova, Cand. Sci. (Law), Associate Professor, Associate Professor, Department of Theory and History of State and Law, Baikal State University (BSU) ul. Lenina, d. 11, Irkutsk, Russia, 664003 svetlana kornakova@yandex ru Более того, акцентируется внимание и на усилении роли государства в качестве гаранта безопасно- сти личности, на повышении результативности в системе профилактики преступлений, преду- преждении преступных проявлений, прежде всего среди несовершеннолетних и молодежи. В Стратегии национальной безопасности Российской Федерации до 2025 года1 сформу- лированы цели политического курса государ- ственной политики в области обеспечения национальной безопасности России на долго- срочную перспективу. Указанный документ яв- ляется базовым документом стратегического планирования, определившим национальные интересы и национальные приоритеты Рос- сийской Федерации, среди которых отмечено «сбережение народа России и развитие чело- веческого потенциала». При этом особое вни- мание уделяется поддержке семьи, материн- ства, отцовства и детства, воспитанию детей, их всестороннему духовному, интеллектуальному, нравственному и физическому развитию. Более того, акцентируется внимание и на усилении роли государства в качестве гаранта безопасно- сти личности, на повышении результативности в системе профилактики преступлений, преду- преждении преступных проявлений, прежде всего среди несовершеннолетних и молодежи. В этой связи представляется целесообраз- ным рассмотрение фактического уровня пре- ступности несовершеннолетних с учетом официальной статистической отчетности на современном этапе. В криминологической литературе отмечается, что подростковая пре- LEX RUSSICA Том 76 № 7 (200) июль 2023 77 Науки криминального цикла JUS CRIMINALE LEX RUSSICA LEX RUSSICA JUS CRIMINALE Диаграмма 1. Удельный вес преступности несовершеннолетних в общем объеме преступности в России, % LEX RUSSICA JUS CRIMI Диаграмма 1. Удельный вес преступности несовершеннолетних в общем объеме преступности в России, % Диаграмма 1. Удельный вес преступности несовершеннолетних в общем объеме преступности в России, % Латентность преступности несовершенно- летних может быть связана, во-первых, с укры- тием преступлений от учета и регистрации, во-вторых, с определенным снижением актив- ности правоохранительных органов по их выяв- лению. В-третьих, с либеральным отношением органов уголовной юстиции к совершаемым несовершеннолетними преступлениям неболь- шой и средней тяжести. И в-четвертых, в ряде случаев латентность связана с категорией пре- ступления и с реакцией потерпевших на его со- вершение. Как следствие, при отсутствии адек- ватного реагирования со стороны государства на первичную преступность несовершеннолет- них с высокой степенью вероятности можно предположить совершение ими повторных пре- ступлений в дальнейшем, но уже относящихся к категории тяжких и особо тяжких. В частно- сти, официальные данные портала правовой статистики свидетельствуют о том, что в 2022 г. каждый четвертый осужденный несовершен- нолетний совершил тяжкое преступление, а каждый двадцатый — преступление особо тяжкое. Причем отмечался темп прироста осо- бо тяжких преступлений: в 2017 г. — +14,2 %; в 2018 — +5,6 %; 2019 г. — +6,9 %, а в 2022 г. 2 Голубичная Л. С. Преступность несовершеннолетних: криминологическая характеристика и пробле- мы предупреждения (по материалам Дальневосточного федерального округа) : автореф. дис. … канд. юрид. наук : 12.00.08. Владивосток, 2011. С. 4. 3 Бастрыкин А. И. Глава Следственного комитета о причинах жестоких преступлений подростков // URL: https://rg.ru/2021/12/28/glava-sledstvennogo-komiteta-o-prichinah-zhestokih-prestuplenij-podrostkov/ (дата обращения: 20.03.2023). 4 Яровенко В. В., Яровенко Е. В. Криминологические проблемы девиантного поведения несовершенно- летних // Всероссийский криминологический журнал. 2013. № 3. С. 62. 5 Сутурин М. А. Некоторые аспекты криминологического анализа личности преступника (по материалам Иркутской и Читинской областей) // Сибирский юридический вестник. 2009. № 1 (44). С. 93. 6 Уголовное дело № 36694. Архив Иркутского областного суда. 7 Уголовное дело № 50981. Архив Иркутского областного суда. Svetlana V. Kornakova, Cand. Sci. (Law), Associate Professor, Associate Professor, Department of Theory and History of State and Law, Baikal State University (BSU) ul. Lenina, d. 11, Irkutsk, Russia, 664003 svetlana kornakova@yandex ru — уже +22,5 %, что указывает на негативную тен- денцию к увеличению особо тяжких преступле- ний, совершаемых несовершеннолетними, по ступность весьма специфична, поскольку обу- словлена социальной средой, личностью несо- вершеннолетних, совершивших преступление, и действующим уголовным законом. Кроме этого, ученые указывают и на ее способность трансформироваться в зависимости от соци- альных изменений, чем определяются особен- ности причинного комплекса, требующие раз- работки специальных мер профилактического воздействия2. ступность весьма специфична, поскольку обу- словлена социальной средой, личностью несо- вершеннолетних, совершивших преступление, и действующим уголовным законом. Кроме этого, ученые указывают и на ее способность трансформироваться в зависимости от соци- альных изменений, чем определяются особен- ности причинного комплекса, требующие раз- работки специальных мер профилактического воздействия2. Между тем данные официальной статисти- ки демонстрируют в последние годы суще- ственное снижение количественных показате- лей подростковой преступности. За шесть лет (с 2017 по 2022 гг.) подростковая преступность снизилась в 1,5 раза (с 45 288 до 30 469). В це- лом по стране в общем объеме преступности удельный вес преступности несовершенно- летних составил менее 2 %. Такие показатели свидетельствуют о наметившейся тенденции существенного снижения количественных ха- рактеристик такой преступности, а именно со- кращение числа зарегистрированных престу- плений и выявленных лиц, их совершивших. Как нам представляется, приведенные на диаграмме 1 данные отражают только реги- стрируемый, но далеко не фактический уровень преступности несовершеннолетних, поскольку она отличается высокой латентностью. Том 76 № 7 (200) июль 2023 78 Корнакова С. В., Корягина С. А. Корнакова С. В., Корягина С. А. Корнакова С. В., Корягина С. А. Современные аспекты насильственной преступности несовершеннолетних стране в целом (см. табл. 1). Отмеченные пока- затели явно свидетельствуют о глубине и стой- кости антиобщественной установки личности несовершеннолетнего, повышении обществен- ной опасности его индивидуального преступно- го поведения. несовершеннолетнего, повышении обществен- ной опасности его индивидуального преступно- го поведения. Та б л и ц а 1 Структура преступности несовершеннолетних по категориям преступлений в России,% Показатель Год 2017 2018 2019 2020 2021 2022 Предварительно расследовано тяжких преступлений, совершенных несовершеннолетними или при их соучастии 8 375 7 748 8 010 7 728 7 214 7 039 Удельный вес в структуре преступности, % 18,5 17,8 19,3 20,5 22,6 23,1 Прирост к предш. году, % –15,4 –7,5 +3,5 –3,5 –5,7 –2,4 Предварительно расследовано особо тяжких преступлений, совершенных несовершеннолетними или при их соучастии 1 863 1 968 2 103 2 069 1 841 2 256 Удельный вес в структуре преступности, % 4,2 4,6 5,1 5,5 5,7 7,4 Прирост к предш. 8 Уголовное дело № 43795. Архив Иркутского областного суда. 9 В. В. Путин высказался о трагедии в Керчи и о тех, кто «сдохнет» // URL: https://vz.ru/ politics/2018/10/18/946780.html/ (дата обращения: 20.03.2023). 10 Дунаева В. И. Проблемы формирования ценностных ориентиров молодежи России // Россия: тенден- ции и перспективы развития : ежегодник, Москва, 1–30 декабря 2016 г. / отв. ред. В. И. Герасимов. М. : Институт научной информации по общественным наукам РАН, 2017. С. 772. 11 Волконская Е. К. Бедность в детерминации рецидива насильственных преступлений // Lex russica (Рус- ский закон). 2013. Т. (95). № 10. С. 1125. 12 Антонян Ю. М. Причины насилия в семье // Пенитенциарная наука. 2020. Т. 14. № 2 (50). С. 171. 13 Дубинин Н. П., Карпец И. И., Кудрявцев В. Н. Генетика, поведение, ответственность : О природе анти- общественных поступков и путях их предупреждения. 2‑е изд., перераб. и доп. М. : Политиздат, 1989. С. 277. LEX RUSSICA но со стороны родителей, в большинстве случа- ев присуще совершение преступлений против жизни и здоровья, поскольку отрицательные последствия детства очень устойчивы и остав- ляют негативный след в неокрепшем сознании несовершеннолетнего. Думается, что, повзро- слев, подросток путем физического примене- ния насилия к другим лицам компенсирует свое ранее неудовлетворительное положение, демонстрирует свое превосходство над ними. Иными словами, «такая взаимосвязь отражена в цикле, предполагающем, что жертвы насилия становятся его виновниками»11. «К. (17 лет) по мотивам ревности из охотни- чьего ружья выстрелил в голову своему одно- класснику…»8. О проблемах агрессивного поведения в под- ростково-молодежной среде указывается и пре- зидентом В. В. Путиным. Так, на заседании дис- куссионного клуба «Валдай» после трагических событий в Керчи (тогда погибло более 20 мо- лодых людей) Президент отметил, что проис- шествие является результатом глобализации, поскольку в соцсетях сформированы целые со- общества, где молодые люди с неустойчивой психикой создают для себя каких-то лжегеро- ев. Это значит, что мы не создаем нужного, ин- тересного и полезного контента для молодых людей, и они хватают этот суррогат героизма, приводящий к трагедиям подобного рода9. По справедливому замечанию Ю. М. Ан- тоняна, «психологические раны, наносимые ребенку насилием в детстве, уходят в глубину психики и в дальнейшем могут проявляться не- ожиданно для самого преступника в деструк- тивных и даже жестоких действиях. Агрессив- ные вспышки чаще всего обнаруживаются в состоянии любого опьянения, когда волевой элемент ослаблен, и лицо менее всего способ- но контролировать свое поведение, что вле- чет за собой его желание избавиться от своих психотравмирующих переживаний, применяя насилие, несовершеннолетний преступник тем самым символически ликвидирует свои дет- ские страдания»12. Это не единственный случай резонансных насильственных противоправных действий, совершенных несовершеннолетними. Можно упомянуть и события, произошедшие в Казани, Перми. Следует согласиться с мнением В. И. Ду- наевой об очевидной тенденции подмены цен- ностных ориентиров молодежи на усредненные образцы массовой потребительской культуры, переориентацию молодых людей на корыст- но-индивидуальные ценности взамен тради- ционных духовных. Более того, негативное вли- яние СМИ, экспансия массовой коммерческой культуры, насаждение психологии общества потребления, по ее мнению, свидетельствует о глубокой и системной социальной деградации значительной части молодежи и, как следствие, о снижении значимости базовых ценностей10. Кроме этого, при определении на индивиду- альном уровне типичных причин совершения насильственных преступлений несовершенно- летними не должно оставаться без внимания наличие у некоторых из них психических ано- малий, которые оказывают значительное вли- яние на их поведение в ситуации совершения преступления, поскольку при таких характери- стиках снижаются адаптационные возможности психики подростка13. Svetlana V. Kornakova, Cand. Sci. (Law), Associate Professor, Associate Professor, Department of Theory and History of State and Law, Baikal State University (BSU) ul. Lenina, d. 11, Irkutsk, Russia, 664003 svetlana kornakova@yandex ru году, % +14,2 +5,6 +6,9 –1,6 –11,01 +22,5 Глава Следственного комитета России А. И. Бастрыкин отмечает, что в насильствен- ных преступлениях, совершенных подростками, проявляется стремление молодежи «любым путем обогатиться либо получить желаемое»3. Иными словами, популяризуется культ насилия, потребления, наслаждения, вседозволенности и безнаказанности, когда абсолютизация сво- боды личности противопоставляется такой, без- условно, гораздо более важной ценности, как жизнь человека. В этой связи следует отметить, что в настоя- щее время в российском обществе разрешение проблем и конфликтов при помощи насилия фактически становится приемлемой формой поведения, что связано именно с различными проблемами трансформирующегося обще- ства — недостатками досуговой деятельности4, «П. (15 лет), вооружившись ножом, нанес множественные удары в живот, горло, левую руку…»6. «М. (17 лет) на почве неприязненных отноше- ний выстрелил из пистолета в своего отчима…»7. LEX RUSSICA Том 76 № 7 (200) июль 2023 79 Науки криминального цикла JUS CRIMINALE LEX RUSSICA Еще одним фактором, детерминирующим насилие в качестве привычной формы поведе- ния подростков, является постоянное его прояв- ление в физическом и психологическом плане в ближайшем окружении, со стороны значимых для них взрослых. Общеизвестно, что подрост- кам, испытывавшим насилие в детстве, особен- Следует почеркнуть, что ни один из указан- ных выше факторов, влияющих на проявление отклоняющегося поведения несовершеннолет- Том 76 № 7 (200) июль 2023 80 Корнакова С. В., Корягина С. А. Современные аспекты насильственной преступности несовершеннолетних р р Современные аспекты насильственной преступности несовершеннолетних Том 76 №7 (200) июль 2023 них, не может являться первопричиной совер- шения ими преступлений. Более того, противо- правное поведение — во многом проявление внешней среды, в которой действует субъект, исходя из взаимодействия его личностных свойств и конкретной ситуации, т.е. внутрен- них установок индивида, благодаря которым в одной ситуации он избирает правомерное поведение, а в другой — противоправное. При этом конкретная ситуация выступает только поводом, но не причиной совершения престу- пления, поскольку в конечном счете преступле- ние совершается при определяющей установке лица на его совершение. В этой связи можно утверждать, что ситуация перед совершением преступления — это всегда ситуация морально- го выбора, служащая индикатором социальной адаптированности индивида. Полагаем, что основной и наиболее распро- страненной на сегодня личностной причиной девиантного поведения подростков является отсутствие у них стабильной, сформированной системы ценностей и идеалов. В этой связи А. И. Осинцев на основании сравнения ценност- ных ориентаций несовершеннолетних право- нарушителей и «правопослушных» подростков выделяет следующие свойственные первым социально и психологически обусловленные отл рал ства воз ное рам узо гол стим лич В вое вер пос под нов ти п и на нир вер А. А вия оце как мис этим нос в св Удельный вес осужденных несовершеннол против личности в составе Показатель 2017 2018 2 Удельный вес осужденных несовершеннолетних, со (ч. 2 ст. 105 УК Совершили преступление в группе 68 74 Удельный вес, % 73,11 77,08 Удельный вес осужденных несовершеннолетних, сове (ч. 2–4 ст. 111 У Совершили преступление в группе 155 154 Удельный вес, % 37,2 42,1 14 Осинцев А. И. Криминологическое обеспечение проф дис. … канд. юрид. наук : 12.00.08. Краснодар, 2000. С. 3 15 Александров А. А. Прекращение досудебного уголовно няемого в порядке, предусмотренном статьей 427 УПК стве. 2004. № 3. С. 293. 16 Данные Управления Судебного департамента при Верх cdep.ru (дата обращения: 21.03.2023). LEX RUSSICA отличия: пренебрежение к общественной мо- рали, проявляющееся в резком снижении чув- ства должного; распространение убеждения в возможности избежать наказания за содеян- ное; практически полное отсутствие осознания рамок дозволенного поведения, ограничений; узость интересов; легкость перехода к алко- гольной и наркотической зависимости; допу- стимость применения насилия для достижения личностно значимых целей14. них, не может являться первопричиной совер- шения ими преступлений. Более того, противо- правное поведение — во многом проявление внешней среды, в которой действует субъект, исходя из взаимодействия его личностных свойств и конкретной ситуации, т.е. внутрен- них установок индивида, благодаря которым в одной ситуации он избирает правомерное поведение, а в другой — противоправное. При этом конкретная ситуация выступает только поводом, но не причиной совершения престу- пления, поскольку в конечном счете преступле- ние совершается при определяющей установке лица на его совершение. В этой связи можно утверждать, что ситуация перед совершением преступления — это всегда ситуация морально- го выбора, служащая индикатором социальной адаптированности индивида. них, не может являться первопричиной совер- шения ими преступлений. Более того, противо- правное поведение — во многом проявление внешней среды, в которой действует субъект, исходя из взаимодействия его личностных свойств и конкретной ситуации, т.е. внутрен- них установок индивида, благодаря которым в одной ситуации он избирает правомерное поведение, а в другой — противоправное. При этом конкретная ситуация выступает только поводом, но не причиной совершения престу- пления, поскольку в конечном счете преступле- ние совершается при определяющей установке лица на его совершение. В этой связи можно утверждать, что ситуация перед совершением преступления — это всегда ситуация морально- го выбора, служащая индикатором социальной адаптированности индивида. Важно указать также, что морально-право- вое сознание подростков гораздо легче под- вергается деформации при действии в группе, поскольку анонимность действий в группе, подчиненность ее общей воле ведет к исчез- новению личной вины, ее размыванию, поч- ти полностью снимая страх ответственности и наказания. Указанный фактор, как детерми- нирующий противоправное поведение несо- вершеннолетних, по справедливому мнению А. А. Александрова, особенно опасен в усло- виях низкой раскрываемости преступлений, оцениваемой по стране лишь в 65–75 %. Тогда как латентность преступлений, по самым опти- мистичным оценкам, составляет 1/3. В связи с этим не должна вызывать удивления убежден- ность части несовершеннолетних преступников в своей безнаказанности15. Полагаем, что основной и наиболее распро- страненной на сегодня личностной причиной девиантного поведения подростков является отсутствие у них стабильной, сформированной системы ценностей и идеалов. В этой связи А. И. LEX RUSSICA LEX RUSSICA Показатель Год 2017 2018 2019 2020 2021 2022, I полугодие Удельный вес осужденных несовершеннолетних, совершивших преступление против половой неприкосновенности половой свободы личности Совершили преступление в группе 147 131 115 77 86 27 Удельный вес, % 42,0 40,8 41,8 30,2 30,1 20,0 вершеннолетний — против половой неприкос- новенности и половой свободы личности. Думается, что приведенные в таблице 2 показатели связаны с типичностью для несо- вершеннолетнего возраста в целом группово- го характера поведения. Даже объединенные сравнительно продолжительной преступной деятельностью группы несовершеннолетних, имеющие определенную иерархию и другие признаки, присущие преступной группе, обыч- но преобразуются из досуговых групп сверстни- ков. Вместе с тем в структуре осужденных несо- вершеннолетних доля осужденных за насиль- ственные сексуальные действия невелика и в среднем по стране составляет 0,9 %, однако темп прироста свидетельствует о росте количества та- ких преступлений: в 2021 г. он составил +18,6 %. Кроме этого, в 2021 г. выявлен негативный по- казатель «омоложения» преступности несовер- шеннолетних, повышение криминальной актив- ности подростков возрастной группы 14–15 лет по данной категории преступлений — +8,9 % (табл. 3), что не может не вызывать тревогу. Комментируя эти данные, укажем, что каж- дый второй осужденный несовершеннолетний совершил преступление в группе против жизни и здоровья и каждый третий осужденный несо- Та б л и ц а 3 Характеристика осужденных несовершеннолетних по возрасту, совершивших преступление против половой неприкосновенности половой свободы личности Показатель Год 2017 2018 2019 2020 2021 Осуждено несовершеннолетних 350 321 275 254 290 Темп прироста к предшествующему году,% — –8,3 –14,3 –7,6 +14,2 14–15 лет 164 167 143 123 134 Удельный вес, % 46,9 52,0 52 49,0 46,1 Темп прироста к предшествующему году, % — 1,8 –14,4 –14,0 +8,9 Характеристика осужденных несовершеннолетних по возрасту, совершивших преступление против половой неприкосновенности половой свободы личности Сто́ит указать, что в материалах уголовной статистики насильственные преступления пред- ставлены лишь несколькими видами престу- плений, которые в уголовно-правовом понима- нии характеризуются внутренним единством и обусловлены в большинстве своем социаль- но-психологическим механизмом преступно- го поведения, что и является основанием для объединения их в одну группу, основу которой составляет применение насилия или угрозы его применения. К числу таких преступлений следу- ет отнести убийства, умышленное причинение различной степени тяжести вреда здоровью, насильственные сексуальные преступления, а также хулиганство, которое зачастую соверша- ется именно несовершеннолетними. LEX RUSSICA Осинцев на основании сравнения ценност- ных ориентаций несовершеннолетних право- нарушителей и «правопослушных» подростков выделяет следующие свойственные первым социально и психологически обусловленные Та б л и ц а 2 Удельный вес осужденных несовершеннолетних, совершивших преступления против личности в составе группы лиц, %16 Показатель Год 2017 2018 2019 2020 2021 2022, I полугодие Удельный вес осужденных несовершеннолетних, совершивших преступление против жизни (ч. 2 ст. 105 УК РФ) Совершили преступление в группе 68 74 64 26 49 14 Удельный вес, % 73,11 77,08 74,4 62,1 73,1 63,6 Удельный вес осужденных несовершеннолетних, совершивших преступление против здоровья (ч. 2–4 ст. 111 УК РФ) Совершили преступление в группе 155 154 153 88 145 70 Удельный вес, % 37,2 42,1 39,9 34,1 47,1 43,2 14 Осинцев А. И. Криминологическое обеспечение профилактики преступности несовершеннолетних : дис. … канд. юрид. наук : 12.00.08. Краснодар, 2000. С. 34. 15 Александров А. А. Прекращение досудебного уголовного преследования несовершеннолетнего обви- няемого в порядке, предусмотренном статьей 427 УПК РФ // Черные дыры в Российском законодатель- стве. 2004. № 3. С. 293. 16 Данные Управления Судебного департамента при Верховном Суде РФ за 2017–2022 г. URL: http://www. cdep.ru (дата обращения: 21.03.2023). Удельный вес осужденных несовершеннолетних, совершивших преступления против личности в составе группы лиц, %16 ьный вес осужденных несовершеннолетних, совершивших преступление против здоровья (ч. 2–4 ст. 111 УК РФ) 16 Данные Управления Судебного департамента при Верховном Суде РФ за 2017–2022 г. URL: http://www. cdep.ru (дата обращения: 21.03.2023). LEX RUSSICA Том 76 № 7 (200) июль 2023 81 Науки криминального цикла JUS CRIMINALE LEX RUSSICA Если говорить о мотивах хулиганских дей- ствий, то криминологами отмечается, что мо- тивы, «определяемые как побуждения, могут иметь двойную основу, во-первых, когда они, являясь неотъемлемой, имманентной чертой совершившего насилие из хулиганских по- буждений, идут изнутри, представляют собой свойство личности. И во-вторых, когда они являются агрессивной реакцией на ситуацию, внешние “раздражители” и окружающих лю- Том 76 № 7 (200) июль 2023 82 Корнакова С. В., Корягина С. А. Современные аспекты насильственной преступности несовершеннолетних дей»17. Агрессивно-хулиганские преступления несовершеннолетних можно проиллюстриро- вать следующим примером: вреда здоровью не планировали и не готовили заранее, противоправные действия были со- вершены в результате сложившейся ситуации, желание возникло во время конфликта на поч- ве личных отношений20. Группа подростков ожидала электричку на вокзале, одна из девушек решила закурить и обратилась к пожилому мужчине с просьбой дать ей прикурить. Пожилой человек отве- тил, что не курит и ей не советует. Девушка подошла к своей группе, после чего все под- ростки накинулись на пожилого мужчину, сби- ли его с ног и стали избивать. Затем бессоз- нательное тело оттащили в кусты, искололи ножами, и закопали18. К числу ситуативных преступлений во мно- гих случаях можно отнести и насильственные действия сексуального характера. Криминоло- ги отмечают, что в 86,2 % случаев у несовер- шеннолетних осужденных желание их совер- шить возникло неожиданно21, т.е. внезапно возникший умысел свойственен большинству осужденных за половые преступления под- ростков. Что побудило подростков совершить данное преступление? Мотивы отвечают на вопросы: «Зачем, почему человек действовал опреде- ленным образом?», «Какую потребность он мог удовлетворить?». Но на эти вопросы подростки, совершившие тяжкое преступление, ответа не нашли. Ситуативный характер, облегчающий совер- шение преступления, свойственен и уличному хулиганству, как правило, в ночное время су- ток, причем повод для проявления агрессив- ного поведения, жестокости, пренебрежения нравственными и правовыми нормами может быть незначительным22. В основе хулиганских побуждений и стремлений лежит удовлетво- рение собственных интересов, при этом иг- норируются интересы других лиц, поскольку виновные грубо и публично нарушают обще- ственный порядок, демонстрируют вседозво- ленность и безнаказанность, стремление к выражению протеста, что характерно для несо- вершеннолетних, совершивших указанное пре- ступление. Вместе с тем доля осужденных за хулиганство подростков невелика и в среднем составляет 1 %. Однако в последние годы так- же наблюдается его прирост: в 2020 г. +31,25 % и в 2021 г. +23,8 %. Анализ данных Судебного департамента при Верховном Суде РФ свидетельствует, что основную долю в структуре осужденных не- совершеннолетних занимают осужденные за насильственные преступления против жизни и здоровья, совершенные при отягчающих обстоятельствах. 17 Антонян Ю. М., Еркубаева А. Ю. Преступления из хулиганских побуждений: объяснение и мотивы. Об- щество и право. 2014. № 2 (48). С. 129–132. 18 Уголовное дело № 30546. Архив Иркутского областного суда. 19 Данные Управления Судебного департамента при Верховном Суде РФ за 2017–2022 г. // URL: http:// www.cdep.ru (дата обращения: 21.03.2023). 20 Варыгин А. Н., Григорян З. М. О некоторых причинах и условиях убийств, совершаемых несовершенно- летними // Юридическая наука. 2012. № 2. С. 97. 21 Смирнов А. М. Общая характеристика несовершеннолетних осужденных к лишению свободы за совер- шение половых преступлений // Человек: преступление и наказание. 2014. № 3. С. 131. 22 Репецкая А. Л., Рыбакова А. С. Региональные особенности детерминации и предупреждения совре- менного хулиганства (на примере Сибирского Федерального округа) // Академический юридический журнал. 2022. Т. 23. № 2 (88). С. 189. 17 Антонян Ю. М., Еркубаева А. Ю. Преступления из хулиганских побуждений: объяснение и мотивы. Об- щество и право. 2014. № 2 (48). С. 129–132. щ р ( ) 18 Уголовное дело № 30546. Архив Иркутского областного суда. 23 Плешаков В. А. Введение в криминологическую теорию ситуаций, или ситуационную криминологию // Человек: преступление и наказание. 2012. № 1 (76). С. 103. 24 Ильин И. С. Особенности ситуативного предупреждения демонстративно-протестных преступлений // Научный вестник Омской Академии МВД России. 2022. № 3. С. 216. 25 Рыбальская В. Я. Проблемы борьбы с преступностью несовершеннолетних : учеб. пособие. Иркутск : Изд-во ИГУ, 1994. С. 166. 26 Фортуныч М. В., Черникова И. А., Яковлев О. В. Предкриминальное поведение несовершеннолетних, совершивших умышленные преступления. М. : ВНИИ МВД России, 2007. С. 80. LEX RUSSICA Ранняя профилактика отклоняющегося по- ведения несовершеннолетних, по мнению уче- ных, должна быть направлена на устранение и нейтрализацию источников криминогенных влияний, еще не успевших негативно воздей- ствовать на несовершеннолетнего, или на сня- тие слабых деформаций его личности, которые еще не переросли в устойчивую позицию26. При этом следует учитывать, что ранняя про- филактика не требует больших материальных вложений, так как в основе своей предполагает меры воспитательно-педагогического характе- ра, оставляя резерв времени для дальнейшего предупредительного воздействия, если в нем возникнет необходимость. проанализировать ситуационные факторы и, как следствие, выработать комплекс мер ситу- ационного предупреждения23. Анализ подходов к ситуационному преду- преждению преступности провел И. С. Ильин. Он указал, что ситуационные меры располагают набором специальных техник предупреждения преступлений, направленных на создание во внешней среде условий, которые существенно затрудняли бы совершение преступлений либо снижали степень заинтересованности в его со- вершении24. Между тем, по справедливому мнению В. Я. Рыбальской, ядром всей системы пред- упреждения преступлений несовершеннолет- них является их ранняя профилактика, и имен- но ею должно определяться преобладание мер социальной защиты детей и подростков, мер воспитательно-педагогического воздей- ствия, помощи и поддержки по сравнению с мерами воспитательного воздействия прину- дительного характера, тем более с уголовным наказанием. Раннее предупреждение доста- точно отдалено от непосредственного преступ- ного поведения и максимально приближено к источникам, которые порождают отклоняюще- еся поведение подростков25. Следовательно, непосредственное значение имеют субъекты профилактики, которые должны располагать и временем, и средствами для предотвращения процесса перехода отклоняющегося от нормы поведения несовершеннолетних в преступное поведение. Таким образом, рассмотренное в статье со- временное состояние насильственной преступ- ности несовершеннолетних показывает в ряде случаев вызывающую тревогу негативную ди- намику. В этой связи общественная опасность насильственных преступлений обуславливает необходимость выработки эффективных мер, к числу которых следует отнести, с одной сторо- ны, комплексный подход к изучению негатив- ных качеств личности, устранение основных де- терминант антиобщественного поведения, что предполагает скоординированность деятельно- сти учреждений и служб разной ведомственной подчиненности (в частности, образовательных, просветительных, спортивных, культурно-до- суговых учреждений, правоохранительных органов), предупреждающей преступные проявления. С другой — меры ситуационного предупреждения, которые бы снижали заин- тересованность в совершении преступлений на ранних этапах формирования преступного замысла соответствующей преступной деятель- ности, повышали риск их раскрытия. Основными субъектами профилактической деятельности являются общественные органи- зации и государственные органы, выполняющие функции общего социального контроля, обязан- ные в своей работе знать и учитывать особен- ности психологии, правового и фактического положения несовершеннолетних. Их основной задачей является удержание подростка в зоне благополучной семейной системы, учебного процесса и стимулирование отказа от асоциаль- ных форм досуга. LEX RUSSICA При этом темп прироста со- вершенных несовершеннолетними квалифи- цированных видов убийств имел негативную динамику: в 2018 г. +3,3 %; в 2021 г. +59,6 %, а количество причинения тяжкого вреда здоро- вью при отягчающих обстоятельствах харак- теризовалось приростом: в 2019 г. +4,07 % и в 2021 г. +18,5 %19. В. А. Плешаков в этой связи отмечает, что си- туационный характер следует учитывать в тех преступлениях, в совершении которых ситуация сыграла решающую роль, т.е. спровоцировала преступника или способствовала реализации его преступного умысла в совершении престу- пления. Знание подобных ситуаций позволяет В проведенном А. Н. Варыгиным и З. М. Гри- горян исследовании указывается, что большин- ство преступлений, совершенных несовершен- нолетними против жизни и здоровья, носит ситуационный характер. Так, 86,7 % опрошен- ных ими осужденных несовершеннолетних ука- зали, что причинение смерти или причинение 22 Репецкая А. Л., Рыбакова А. С. Региональные особенности детерминации и предупреждения совре- менного хулиганства (на примере Сибирского Федерального округа) // Академический юридический журнал. 2022. Т. 23. № 2 (88). С. 189. LEX RUSSICA 83 Том 76 № 7 (200) июль 2023 Науки криминального цикла JUS CRIMINALE LEX RUSSICA Полагаем, что молодежная политика госу- дарства должна быть направлена на повыше- ние ценности здорового образа жизни, обра- зования и культуры, а основным ее вектором в этой сфере должно быть дальнейшее усиле- ние мер по нравственному и идеологическо- Том 76 № 7 (200) июль 2023 84 Корнакова С. В., Корягина С. А. Современныеаспектынасильственнойпреступностинесовершеннолетних Корнакова С. В., Корягина С. А. Современные аспекты насильственной преступности несовершеннолетних му27 воспитанию несовершеннолетних, повы- шению престижа семьи, а также организации доступной досуговой деятельности, поскольку обусловленную возрастом поведенческую ак- тивность несовершеннолетних следует не по- давлять, а переориентировать. му27 воспитанию несовершеннолетних, повы- шению престижа семьи, а также организации доступной досуговой деятельности, поскольку обусловленную возрастом поведенческую ак- тивность несовершеннолетних следует не по- давлять, а переориентировать. му27 воспитанию несовершеннолетних, повы- шению престижа семьи, а также организации доступной досуговой деятельности, поскольку обусловленную возрастом поведенческую ак- тивность несовершеннолетних следует не по- давлять, а переориентировать. 27 В этом отношении следует положительно оценить введение с 2022 г. в учебный процесс обязательного патриотического и гражданского воспитания школьников и студентов. БИБЛИОГРАФИЯ 1. Александров А. А. 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Problemy borby s prestupnostyu nesovershennoletnikh [Problems of combating delinquency]. Irkutsk: Publishing House of the ISU; 1994. (In Russ.). 15. Smirnov AM. Obshchaya kharakteristika nesovershennoletnikh osuzhdennykh k lisheniyu svobody za sovershenie polovykh prestupleniy [General characteristics of minors sentenced to imprisonment for sexual crimes]. Chelovek: prestuplenie i nakazanie [Man: Crime and punishment]. 2014;3:130-135. (In Russ.). 15. Smirnov AM. Obshchaya kharakteristika nesovershennoletnikh osuzhdennykh k lisheniyu svobody za sovershenie polovykh prestupleniy [General characteristics of minors sentenced to imprisonment for sexual crimes]. Chelovek: prestuplenie i nakazanie [Man: Crime and punishment]. 2014;3:130-135. (In Russ.). 16. Suturin MA. Nekotorye aspekty kriminologicheskogo analiza lichnosti prestupnika (po materialam Irkutskoy i Chitinskoy oblastey) [Some aspects of criminological analysis of the criminal’s personality (Case Sudy of the Irkutsk and Chita regions)]. Sibirskiy yuridicheskiy vestnik [Siberian Law Herald]. 2009;1:90-93. (In Russ.). 16. Suturin MA. Nekotorye aspekty kriminologicheskogo analiza lichnosti prestupnika (po materialam Irkutskoy i Chitinskoy oblastey) [Some aspects of criminological analysis of the criminal’s personality (Case Sudy of the Irkutsk and Chita regions)]. Sibirskiy yuridicheskiy vestnik [Siberian Law Herald]. 2009;1:90-93. (In Russ.). 17. Fortunych MV, Chernikova IA, Yakovlev OV. Predkriminalnoe povedenie nesovershennoletnikh, sovershivshikh umyshlennye prestupleniya [Pre-criminal behavior of minors who have committed intentional crimes]. Moscow: Research Institute of the Ministry of Internal Affairs of Russia; 2007. (In Russ.). 17. Fortunych MV, Chernikova IA, Yakovlev OV. Predkriminalnoe povedenie nesovershennoletnikh, sovershivshikh umyshlennye prestupleniya [Pre-criminal behavior of minors who have committed intentional crimes]. Moscow: Research Institute of the Ministry of Internal Affairs of Russia; 2007. REFERENCES nauk [Juvenile delinquency: Criminological characteristics and problems of prevention (Far Eastern Federal District Cases). The Author’s Abstract of the Cand. Sci. (Law) Thesis]. Vladivostok; 2011. (In Russ.). 8. Dubinin NP, Karpets II, Kudryavtsev VN. Genetika, povedenie, otvetstvennost: O prirode antiobshchestvennykh postupkov i putyakh ikh preduprezhdeniya [Genetics, behavior, responsibility: On the nature of antisocial acts and ways to prevent them]. Moscow: Politizdat Publ.; 1989. (In Russ.). 8. Dubinin NP, Karpets II, Kudryavtsev VN. 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Kriminologicheskoe obespechenie profilaktiki prestupnosti nesovershennoletnikh: dis. … kand. yurid. nauk [Criminological support for the prevention of juvenile delinquency. Cand. Sci. (Law) Thesis]. Krasnodar; 2000. (In Russ.). 11. Osintsev AI. Kriminologicheskoe obespechenie profilaktiki prestupnosti nesovershennoletnikh: dis. … kand. yurid. nauk [Criminological support for the prevention of juvenile delinquency. Cand. Sci. (Law) Thesis]. Krasnodar; 2000. (In Russ.). 12. Pleshakov VA. Vvedenie v kriminologicheskuyu teoriyu situatsiy, ili situatsionnuyu kriminologiyu [Introduction to the criminological theory of situations, or situational criminology]. БИБЛИОГРАФИЯ Криминологические проблемы девиантного поведения несовершенно- летних // Всероссийский криминологический журнал. — 2013. — № 3. — С. 60–65. Материал поступил в редакцию 16 февраля 2023 г. Материал поступил в редакцию 16 февраля 2023 г. Материал поступил в редакцию 16 февраля 2023 г. 27 В этом отношении следует положительно оценить введение с 2022 г. в учебный процесс обязательного патриотического и гражданского воспитания школьников и студентов. LEX RUSSICA Том 76 № 7 (200) июль 2023 85 Науки криминального цикла JUS CRIMINALE Науки криминального цикла JUS CRIMINALE REFERENCES (In Russ.). 18. Yarovenko VV, Yarovenko EV. Kriminologicheskie problemy deviantnogo povedeniya nesovershennoletnikh [Criminological problems of deviant behavior of minors]. Vserossiyskiy kriminologicheskiy zhurnal [Russian Journal of Criminology]. 2013;3:60-65. (In Russ.). 18. Yarovenko VV, Yarovenko EV. Kriminologicheskie problemy deviantnogo povedeniya nesovershennoletnikh [Criminological problems of deviant behavior of minors]. Vserossiyskiy kriminologicheskiy zhurnal [Russian Journal of Criminology]. 2013;3:60-65. (In Russ.). 18. Yarovenko VV, Yarovenko EV. 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https://openalex.org/W2170057104	https://figshare.com/articles/journal_contribution/Tasman_Leakage_of_intermediate_waters_as_inferred_from_Argo_floats/22914887/1/files/40649507.pdf	English	null	Tasman Leakage of intermediate waters as inferred from Argo floats	Geophysical research letters	2,013	cc-by	4,442	GEOPHYSICAL RESEARCH LETTERS, VOL. 40, 5456–5460, doi:10.1002/2013GL057797, 2013 GEOPHYSICAL RESEARCH LETTERS, VOL. 40, 5456–5460, doi:10.1002/2013GL057797, 2013 Tasman Leakage of intermediate waters as inferred from Argo ﬂoats Miquel Rosell-Fieschi,1 Stephen R. Rintoul,2,3,4,5 Jeröme Gourrion,1 and Josep L. Pelegrí1 Received 26 August 2013; revised 4 October 2013; accepted 6 October 2013; published 23 October 2013. subsurface ﬂow south of Tasmania [Rintoul and Bullister, 1999; Rintoul and Sokolov, 2001] but say nothing about its pathway to the Indian Ocean. Model studies and observations suggest that the TL provides a Paciﬁc-to-Indian supply, a key link in a “supergyre” connecting the subtropical gyres of all Southern Hemisphere basins [Rintoul and Sokolov, 2001; Sloyan and Rintoul, 2001; Speich et al., 2002; Ridgway, 2007; Ridgway and Dunn, 2007]. [1] We use Argo ﬂoat trajectories to infer ocean current velocity at the sea surface and 1000 dbar near Australia. The East Australian Current ﬂows southward along the east coast of Australia at both surface and intermediate levels, but only the intermediate waters leak round the southern tip of Tasmania and cross the Great Australian Bight. We calculate the transport of Antarctic Intermediate Water (AAIW) between the southern Australian coast and the Antarctic Circumpolar Current (ACC) as the velocity at 1000 dbar times the layer thickness. Between March 2006 and December 2012, the Eulerian AAIW transport through 147°E ranges between 0 and 12.0sverdrup (Sv). The mean Tasman Leakage of intermediate waters from the Paciﬁc Ocean into the Indian Ocean, obtained using all Argo data until March 2013, is 3.8± 1.3Sv. The mean intermediate water transport into the Indian Ocean through 115°E increases to 5.2± 1.8Sv due to contributions from the westward recirculation of ACC waters. Citation: Rosell-Fieschi, M., S. R. Rintoul, J. Gourrion, and J. L. Pelegrí (2013), Tasman Leakage of intermediate waters as inferred from Argo ﬂoats, Geophys. Res. Lett., 40, 5456–5460, doi:10.1002/2013GL057797. 2007; Ridgway and Dunn, 2007]. [3] The westward ﬂowing South Equatorial Current (SEC) reaches the western boundary of the Paciﬁc Ocean and bifur- cates between 15°S and 20°S into northern (North Vanuatu and North Caledonia) and southern (South Caledonia) jets (Figure S1 in the supporting information). The North Caledonia (near surface) and South Caledonia (at intermedi- ate levels) jets turn south at the Australian coast to form the East Australian Current (EAC), the western boundary current of the South Paciﬁc subtropical gyre [Godfrey et al., 1980; Ganachaud et al., 2008]. In the near-surface layers, the EAC separates from the coast on its southward trip, either recirculating to form a double-cell structure [Ridgway and Dunn, 2003] or turning offshore at the Tasman Front or near Tasmania. Tasman Leakage of intermediate waters as inferred from Argo ﬂoats Miquel Rosell-Fieschi,1 Stephen R. Rintoul,2,3,4,5 Jeröme Gourrion,1 and Josep L. Pelegrí1 Surface waters of the EAC extend farther south in summer but do not breach the southern tip of Tasmania [Ridgway and Godfrey, 1997]. At intermediate depths, on the other hand, part of the EAC continues south along the coast and turns west, between Tasmania and the South Tasman Rise, as the TL (Figure S1 in the supporting informa- tion). The extension of the TL into the Great Australian Bight (GAB) is known as the Flinders Current [Middleton and Cirano, 2002]; this subsurface transport increases as it pro- gresses west because a fraction of the water transported by the Antarctic Circumpolar Current (ACC) recirculates westward through a large anticyclonic loop located between about 139°E and 146°E [Rintoul and Sokolov, 2001]. Along the southern coast of Australia, an extension of the Leeuwin Current forms a weak eastward ﬂow of surface wa- ters extending from Western Australia to Tasmania [Ridgway and Condie, 2004]. 1. Introduction [2] The global overturning circulation largely determines the capacity of the ocean to store and transport heat and carbon and thereby inﬂuence climate. Sinking of dense water in the North Atlantic and the Southern Ocean is balanced by a return ﬂow of lighter water in the upper ocean. The existence of such a global-scale overturning circulation depends on the exchange of water masses between basins, both in the deep and upper levels of the ocean. For the upper limb, most attention has been placed on two primary pathways: the cold route through the Drake Passage [Rintoul, 1991] and the warm route following the Indonesian Throughﬂow and the Agulhas Current [Gordon, 1986]. A third route, the Tasman Leakage (TL), has been identiﬁed more recently [Speich et al., 2001, 2002, 2007]. Repetitions of section SR3, a World Ocean Circulation Experiment (WOCE) hydrographic section between Tasmania and Antarctica, have conﬁrmed a westward , ] [4] Previous studies of the EAC and the TL have relied on hydrographic data or model output. In this work we use Argo ﬂoat data to infer the velocity at the sea surface and 1000 dbar (the parking pressure of most Argo ﬂoats) and to estimate the thickness of the Antarctic Intermediate Water (AAIW) layer. We then combine the velocity and thickness information to quantify the variability in water transport south of Tasmania and the mean transport reaching the Indian Ocean. Additional supporting information may be found in the online version of this article. 1Institut de Ciències del Mar, CSIC, Barcelona, Spain. 2CSIRO Marine and Atmospheric Research, Hobart, Tas, Australia. 3Centre for Australian Weather and Climate, Hobart, Tas, Australia. 4Antarctic Climate and Ecosystems Cooperative Research Centre, Hobart, Tas, Australia. 5CSIRO Wealth from Oceans National Research Flagship, Hobart, Tas, Australia. Additional supporting information may be found in the online version of this article. 1Institut de Ciències del Mar, CSIC, Barcelona, Spain. 2CSIRO Marine and Atmospheric Research, Hobart, Tas, Australia. 3Centre for Australian Weather and Climate, Hobart, Tas, Australia. 4Antarctic Climate and Ecosystems Cooperative Research Centre, Hobart, Tas, Australia. 5CSIRO Wealth from Oceans National Research Flagship, Hobart, Tas, Australia. Corresponding author: J. L. Pelegrí, Institut de Ciències del Mar, CSIC, Barcelona ES-08003, Spain. (pelegri@icm.csic.es) ©2013. American Geophysical Union. All Rights Reserved. 0094-8276/13/10.1002/2013GL057797 ©2013. American Geophysical Union. All Rights Reserved. 0094-8276/13/10.1002/2013GL057797 2. Data and Methods [5] The Argo ﬂoat trajectories have been used to generate climatological velocity ﬁelds [Lebedev et al., 2007] and to infer the current velocity in different regions [Nuñez-Riboni et al., 2005; Park et al., 2005; Ollitrault et al., 2006; Xie Corresponding author: J. L. Pelegrí, Institut de Ciències del Mar, CSIC, Barcelona ES-08003, Spain. (pelegri@icm.csic.es) ©2013. American Geophysical Union. All Rights Reserved. 0094-8276/13/10.1002/2013GL057797 5456 5456 ROSELL-FIESCHI ET AL.: TASMAN LEAKAGE FROM ARGO FLOATS Figure 1. (a) Central level and (b) thickness of the AAIW layer (in meters), deﬁned as having neutral densities between 27.125 and 27.6 kg m3. Figure 1b also shows the sections used to estimate the error in the transport associated to the TL. parcels, so the velocity vectors inferred from the ﬂoat displacements shall be understood as an Eulerian velocity measure (see the Appendix in the supporting information). [6] While the error in surface velocity depends only on the positioning accuracy, the subsurface velocity estimates are inﬂuenced by the horizontal displacements experienced by the ﬂoat during its vertical migration. Lebedev et al. [2007] did a detailed analysis of velocity errors at a parking pressure of 1000 dbar and concluded that the most probable relative error is 3% of the inferred value; they also concluded that 54.0% (97.6%) of the deep velocity data have relative errors less than 10% (100%) of the calculated velocity value. ( ) y [7] Here we use all delayed-mode Argo data up to March 2013, with a quality control label of “good,” downloaded from the Coriolis Operational Oceanography center (www.coriolis. eu.org). We use Chauvenet’s criterion [Taylor, 1997] to iden- tify, and reject, spurious velocity vectors as those with either zonal or latitudinal components exceeding ﬁve standard devi- ations. This gives a total of 69,115 velocity vectors for the sur- face and 58,757 velocity vectors at 1000 dbar. The annual mean velocity is interpolated on a 1/2° × 1/2° grid, calculated from all data contained in a 100 km radius; similarly, the sea- sonal values are interpolated on a 1° × 1° grid, using a 150 km search radius. This generates considerable overlap between adjacent cells but increases signiﬁcantly the number of veloc- ity vectors available in each cell. Figure 1. (a) Central level and (b) thickness of the AAIW layer (in meters), deﬁned as having neutral densities between 27.125 and 27.6 kg m3. 3. Results [12] The surface EAC has seasonal variations greater than most midlatitude western boundary currents [Ridgway and Godfrey, 1997], strengthening and extending farther south during the austral summer and weakening in winter (Figures 3a, 3b, and S4). The greatest variability is associated with the northward offshore countercurrent, to the extent that the annual mean ﬂow may be largely accomplished by eddies [Ridgway and Godfrey, 1997; Ridgway and Dunn, 2003]. One cause for such variability may be the seasonal cycle of the impinging SEC, which feeds the EAC [Sokolov and Rintoul, 2000]. Another reason may be the variable extension of the Leeuwin Current across the GAB [Ridgway and Godfrey, 1997]: During the austral winter and spring, this current rounds the southern tip of Tasmania and ﬂows along its eastern coast as the northward extension of the Zeehan Current, possi- bly blocking the southward progression of the EAC. 2. Data and Methods Figure 1b also shows the sections used to estimate the error in the transport associated to the TL. y [8] The AAIW layer is deﬁned to lie between neutral den- sities of 27.125 and 27.6 kg m3 [Speich et al., 2002]. The depths of the neutral surfaces, calculated from the salinity and temperature proﬁles of those same Argo cycles used to compute the velocity ﬁelds, are projected on a grid following the same procedure as for the annual mean velocities. The results show that the central depth point of the AAIW layer is reasonably constant and close to 1000 dbar for the whole study area (Figure 1a), conﬁrming that ﬂoats parked near this depth level indeed track this water mass. and Zhu, 2008]. Here we use the Argo ﬂoat data to produce our own velocity estimates, following the same methodology as in Lebedev et al. [2007]. The ﬁrst and last positions of a ﬂoat cycle are used to calculate the sea surface and parking depth velocities. The individual velocity estimates are spa- tially and temporally averaged to construct the velocity maps, with the spatial and temporal resolution depending on the amount of available data. The alternation between surface and deep drifts means that the Argo ﬂoats do not follow water Figure 2. Mean current speed (color-coded, m s1) and current velocity vectors near Australia, both at (a) the sea surface and (b) 1000 dbar. The inset illustrates the available number of data point for each 0.5° × 0.5° cell element. Figure 2. Mean current speed (color-coded, m s1) and current velocity vectors near Australia, both at (a) the sea surface and (b) 1000 dbar. The inset illustrates the available number of data point for each 0.5° × 0.5° cell element. Figure 2. Mean current speed (color-coded, m s1) and current velocity vectors near Australia, both at (a) the sea surface and (b) 1000 dbar. The inset illustrates the available number of data point for each 0.5° × 0.5° cell element. 5457 Figure 3. (a, b) Surface and (c, d) 1000 dbar monthly current speed (color-coded, m s1) and current velocity vectors for the region with the EAC and the TL; the monthly currents are calculated as 3 month averages centered on January for Figures 3a and 3c and on July for Figures 3b and 3d. 2. Data and Methods ROSELL-FIESCHI ET AL.: TASMAN LEAKAGE FROM ARGO FLOATS ROSELL-FIESCHI ET AL.: TASMAN LEAKAGE FROM ARGO FLOATS ROSELL-FIESCHI ET AL.: TASMAN LEAKAGE FROM ARGO FLOATS Figure 3. (a, b) Surface and (c, d) 1000 dbar monthly current speed (color-coded, m s1) and current velocity vectors for the region with the EAC and the TL; the monthly currents are calculated as 3 month averages centered on January for Figures 3a and 3c and on July for Figures 3b and 3d. [9] The Eulerian transport is estimated by multiplying the thickness of the AAIW layer (Figure 1b) by the velocity at 1000 dbar. The numerical results of Cirano and Middleton [2004] endorse the assumption of a nearly constant velocity throughout the AAIW layer. In the Appendix in the supporting information, we use the divergence of the inferred velocity ﬁeld to estimate a potential error of 35% in our trans- port estimates. We also show that this error bar is large enough to take into account possible uncertainties associated with the vertical shear of horizontal velocity within the AAIW layer and the limited sampling of relatively narrow boundary currents. [9] The Eulerian transport is estimated by multiplying the thickness of the AAIW layer (Figure 1b) by the velocity at 1000 dbar. The numerical results of Cirano and Middleton [2004] endorse the assumption of a nearly constant velocity throughout the AAIW layer. In the Appendix in the supporting information, we use the divergence of the inferred velocity ﬁeld to estimate a potential error of 35% in our trans- port estimates. We also show that this error bar is large enough to take into account possible uncertainties associated with the vertical shear of horizontal velocity within the AAIW layer and the limited sampling of relatively narrow boundary currents. west. The AAIW contains two different water types: a cooler and fresher variety coming from the ACC and a warmer and saltier variety supplied by the EAC. An analysis of tempera- ture-salinity diagrams from the east and south coasts of Australia conﬁrms the continuity of the EAC variety all the way from east to west Australia (Figure S2 in the supporting information). It also conﬁrms that part of the AAIW carried east by the ACC recirculates to the west at lower latitudes, as observed south of Tasmania by Rintoul and Sokolov [2001], thereby enhancing the westward AAIW ﬂow in the northern GAB (Figure S3). 3.1. Annual Mean [10] The annual mean sea surface velocity map (Figure 2a) shows a double anticyclone associated with recirculations of the EAC, with a weak separation from the coast at about 32°S and a much stronger one at about 34°S, associated to the Tasman Front [Ridgway and Dunn, 2003]. The ultimate offshore diversion of the surface ﬂow, however, takes place at about 40°S, so that no ﬂow goes round the southern tip of Tasmania. The annual mean AAIW velocity map (Figure 2b) also exhibits some partial retroﬂection at about 34°S, but a major portion continues south and then turns west, between the Tasmania coast and the South Tasman Rise, as the TL [Rintoul and Sokolov, 2001; Ridgway and Dunn, 2007]. [11] South of Australia, the surface waters have a weak east- ward ﬂow, while the AAIW stratum predominantly moves 5458 Figure 4. (a) Annual mean AAIW streamlines south of Australia; zero is taken at the coast, and increasing negative values represent westward transport (Sv). Sections S1 (115°E) and S2 (147°E) are shown as red dotted lines; the blue dotted lines coincide with 145°E, where Speich et al. [2007] did their transport calculations. (b) AAIW transport across 147°E (Sv), integrated south from Tasmania, as a function of time and latitude; values are obtained using a 5 month running ﬁlter with a search radius of 150 km and gridding on 1° cells. (c) Time series of the maximum westward transports (negative values, Sv) in Figure 4b. Tick marks in Figures 4b and 4c indicate the beginning of the calendar year. ROSELL-FIESCHI ET AL.: TASMAN LEAKAGE FROM ARGO FLOATS ROSELL-FIESCHI ET AL.: TASMAN LEAKAGE FROM ARGO FLOATS ROSELL-FIESCHI ET AL.: TASMAN LEAKAGE FROM ARGO FLOATS Figure 4. (a) Annual mean AAIW streamlines south of Australia; zero is taken at the coast, and increasing negative values represent westward transport (Sv). Sections S1 (115°E) and S2 (147°E) are shown as red dotted lines; the blue dotted lines coincide with 145°E, where Speich et al. [2007] did their transport calculations. (b) AAIW transport across 147°E (Sv), integrated south from Tasmania, as a function of time and latitude; values are obtained using a 5 month running ﬁlter with a search radius of 150 km and gridding on 1° cells. (c) Time series of the maximum westward transports (negative values, Sv) in Figure 4b. Tick marks in Figures 4b and 4c indicate the beginning of the calendar year. 3.1. Annual Mean [13] In contrast to the strong seasonality of the surface ﬂow, the EAC and its westward TL extension are remarkably steady at the AAIW level during all seasons (Figures 3c, 3d, and S4). Nevertheless, the subsurface EAC also experiences seasonal changes, as its northern portion strengthens during austral spring and autumn and the southern portion gains in- tensity during austral winter and summer. This may be re- lated to the alternation in the intensity of those jets (North Vanuatu, North Caledonia, and South Caledonia) feeding the EAC [Ganachaud et al., 2008]. AAIW layer shoals rapidly to the south (Figure 1a). The accumulated annual mean zonal transport is calculated every 0.5° between sections S2 (147°E) and S1 (115°E), i.e., along the whole GAB, integrating south from the Australian coast (Figure 4a). These accumulated transports, equivalent to streamlines for the AAIW layer, reveal the path of the TL from the Paciﬁc Ocean to the Indian Ocean and the appearance of contributions due to the recirculation of ACC. We ﬁnd an Eulerian mean transport of 3.8 ± 1.3 Sv across 147°E. A signif- icant ACC input (1.2 Sv) occurs between 145°E and 147°E, provided by the eastern limb of a large anticyclonic gyre which extends from about 123°E to 146°E. The transport entering the Indian Ocean is estimated as 5.2 ± 1.8 Sv, being the result of three contributions: the TL through 147°E (3.8 Sv), water escaping westward from the anticyclone between 123°E and 146°E (0.4 Sv), and water recirculating in the southwestern end of our domain (1.0 Sv). 5. Conclusions Condie (2004), The 5500-km-long boundary ﬂow off western and southern Australia, J. Geophys. Res., 109, C04017, doi:10.1029/2003JC001921. Ridgway, K. R., and J. R. Dunn (2003), Mesoscale structure of the mean East Australian Current system and its relationship with topography, Progr. Oceanogr., 56, 189–222. Ridgway, K. R., and J. R. Dunn (2007), Observational evidence for a south- ern hemisphere oceanic supergyre, Geophys. Res. Lett., 34, L13612, doi:10.1029/2007GL030392. Ridgway, K. R., and J. S. Godfrey (1997), Seasonal cycle of the East Australian Current, J. Geophys. Res., 102, 22,921–22,936. Rintoul, S. R. (1991), South Atlantic interbasin exchange, J. Geophys. Res., 96, 2675–2692. Rintoul, S., and J. Bullister (1999), A late winter hydrographic section from Tasmania to Antarctica, Deep Sea Res., Part I, 46, 1417–1454. Rintoul, S., and S. Sokolov (2001), Baroclinic transport variability of the Antarctic Circumpolar Current south of Australia (WOCE repeat section SR3), J. Geophys. Res., 106, 2815–2832. [19] Acknowledgments. Funding for this work comes from the Spanish Ministerio de Ciencia e Innovación through project “Tipping Corners in the Meridional Overturning Circulation” (TIC-MOC, reference CTM2011- 28867). Miquel Rosell-Fieschi would also like to acknowledge the Ministerio de Ciencia e Innovación for funding through a FPU grant. This work was supported in part by the Australian Government’s Cooperative Research Centres Program, through the Antarctic Climate and Ecosystems Cooperative Research Centre (ACE CRC), and by the Department of Climate Change and Energy Efﬁciency, through the Australian Climate Change Science Program. [ ] Sloyan, B., and S. Rintoul (2001), The Southern Ocean limb of the global deep overturning circulation, J. Phys. Oceanogr., 31, 143–173. Sokolov, S., and S. Rintoul (2000), Circulation and water masses of the southwest Paciﬁc: WOCE Section P11, Papua New Guinea to Tasmania, J. Mar. Res., 58, 223–268. Speich, S., B. Blanke, and G. Madec (2001), Warm and cold water routes of an OGCM thermohaline conveyor belt, Geophys. Res. Lett., 28, 311–314. Speich, S., B. Blanke, P. de Vries, S. Drijfhouté, K. Döös, A. Ganachaud, and R. Marsh (2002), Tasman leakage: A new route in the global ocean conveyor belt, Geophys. Res. Lett., 29(10), 1416, doi:10.1029/ 2001GL014586. [20] The Editor thanks Sabrina Speich and an anonymous reviewer for their assistance in evaluating this paper. [20] The Editor thanks Sabrina Speich and an anonymous reviewer for their assistance in evaluating this paper. Speich, S., B. Blanke, and W. Cai (2007), Atlantic meridional overturning circulation and the Southern Hemisphere supergyre, Geophys. Res. 5. Conclusions Lebedev, K. V., H. Yoshinari, N. Maximenko, and P. W. Hacker (2007), YoMaHa’07: Velocity data assessed from trajectories of Argo ﬂoats, IPRC Tech. Note 4(2), 20 pp. [17] Velocity data inferred from Argo ﬂoat trajectories are used to characterize the currents near Australia at the sea sur- face and 1000 dbar levels. The inferred ﬂow ﬁeld agrees with the geostrophic circulation as estimated from a hydrographic climatology and a level of no motion [Ridgway and Dunn, 2003]. The circulation off eastern Australia is dominated at both levels by the EAC. However, only the intermediate wa- ters turn around the southern tip of Tasmania and ﬂow west into the GAB. We ﬁnd that the subsurface extension of the EAC supplies the TL and, ultimately, the Paciﬁc-to-Indian link of the Southern Hemisphere supergyre. Middleton, J. F., and M. Cirano (2002), A northern boundary current along Australia’s southern shelves: The Flinders Current, J. Geophys. Res., 107(C9), 3129, doi:10.1029/2000JC000701. ( ) Nuñez-Riboni, I., O. Boebel, M. Ollitrault, Y. You, P. L. Richardson, and R. Davis (2005), Lagrangian circulation of Antarctic Intermediate Water in the subtropical South Atlantic, Deep Sea Res., Part II, 52, 545–564. Ollitrault, M., M. Lankhorst, D. Fratantoni, P. Richardson, and W. Zenk (2006), Zonal intermediate currents in the equatorial Atlantic Ocean, Geophys. Res. Lett., 33, L05605, doi:10.1029/2005GL025368. p y Park, J. J., K. Kim, B. A. King, and S. C. Riser (2005), An advanced method to estimate deep currents from proﬁling ﬂoats, J. Atmos. Oceanic Technol., 22, 1294–1304. Ridgway, K. R. (2007), Seasonal circulation around Tasmania—An inter- face between eastern and western boundary dynamics, J. Geophys. Res., 112, C10016, doi:10.1029/2006JC003898. p p gy [18] These observations are the ﬁrst direct measurements of the intermediate-depth circulation near Australia with ade- quate spatial resolution to resolve boundary currents and their recirculations and sufﬁcient temporal resolution to account for the seasonal cycle. The westward Eulerian transport south of Tasmania displays substantial variability, likely due to the presence of eddy activity, between times of no leakage to maximum values of 14.5 Sv. We estimate the mean leakage of AAIW into the Indian Ocean to be 3.8 ± 1.3 Sv. Between 147°E and 145°E, the mean water transport increases to 5.0 ± 1.8 Sv because of the contribution from an anticyclonic gyre north of the ACC, and at 115°E, the mean net westward transport is 5.2 ± 1.8 Sv. Ridgway, K. R., and S. A. 5. Conclusions Lett., 34, L23614, doi:10.1029/2007GL031583. ROSELL-FIESCHI ET AL.: TASMAN LEAKAGE FROM ARGO FLOATS ROSELL-FIESCHI ET AL.: TASMAN LEAKAGE FROM ARGO FLOATS 2004 from a hydrographic inverse box model, J. Phys. Oceanogr., 38, 2072–2084. ﬂowing waters, so that the cross-sectional transport area is A=h d, we may decompose the mean transport Au ¼ 4.4Sv as contributions from the mean values and their ﬂuctuations, Au ¼ Au þ A′u′ . Computing Au ¼ 2.7 Sv, we estimate the eddy contribution to be A′u′ ¼ 1.7 Sv, or 39% of the total transport. Godfrey, J. S., G. R. Cresswell, T. J. Golding, and A. F. Pearce (1980), The separation of the East Australian Current, J. Phys. Oceanogr., 10, 430–440. Gordon, A. L. (1986), Interocean exchange of thermocline water, J. Geophys. Res., 91, 5037–5046. Herraiz-Borreguero, L., and S. R. Rintoul (2011), Regional circulation and its impact on upper ocean variability south of Tasmania, Deep Sea Res., Part II, 58, 2071–2081. 4. Intermediate Water Mass Transport by the TL [14] There have been several attempts at estimating the magnitude of the TL. The ﬁrst geostrophic calculations, from repeated SR3 WOCE sections, gave a transport of 8 ± 13 sver- drup (Sv) for the whole water column [Rintoul and Sokolov, 2001]. Early results by Speich et al. [2002] quantiﬁed the TL as the result of 13 ± 3 Sv of Subantarctic Mode Water (SAMW) and 26 ± 4 Sv of AAIW; however, only a fraction of this transport, 3.7 ± 2.5 Sv of SAMW and 10 ± 3 Sv of AAIW, reached the Indian Ocean. Later model computations by Speich et al. [2007] found a Lagrangian mean transport of 3.2Sv of intermediate waters all the way to the North Atlantic. Davis [2005], from a limited number of ﬂoat trajecto- ries near 1000 dbar and assuming an AAIW thickness of 500 m, estimated a leakage of 7.5 Sv from the Paciﬁc Ocean to the Indian Ocean. ( ) [16] The number of Argo ﬂoats south of Tasmania allows estimating changes in water transport through section 147°E, at temporal scales inﬂuenced by eddies. We calculate the accu- mulated transports south of Tasmania, every 15 days, between March 2006 and December 2013; for this calculation, we use a running interval of 150 days (5 months) as a characteristic time scale for eddies in this region [Herraiz-Borreguero and Rintoul, 2011] (Figure 4b). The negative values are a measure of the Eulerian TL transport at each time, which varies be- tween 0 and 12.0 Sv, with a mean value of 4.4 Sv and a standard deviation of 2.8 Sv (Figure 4c); this variability reﬂects the high eddy activity along 147°E, as suggested by van Sebille et al. [2012] from numerical models. Given the thickness h, width d, and speed u of the westward AAIW [15] Here we estimate both the TL and ACC contributions to the intermediate water transport into the Indian Ocean. We limit our calculations to the region north of the ACC, as the 5459 Xie, J., and J. Zhu (2008), Estimation of the surface and mid-depth currents from Argo ﬂoats in the Paciﬁc and error analysis, J. Mar. Syst., 73, 61–75. References Cirano, M., and J. F. Middleton (2004), Aspects of the mean wintertime circulation along Australia’s southern shelves, J. Phys. Oceanogr., 34, 668–684. Taylor, J. (1997), An Introduction to Error Analysis: The Study of Uncertainties in Physical Measurements, 2nd ed., pp. 166–168, Univ. Sci. Books, Sausalito, Calif. Davis, R. E. (2005), Intermediate-depth circulation of the Indian and South Paciﬁc Oceans measured by autonomous ﬂoats, J. Phys. Oceanogr., 35, 683–707. Van Sebille, E., M. H. England, J. D. Zika, and B. N. Sloyan (2012), Tasman leakage in a ﬁne-resolution ocean model, Geophys. Res. Lett., 39, L06601, doi:10.1029/2012GL051004. Ganachaud, A., L. Gourdeau, and W. Kessler (2008), Bifurcation of the sub- tropical South Equatorial Current against New Caledonia in December Xie, J., and J. Zhu (2008), Estimation of the surface and mid-depth currents from Argo ﬂoats in the Paciﬁc and error analysis, J. Mar. Syst., 73, 61–75. 5460
https://openalex.org/W4231715780	https://curationis.org.za/index.php/curationis/article/download/616/555	Afrikaans	null	Die Rouproses	Curationis	1,985	cc-by	7,384	SUMMARY In offering support to mourners one should be there, allow them to express their emotions in their own way and to talk about the deceased and their feelings of guilt. One should also lead them to understand that their reaction is normal and that the work of mourning must be completed. After considerable time sensi tive attempts may be made to direct the mourner to the future. Normale treursmart Dit is die nor male fisiese en psigiese reaksies wat ’n persoon tydens die afsterwe van ’n dierbare beleef. Normale treursmart Dit is die nor male fisiese en psigiese reaksies wat ’n persoon tydens die afsterwe van ’n dierbare beleef. Dit is belangrik om daarop te let dat rou nie net tot die dierbares beperk is wat direk aan die ster- wende verwant is nie. Die ster- wende persoon treur self asook sy familie, vriende, geneeshere en ver- pleegkundiges, predikante en ander gesondheidswerkers wat in sy ver sorging betrokke is, en in sommige gevalle gemeenskappe en die same- lewing in die geheel (bv. tydens groot rampe en die dood van ’n Staatsleier) (Barton, ibid: 107). Abnormaal verlengde rouproses wat dikwels met ’n intensifisering van een of meer van die manifestasies van normale treursmart gepaard gaan, soos oormatige skuld en iden- tifiseringsimptome. ’n Verkorte rouproses. n Kort, deur- leefde rousmart wat eg is, weens die onmiddellike verplasing van die ver lore dierbare (soos in die geval van ’n nuwe huwelik kort na afsterwe van die eggenoot/e). SUMMARY Die rouproses is ’n normale en uni- versele reaksie op verlies, wat inhe rent in menswees is. Terwyl die ma- nifestasies verskil van persoon tot persoon, van familie tot familie, en in verskillende sosiale en kulturele verbande, is die proses van rou ’n noodsaaklike deel, of die reaksie op, alle vlakke van verlies, (dood ingesluit) (Barton, 1977:107). V er lies is ’n natuurlike deel van ons be- staan : ons verloor byvoorbeeld troeteldiere, vriende, besittings, lig- gaamsdele, werk, en ’n dierbare deur die dood of egskeidings. Elkeen van hierdie verliese het ’n impak op die mens en sal in min- dere of meerdere mate treursmart m e e b rin g . D ie b e te k e n is en omvang van die verlies is hoogs ver- anderlik en gei'ndividualiseerd af- hangende van die individualiteit en tipe persoonlikheid van die treu- rende, die verlore verhouding en verwantskap, die sosiale milieu en die kultuur waarin hy hom bevind (Hospice Education Program for Nurses, 1981:129). The mourning process is a normal and universal reaction to loss. Awareness of the loss, confrontation and adaption are the main phases of the mourning pro cess, although each mourner’s reactions are highly individulised. , g g y Mourning can be regarded as essential “work” — which can never be escaped. The bond with the deceased must be untied, the mourner must adapt to the new environment without the loved one and then form new relationships. In offering support to mourners one should be there, allow them to express their emotions in their own way and to talk about the deceased and their feelings of guilt. One should also lead them to understand that their reaction is normal and that the work of mourning must be completed. After considerable time sensi tive attempts may be made to direct the mourner to the future. Mourning can be regarded as essential “work” — which can never be escaped. The bond with the deceased must be untied, the mourner must adapt to the new environment without the loved one and then form new relationships. The bond with the deceased must be untied, the mourner must adapt to the new environment without the loved one and then form new relationships. DIE VERSKILLENDE TIPES VAN ROU-REAKSIES Navorsers onderskei dikwels tussen variante van rou-reaksies soos onder andere normale rousmart, abnormale of patalogiese rousmart, tipiese of a-tipiese. Dit is egter be langrik om te onthou dat die onder- skeie variante so nou vervleg is dat dit dikwels baie moeilik is om hulle in aparte entiteite te onderskei. Dit is duidelik dat daar ’n baie fyn grens tussen normale en patalogiese rou smart bestaan en dat baie van die manifestasies van patalogiese rou smart dikwels net ’n intensifisering, verlenging, of distorsie van die gewone en verwagte rouproses is. Barton (ibid: 115) verwys na Engel wat eerder die begrip onge- kompliseerde rouproses gebruik as om na normale rousmart te verwys. Hoe dit ook al is, rousmart kan ge- kompliseer raak. Daar is voldoende feite wat daarop dui dat die rou proses dikwels met fisiese en/of psi- giese disfunksies gepaard gaan. Navorsers onderskei dikwels tussen variante van rou-reaksies soos onder andere normale rousmart, abnormale of patalogiese rousmart, tipiese of a-tipiese. Dit is egter be langrik om te onthou dat die onder- skeie variante so nou vervleg is dat dit dikwels baie moeilik is om hulle in aparte entiteite te onderskei. Dit is duidelik dat daar ’n baie fyn grens tussen normale en patalogiese rou smart bestaan en dat baie van die manifestasies van patalogiese rou smart dikwels net ’n intensifisering, verlenging, of distorsie van die gewone en verwagte rouproses is. Dit impliseer dat elke persoon se rouproses uniek is en dat dit nie ge- konformeer kan word tot ’n sekere konseptuele model van manifesta- sies en ’n bepaalde tydsverloop nie. In die proses van rou is daar egter sekere fenomenale verskynsels wat universeel in alle treurendes tot uit- drukking sal kom. Die wyse waarop uitdrukking hieraan gegee word en intrapersoonlike, interpersoonlike, sosiokulturele faktore en die mens se behoeftes bepaal die uiteindelike aanpassings- of wanaanpassings- kwaliteite van die prognose van die rouproses. Onderdrukte rousmart. Daar is veral ’n onderdrukking van baie van die emosionele reaksies van normale rousmart, maar veral liggaamlike klagtes kom te voorskyn (Hospice educational program for nurses, ibid: 143). Onderdrukte rousmart. Daar is veral ’n onderdrukking van baie van die emosionele reaksies van normale rousmart, maar veral liggaamlike klagtes kom te voorskyn (Hospice educational program for nurses, ibid: 143). Vertraagde of uitgestelde treursmart. Die vertraging of uitstelling kan wissel vanaf ’n kort tydperk tot twee jaar (Lindemann, 1944:141-148). Antisiperende treursmart. DIE VERSKILLENDE TIPES VAN ROU-REAKSIES Treur smart vind nie net plaas na die af sterwe van ’n geliefde nie. Dit kom ook voor in voorbereiding vir die dood, jou eie dood of dié van ’n ster- wende dierbare. Die rouproses begin wanneer die komende verlies van die dierbare erken word en dit strek dan tot die periode na die afsterwe (Hos pice educational program for nurses, ibid: 143; Barton, ibid: 116). Barton (ibid: 115) verwys na Engel wat eerder die begrip onge- kompliseerde rouproses gebruik as om na normale rousmart te verwys. Hoe dit ook al is, rousmart kan ge- kompliseer raak. Daar is voldoende feite wat daarop dui dat die rou proses dikwels met fisiese en/of psi- giese disfunksies gepaard gaan. Prof F.M.J. de Villiers, BA (Unisa) B.Ed (Rau), MEd (Unisa), DEd (PU vir CHO), GAV, GVV, DVO. DVA, GGV. Hoof, Departement Verpleeg kunde, Potchefstroomse Universiteit vir Christelike Hoër Onderwys. 38 CURATIONIS SEPTEMBER 1985 SEPTEMBER 1985 Bowlbys Bowlbys (1961) differensieer sy teorie ook in drie fases. • Die eerste fase is die protes-fase w at g e k e n m e rk w o rd d e u r woede, hunkering na die afge- storwe dierbare, ontkenning en ween. • Woede en opstandigheid • Onderhandeling • Die tweede fase is die desorgani- serende fase waarin daar wan- hoop en terneergedruktheid is. • Terneergedruktheid • Aanvaarding. p g • Die derde fase is die herorganise- ringsfase waartydens die treu rende persoon hom ontkoppel van sy bindinge met die afgestor wene dierbare en begin om nuwe bindinge aan te gaan. Parkes Parkes het gedurende 1970 die psigo-emosionele aspekte van die rouproses beskryf as ’n fase-proses wat begin met die gevoelloosheid- fase. Dit word gevolg deur die tweede fase wat bestaan uit ’n hun kering en ’n soeke na die afgestor wene dierbare en protes, wat verge- stalt in die rusteloosheid en gei'rre- teerdheid met homself of m et ander. Hiermee gepaard is daar ook huile- righeid, angs, spanning en ’n nei- ging om baie waarde te heg aan herinneringe aan die ontslapene. Die derde fase is die desorganise- rende fase wat gekenmerk word deur onvermoë om na die toekoms te kyk of om enige doel in die lewe te sien. H ierna is daar ’n geleidelike terugkeer van belangstellings en eetlus. Aan die ander kant het hierdie eensydige kritiek teenoor die fase- teorie ook tot die misinterpretering van die doel van die teorie gelei. Al die wisselende kognitiewe en affek- tiewe reaksies is gesistematiseer in ’n konseptuele model, en voorsien ’n algemene en bruikbare beeld/pa- troon (Hospice program , ibid: 138; Gram, 1982). Dit is ’n fase van verwarring wat met gevoelens van skok, ontken ning, ongeloof en onwerklikheid gepaard gaan. Aangesien elke treursmart uniek is en elke individu dus op sy eie manier treur sal ween- reaksies by sommige op die voor- grond staan, ander sal moontlik his- teries raak of flou word, terwyl ander moontlik sal swyg in geskokte ongeloof. De Klerk (1966:126-127) wys daarop dat alles verwarrend en flitsend is — wat verbystering, gela- tenheid of wanhoop tot gevolg kan hê. Dit word veral duidelik met uit- drukkings soos: hy kan nie dood wees nie, dit kan nie waar wees nie, wat sal ek doen?, asook klein kort gesprekke wat rondom die geliefde persoon sentreer. Die verloop van die rouproses Die bewuswordende fase Rousmart begin op die moment wanneer kennis van die naderende dood óf die afsterwe van ’n geliefde persoon geneem word. Die wyse waarop dit meegedeel word, die wyse waarop hy/sy gesterf het en die graad van betrokkenheid van die gesin in die versorging asook die kwaliteit van die verhouding met die oorledene, en die teenwoordig- heid of afwesigheid by die sterfbed, beinvloed in mindere of meerdere mate die familie se rouproses. Kubler-Ross Elizabeth Kiibler-Ross (1969) het tussen vyf fases in die sterwensdina- mika onderskei, haar fasemodel word ook gebruik om die rouproses in treurende individue te identifi seer: Daar is ’n groot verskeidenheid konseptuele modelle van die dina- mika wat die rouproses ten grond- slag lê. Alhoewel hierdie teorië of modelle in uitgangspunte, termino- logië en so m eer verskil, fokus almal op die verskynsel verlies en die reaksies daarop. So kry ons die sogenaamde fase -teorie, wát tans skerp onder die loep staan. Die grootste kritiek wat rondom die fase-teorie sentreer is dat fases ko- noteer dat daar ’n nie-variante en sekwensiële proses bestaan. H ier die fase-teorie word ook deur talle gesondheidswerkers misbruik — deur die treurende en sterwende mens in die verskillende fases te wil forseer in plaas daarvan om die fase-teorie te gebruik om ’n beter begrip van die betrokke individue te verkry. D aar m oet onthou word dat elke sterfbed en elke rousmart uniek is. Die een mens kan daarom nie m et ’n ander vergelyk word nie en nog minder mag dit tot norm verhef word waarteenoor ander geëvalueer word. • Ontkenning en isolasie, wat ’n fase van skok is en as ’n buffer teen die oorweldiging van die realiteit van die situasie dien en dan geleidelik die weg tot minder radikale verdedigingsm ega- nismes baan. DIE DINAMIKA VAN TREURSMART/DIE ROUPROSES • Die derde fase is die resolusie van die rouproses wat geken merk word deur die geleidelike terugkeer na die daaglikse akti witeite van die lewe en ’n toene- mende afname in die beheptheid met die beeld van die geliefde. • Die derde fase is die resolusie van die rouproses wat geken merk word deur die geleidelike terugkeer na die daaglikse akti witeite van die lewe en ’n toene- mende afname in die beheptheid met die beeld van die geliefde. Lindman Lindman (1944) beskryf drie fases Lindman (1944) beskryf drie fases • Die inisiële fase is een van skok en ongeloof. Dit word geken- merk deur ’n onvermoë om die verlies te aanvaar en by tye die absolute ontkenning dat die ver lies plaasgevind het. Kavanaugh Kavanaugh het gedurende 1972 sewe fases in sy rouprosesmodel onderskei: • Die tweede fase is die akute treursmart-fase wat gekenmerk word deur die aanvaarding van die verlies, verlies aan belang- stelling in daaglikse aktiwiteite, ween, gevoelens van eensaam- heid, slapeloosheid, en verlies van eetlus. D aar is ’n intense preokkupasie m et die beeld van die afgestorwene. Die heraanpassing W anneer die treurende die hede en die verlede deurworstel het dan is hy eers gereed om hom op die toe- koms te rig. Gedurende hierdie fase is daar ’n geleidelike afname in die treursmart en die intensiteit van die vorige fases verswak. Akute aan- valle van hartseer kan egter nog voorkom, maar tree geleidelik op die agtergrond en die verlangde emosie tree weer op die voorgrond. De Klerk (ibid: 128) wys daarop dat die treurende familie emosio neel deurtastend in verskillende rig- tings soek, talle situasies rakende die oorledene word weer beleef en oor en oor herhaal. Daar word veral baie waarde aan sy laaste woorde of opdragte geheg. Hulle soek ook emosioneel deurtastend na die sin van die gebeure en vir die gelowige staan die godsdienstige in die soeke voorop. ’n Algemene uit- drukking tydens hierdie fase is Wat was God se wil met hierdie gebeure, waarom juis hy? Woede en opstandigheid is ook ’n normale reaksie tydens die rou proses. Hierdie opstandigheid en woede word dikwels geprojekteer op God, geneeshere, verpleeg- kundiges, predikante, die afge- storwe dierbare. Dikwels word daar in treurendes se smart en verdriet gehoor: Ja, hy is nou weg en hier sit ek alleen met al die ellende, hulle voel só hartseer, só skuldig en dan verkwalik hulle nog hul geliefde wat nie meer daar is nie. Die woede kan ook op hulleself gerig word weens die skuldgevoelens, die frustrasie en verlies van beheer. Hierdie fase kan oor weke en maande strek. Daar is ’n geleidelike verskuiwing van die gerigtheid op die gestorwene, na gerigtheid op die toekoms en ander mense. Die emosionele verbintenis met die dierbare word geleidelik losge- knoop. Die werklikheid sonder die geliefde persoon word aanvaar en ten spyte daarvan dat die pyn en verlange mettertyd gestel word (De Klerk, ibid: 12), word die geliefde persoon nooit vergeet nie — hy/sy word net op ’n spesiale plekkie in hulle hart gebêre. Die treurende word ook bevry om nuwe bindinge aan te gaan sonder om patalogies aan die ou bande gebind te wees (Hospice educational program for nurses, ibid: 141). Die aanpassing en aanvaarding word geleidelik af- gerond, daar kom ’n heen wending tot die lewe en balans in die per- soonlikheid (De Klerk, ibid: 129). • Skok • Skok • Desorganisasie • Wisselende gevoelens van woede en vyandigheid • Skuld Barton (ibid: 109) is van mening dat hierdie intense emosionele bele- wenisse ’n tipe van ’n vloedgolf reaksie het wat telkens neig om die treurendes emosioneel te oorspoel. • Eensaamheid en verlorenheid • Eensaamheid en verlorenheid • Verligting • Verligting • Hervestiging 39 CURATIONIS VOL8 NO3 Dit moet egter onthou word dat skuldgevoelens ’n normale reaksie van die rouproses is. Selfs in die mees ideale verhoudinge tussen mense is daar altyd ’n mate van am- bivalensie (daar is altyd positiewe en negatiewe gevoelens teenwoor- dig). Daar sal ook altyd iets wees wat ons vir die ander persoon kon gedoen het. Dit is as gevolg van ons sondeval en die gebroke werklik- heid waarin ons lewe. Dit is dus ’n algemene neiging om na die af sterwe van ’n dierbare te dink oor wat gedoen kon word en wat be- hoort te gewees het. D aar sal altyd skuldgevoelens wees oor die nega tiewe (gevoelens) wat in die ambi- valente gevoelens van elke mens bestaan (Hospice educational pro gram for nurses, ibid: 139). Lindemann (ibid.T41-148) verwys ook na liggaamlike simptome wat soos golwe oor hulle heen spoel, o.a. sugtende asemhaling, uitput- ting, ’n gebrek aan sterkte, verlies van eetlus, slegte spysvertering, slapeloosheid, ens. D aar is ook ’n emosionele distansiëring van ander en ’n merkbare beheptheid met die gestalte van die gestorwe dierbare. Verskeie navorsers wys ook daarop dat baie van die treurendes kort na die afsterwe van hul dierbare ’n gevoel van verligting ervaar, soms in so ’n mate dat dit die smartgevoel vir ’n kort tydjie oorskadu. Hierdie bevindinge korreleer met talle be- droefde persone w aarm ee die skrywer te doen het asook met haar eie ondervinding tydens die af sterwe van haar vader. Dit wat ge- vrees was het nou gebeur — die ge- beure van dood is verby. Almal is dankbaar dat die geliefde persoon uit sy lyding verlos is. Daar is ook ’n verlies aan warmte in hulle verhoudinge en ’n neiging tot gei'rriteerdheid, hulle wil ook nie deur ander gehinder word nie. Hulle is rusteloos en soek altyd iets om hulle mee besig te hou. Hulle het ook ’n gevoel van nie in staat wees om roetine-aktiwiteite mee te begin of om te handhaaf nie. Die fase van konfrontasie In dié fase ontwikkel ’n toene mende bewuswording van die ver- lies Dan is hy werklik dood. Waarom juis hy? is algemene uit- drukkings tydens hierdie fase (Was- serman, 1972:10). In hierdie hoogs emosionele fase word treursmart intens beleef. Die treurende per- soon/e is minder geskok en deurleef talle nuwe reaksies wat ontstaan uit die konfrontasie met die verlies deur afsterwe en die implikasies daarvan (Hospice educational program for nurses, ibid: 139). Daar is ook individue wat skuldig sal voel oor die uiting van hulle ge voelens tydens afsterwe van ’n dierbare — hulle voel dat hulle beheer oor hul gevoelens verloor het. Dit is ’n basiese behoefte van die mens om te praat en uiting te gee aan iets wat ons gelukkig of on- gelukkig en hartseer maak. Ons moet ook onthou dat waar daar liefde is sal daar altyd hartseer wees, voor die sterwe, daartydens en ook daarna (Fox, 1969:75). • Skok Elke taak lê soos ’n berg voor hulle en die geringste takie word met groot moeite afgehandel. Behoeftes van die treurende dierbares Fase 4: Depressiwiteit op drie maande tot op, maar nie ingesluit, agt maande. Die mediaantelling van die ongunstige houdings teen oor die dood: 2.80, 2.70, 2.80 en 2.70 toon respektiewelik, ’n fase v a n t e r n e e r g e d r u k t h e i d / depressiwiteit vir ongeveer vyf maande. Die fluktuasies in die me- diaantellings toon fluktuasie in die gedagteprosesse ged u ren d e die depressiewe fase. deel was, aan te pas. Die aanknoop van nuwe verhoudinge binne of buite hierdie omgewing speel ook ’n rol in die temporale dimensie. Ver- dere veranderlikes in die duur van die rouproses is, onder andere, die ouderdom van die treurende, emo sionele verhouding, die verhouding met die gestorwene en die treu rende se algemene houding teenoor die dood. • ’n Behoefte om te treur, te ween en te praat oor wat hulle rou smart ten grondslag lê • ’n behoefte om vertroos te word • ’n behoefte om uiting aan al hulle emosies te gee • ’n behoefte om nuwe binding aan te gaan • ’n behoefte om hulle geloof te versterk. p Fase 5: H ero rg an iserin g /aan - vaarding op agt maande en langer. Vanaf ongeveer die agste maand begin die treurende individu die dood van sy dierbare aanvaar. Die mediaantelling, alhoewel dit effens fluktueer vanaf die agtste maand plat af tot ’n mediaantelling van 3.0. Die treurende sien sy verlies van die dierbare nou in ’n nuwe perspek- tief. Hy kan nou ook duidelik onderskei tussen die verlede en die toekoms. Sommige ervaar ’n gevoel van verligting noudat hulle die rouproses deurwerk het. Hy is nou gereed om die toekoms en sy lewe sonder sy geliefde voort te sit. Vol gens hierdie temporale model is dit dus duidelik dat die akute rou proses tot ongeveer die agtste maand strek. Kennis van hierdie wisse- lende fases bring ons tot ’n beter besef van die gevoelens en reaksies van die treurendes. Die tydsfaktor is nie die belangrikste as sodanig nie, maar wel die fluktuasies van skok, tot valse aanvaarding, tot valse herorganisering, tot terneer- gedruktheid, en dan uiteindelik die finale herorganisering en aanvaar ding. Dit stel ons in staat om hulle gedrag te verstaan sodat ons hulle gedurende hierdie fases van die rouproses kan help. Dit stel ons ook in staat om onsself ook beter te ver staan. Behoeftes van die treurende dierbares In die ontwerp van die model het Paul en Bowlby ’n houdingskaal ge bruik wat ontwerp is om die hou ding teenoor die dood objektief te meet oor ’n tydsverloop van dertien maande. Hulle het tot die gevolg- trekking gekom dat nam ate ’n per soon deur die rouproses vorder, sy houdings teenoor die dood ook ver ander. Van die inligting wat hulle gedurende die ondersoek verkry het konseptualiseer hulle dat die rouproses geïdenifiseer kan word as bestaande uit ’n reeks van vyf fases wat ongeveer agt maande duur voordat herorganisasie of aanvaar ding van die dood van ’n dierbare identifiseer kan word. Die heraanpassing Wanneer die familie eers die ver- skriklike en hartseer gebeure van die nou ondersoek en deurleef het is hulle gereed om hulle ondersoeke op die verlede te werp. Nou neem hulle hul verhouding met die oorle dene in oënskou en baie gou vind hulle allerlei redes waaroor hulle hulself verwyt, soos waarom hulle nie vir hom veel meer gedoen het nie, waarom hulle hom so verwaar- loos het en so meer. Dit veroorsaak allerlei skuldgevoelens en in som- mige gevalle is daar werklike redes en die ergste van alles is dat daar geen tyd vir restitusie is nie. T ern eerg ed ru k th eid en wan- hoop, angs en spanning is ook alge mene reaksies. Dit lei tot onttrek- king, apatie, afhanklikheid, gevoel- loosheid, hulpeloosheid, eensaam- heid, ’n gebrek aan konsentrasie, verwarring, ’n gevoel van verlaten- heid, onpersoonlikheid en ’n gevoel van verlies van vreugde en blyd- skap, hartseer en gedisorganiseerde optrede. 40 SEPTEMBER 1985 SEPTEMBER 1985 CURATIONIS DIE TEM PO R ELE DIMENSIE/ TYD SVERLO O P VAN DIE ROUPRO SES Lindemann (ibid: 141-148) is van mening dat die duur of tydsverloop van die rouproses afhang van die sukses waarmee ’n treurende per soon sy treurwerk doen, naamlik: • emansipasie van die gebonden- heid aan die oorlede dierbare • heraanpassing tot die omgewing sonder die geliefde persoon • vorming van nuwe bindinge/ver- houdinge. Fase 1: Skok/ontkenning kom voor vanaf die periode van dood, op tot een maand, m aar nie die maand insluitend nie. Die mediaan- telling van 2,90 van die houding teenoor die dood is ’n aanduiding van onaanvaarding van die dood van ’n dierbare — vandaar die ge- skokte ongeloof en ontkenning. Hierdie treurw erk verg beide fi siese en emosionele energie, net soos enige ander werk. Die feit dat die rouproses nie oor die algemeen as werk beskou word nie, veroor- saak dikwels problem e vir die treu- rendes. Hulle is dikwels nie gereed vir die intensiteit van hulle gevoe- lens of reaksies nie, en besef ook nie die belangrikheid om dit te aan- vaar en uitdrukking daaraan te gee nie. Al te dikwels is hulle geneig om die intense ontsteltenis te probeer vermy. Hulle kry ook dikwels nie die sosiale en emosionele onder- steuning wat noodsaaklik is gedu rende hulle treurw erk en rousm art nie, om dat hulle dierbares en vriende ook daarvan onbewus is dat treur werk is. Fase 2: Valse aanvaarding kom voor vir een m aand, op tot, maar nie ingeslote, die tweede maand. Die mediaantelling van 3.00 dui ’n gemiddelde houding aan teenoor die dood. Dit kan verder beskryf word as ’n waarskynlike aanvaar ding van die dood. D ie treurende se houding het verbeter in vergelyking met die eerste fase, m aar omrede die fases wat volg kan hierdie fase die beste beskryf word as valse aan vaarding. Die sterwensmoment Hulle moet nooit alleen gelaat word nie en indien ons eie gemoed ons vol- skiet, moet ons nie bang wees dat ons oë dof word van die trane by die aanskoue van die smart en lyding van ons medemens nie. Om mens te wees is geen wandaad nie. Dit is ook belangrik om te onthou dat wanneer mens herhaaldelik met sterwende en treurende mense in aanraking kom daar ook ’n ekspan- sie in die empatiese horisonne is. D aar is ’n innerlike meegevoel sonder uiterlike vertoon. Riglyne wat hier van belang is, is dat die begeleier/hulpverlener be- skikbaar moet wees en die familie asook die sterwende dierbare, hulle in hul wisselende gevoelens en emo sies volg en die uitdrukking daarvan aanmoedig. Daar moet egter nie getrag word om hulle te oorreed om die onvermydelike nou al te aan- vaar nie, geen mens sal op hierdie stadium daarvoor gereed wees nie. Daar moet egter gewaak word teen verharding en koudheid, want dit is veroordeelbaar. Ons moet onthou dat geen smart so groot is dat ons nie die Man van smarte daaragter kan sien nie. Al gaan ons ook deur rousmart of die dal van doodskaduwee hoef ons geen ont- heil te vrees nie, want Hy verseker ons dat Hy ons nooit sal begewe of verlaat nie. Die ondersoek spreekbaar maak van vrese is nodig. Die onsekerheid oor die toekoms, die gevoel van hulpeloosheid en angs oor die ko- mende verlies van die dierbare, die vrees vir die sterwensgebeure as so- danig — al hierdie dinge open die hart van die een mens vir ’n ander, en bied ’n brug om liefde en hoop en geloof te herwin. Beklemtoning van lewe en hoop vir die toekoms, en die hulp, bystand en lyding van ons Hemelse Vader is by alle per sone wat by die probleme van sterwe en rou betrokke is, nood- saaklik. Met die tyd sal die kennis en erva- ring van die liefdevoile, versor- gende, luisterende en beskikbare p e rso o n d ie n o d i g e v e r t r o u e , ondersteuning en onderskraging bied wat die treurendes tydens die oorweldigende hartseer en emosio- nele ontwrigting nodig het. Die sterwensmoment By die intrede van die sterwensmo ment is die skaal van reaksies uit- eenlopend. Baie van die familie wil die afgestorwe dierbare sien, som- mige wil die liggaam soen of lief- koos, praat, wegloop en terugkom asof hulle dit nie kan glo nie, som- mige wil hulle nie van die sterwende se bed wegskeur nie. Dit is ’n moment van totale betrokkenheid met die sterwende (Fox 1969:79). Die ideaal is dat die hantering van treursmart wat met terminaal siekes geassosieër word reeds begin voor die dood ingetree het. Gedurende hierdie fase is die begeleier in staat om die voorbereidingswerk te doen vir die vrye uitdrukking van gevoe lens en emosies wat aan treursmart, verwant is. Sy kan ook eerlike oop gesprekke voer, die legimiteit van die proses van rousmart bevestig en aan die treurende familie die doel- treffendheid van ’n deernisvolle en caring verhouding demonstreer ten einde hulle by te staan gedu rende hulle aanpassing by die ver lies van hulle geliefde. Dit bevorder ook die proses van antisiperende treursmart, en daardeur berei dit die sterwende persoon asook sy fa milie voor om met die rouproses te begin. W anneer dit voor die aktuele dood gebeur vorder die familie na afsterwe van hul dierbare meer na- tuurlik deur die rouproses. Gepaard hiermee is ’n stadiger, maar toenemende betrokkenheid in die versorging van die sterwende dierbare baie belangrik. Dikwels voel die familie baie ambivalent hieroor — aan die een kant wil hulle graag iets doen maar aan die ander kant is hulle bang en onseker. Hulle kan hul geliefde gemaklik maak, sy rug-en-drukdele vryf, help om hom te voed en te bad wanneer hy swak en hulpeloos is. Dit skep brúe wat mense verbind namate die sterwensproses vorder. D eur hier die take verkry die familie gelykty- dig vaardigheid en vertroue, en voel ook nie so hulpeloos en hopeloos nie. Hulle besef ook dikwels dat die wêreld van verantwoordelikheid wat hulle betree nie so vreesvol is as wat hulle dit verwag het nie. Hulle grootste konflik kom aan die einde wanneer hulle hul nie van die ster wende se bed wil wegskeur nie. Dit lei tot totale betrokkenheid met hom/haar. Dit is dan vir almal be langrik om ten spyte van uitputting weens liggaamlike spanning, naby mekaar te wees (Kutscher, 1969: 78). Hierdie gebeurlikheid om die sterfbed moet ongehinderd en pri- vaat begelei word, want dit is deel van die ontkoppelingsproses. HULP AAN DIE TREURENDES Ter inleiding moet daarop gewys word dat die begeleiers oor ’n ge- stalte van liefde sal beskik, die liefde van Christus wat ons ganse wese deurstraal. D aar moet ook onthou word dat die rouproses ’n baie ingewikkelde gebeure is wat die mens in totaliteit tref— na gees, siel en liggaam — asook die feit dat elke rouproses uniek is, daar elke mens op sy eie unieke manier sal treur. Fase 3: Valse reorganisasie op twee maande, tot op, maar nie inge- sluit, die derde maand. Die medi aantelling gedurende hierdie tyd span is deurgaans hoog naamlik 3.10. In hierdie fase blyk dit asof die treurende sy/haar lewe herorga- niseer het. Die inligting toon egter dat die gevoelens tydens hierdie fase net tydelik is. H ardt (1979:134-137) verwys na Paul en Bowlby se konseptualise- ring van die tydsverloop van die rouproses. Dit is duidelik dat met die verloop van tyd, ook heling van die wonde van rousm art kom. Hoe lank hierdie tydspan is blyk afhank lik te wees van die individu se vermoë om by die omgewing waar- van die gestorwe dierbare vroeër VOL8 NO3 41 CURATIONIS maak van die emosionele dilemma waarin hulle hul bevind. Die sterwensmoment Die sterwensmoment Die ge- borgenheid wat hulle in so ’n ver- trouensverhouding en situasie belewe verskaf aan hulle ’n gevoel van veiligheid en sekuriteit, wat vrye gesprek en uitdrukking aan emosies en oorweldigende gevoe lens moontlik maak. Dit is van groot terapeutiese waarde vir almal (Barton, ibid: 118). Nadat afskeid geneem is is die hartseer soms so diep dat dit lyk asof niks behalwe ’n gevoel van lief- devolle aanraking deurdring nie. Dit is soms nodig om die bedroefde in jou arms te hou, te vertroos soos ’n moeder haar kind, medelydend mee te gaan, teer en geduldig met hulle om te gaan en nie die vryelike vloei van emosies direk na afsterwe te verhinder nie. Hulle moet ook aangemoedig word om te ween. Indien dit moontlik is, moet die familie aangemoedig word om tot die einde toe by die sterwende te bly. Die afwesigheid by die sterfbed kan skuldgevoelens verskerp. Vir die uiteindelike verwerking van rousmart is dit ook soms van waarde as die sterwe meegemaak is — dit is instrum ented vir die besef v a n d ie v e r l i e s ( D e K l e r k , ibid: 181). Daar moet egter getrag word om histerie te bekamp. D aar moet gereik word na geliefdes — raak hulle teer aan, plaas u arm om hulle en help so om die bedroefdes deur die inisiële moment wat op die dood volg te begelei (Fox, ibid:80). Daar vind ook toename in inner- like groei plaas wat betref die treu rendes se oopheid in die spreekbaar 42 CURATIONIS CURATIONIS SEPTEMBER 1985 Na die vrylike vloei van emosies kan die gelowige vriend, predikant, versorgers/begeleiers ’n Bybelversie genuanseerd gebruik om hulle te troos en te versterk, asook ’n kort gebed doen om aan die familie mee- gevoel te betuig en vertroosting in Christus te soek. D it is sinvol om vir enkele oomblikke oor die positiewe aspekte van die dood te praat. Roep aanmerkings wat deur die af- gestorwe dierbare gemaak is en wat van besondere betekenis vir die fa milie kan wees in herinnering. D ik wels heg die naasbestaandes groot waarde aan, en put hulle troos uit, die laaste woorde en belydenisse van die afgestorwene (De Klerck, ibid: 186). rouproses konstruktief te verwerk. Die sterwensmoment Dit impliseer dat hulle die ongemak en al die ander emosionele erva- ringe en fisiese ongemak wat deur die dood van ’n dierbare opgewek word moet aanvaar en realisties na die verlies moet kyk. Die treurende moet aangemoedig word om aktief deel te neem in die verwerking van die rouproses in plaas daarvan om te probeer om dit te ontken of dit te ontsnap. Hy moet besef dat die rou- periode tot ’n mate vertraag maar nooit uitgestel kan word nie, daar dit direk of indirek steeds voort- gaan. Fisiese en emosionele ondersteu- ning deur die totale rouproses is noodsaaklik. Dit is egter veral van kardinale belang in die periode wanneer die ware implikasies en realiteit van die verlies van hul dierbare tot treurendes se voile be- wussyn deurdring. Dit kan weke of maande na afsterwe plaasvind. Dit is die tyd wanneer meeste van die sosiale ondersteuning na die begraf- nis verdwyn het en baie mense ver- onderstel dat die familie oor die ergste is. Vir baie treurendes is hierdie periode een van die moei- likste tye in hulle lewe. Dan is die belewing van pyn en treursm art die akuutste want hulle word nou let- terlik geforseer om die lewe sonder hul dierbare tegemoet te gaan en voort te sit (Hospice educational program for nurses, ibid: 156-157). Lindeman (1944) het die vol- gende riglyne gegee oor hoe die treurendes hulle verlies en treur smart moet konfronteer: ondersteu- ning en gerusstelling voorsien nie voldoende bystand in die mens se rouwerk nie. Hulle m oet hul ver houding met die ontslape geliefde in heroorweging neem en m oet ver- troud raak met die veranderinge in hulle eie modus van emosionele reaksies. Hulle vrees vir kranksin- nigheid, en die vrees om die verras- sende verandering in hulle gevoe lens te aanvaar, veral die oorweldi- gende gevoelens van vyandigheid en o p stan d , m oet deu rg ew erk word. Hulle moet uiting gee aan hulle hartseer en treursm art, asook die gevoel van verlies van hulle dierbare. Hulle sal ook uiting aan gevoelens van skuld en opstandig heid moet gee, asook nuwe persone vind met wie emosionele bindinge aangeknoop kan word. Hulle sal ook ’n aanvaarbare formulering van hul toekomstige verhouding m et die oorledene moet vind. Voortgesette hulpverlening W at treurende persone die meeste benodig is om toegelaat te word om die treuremosies as normaal te aan- vaar en om uiting daaraan te gee. Hulle moet dus hul treursm art kan verwoord en hul verhouding met die afgestorwe dierbare hersien. Pogings om die familie in rigtings weg van die afgestorwe dierbare te beweeg moet dus baie sensitief en op die regte moment gedoen word om van enige waarde te wees. W eer eens is dit net die stille teenwoor- digheid van die begeleier en ak- tiewe luisteraar wat in hierdie sta dium van groot terapeutiese waarde is in die vordering van die rou proses. Die sterwensmoment W anneer alles wat moontlik is, gedoen is om die sterwensproses ’n periode van groei in liefde en be- g r y p i n g te m a a k , sal h i e r d i e moment vir die familie ’n nuwe toe- koms inhou — ’n tyd van hoop ten spyte van hartseer. Dit moet onthou word daar is altyd, of dit hartseer of vreugde is, die drang om sterk emosionele ervarings met iemand wat sal verstaan te deel. Die bedroefde familie benodig dus ’n voile m aat van m edem enslike gevoel en medelye veral na die be- grafnis. Die familie onthou ook nie altyd die tegniese en kliniese vaar dighede nie, m aar wel die warmte, deernis en liefde wat hulle tydens hierdie groot beproewing ontvang, dit is ook dikwels die m aatstaf waarmee die kwaliteit van die be trokke dienste mee gemeet word. • Ondersteun hulle in die aanvaar ding, uiting en identifisering van ge voelens. Daar moet egter gewaak word teen premature pogings om die treurende familie se aandag op ander bedrywighede, werksaam- hede, sosiale interaksies, en so meer te fokus, of om hulle te pro beer forseer om te vergeet en om aan te gaan. Dikwels hoor ons mense sê jy moet vergeet want wat verby is is verby, en die lewe moet voortgaan. Sodanige optrede kan met aggressie en woede aangehoor word, selfs met verontwaardiging, want geen mens is op hierdie sta dium daarvoor gereed nie. Dit kan ook vertroebeling van die ver- trouensverhouding teweegbring wat die onderlinge verhoudinge en ge- spreksvoering kan rem. Basiese riglyne vir verdere hulp verlening Sekere drogerye mag die treurende toelaat om die smart en hartseer van die rouproses te vermy of te onder druk, en dit dus rem en sodoende verleng. Die oordeelkundige ge bruik van klein dosisse van kal- meermiddels en slaapmiddels kan van groot terapeutiese waarde wees veral gedurende die vroeë fase van die rouproses. Indien behoorlike begeleiding van die sterwendes en hulle familie in die kurrikula van die professionele gesondheidwer- kers geïntegreer word dan sal die hand van die geneesheer nie so dik wels uitreik na ’n voorskrif nie, maar sal daar eerder aandag aan die beginsels van begeleiding gegee word. Daar moet ook rekening gehou word met die feit dat sommige treu rendes by tye alleen wil wees, om alleen en stil oor hulle gevoelens, herinneringe en toekoms te besin. Begeleiers moet hiermee rekening hou en hulle ook die geleentheid bied om alleen te wees. Verseker hulle egter van u beskikbaarheid en gewilligheid om te help, indien hulle dit sou benodig (Barton, ibid: 118-120). • Voorsien die treurendes van kennis oor die dinamika van die rouproses, sodat hulle insig in hulle gedrag kan kry. Dit vergemaklik die normale proses van rou en voor kom ’n distorsie/verdraaiing daar van. Verduidelik aan hulle dat daar in elke verhouding ’n mate van am- bivalensie is. Moedig hulle ook aan om oor hulle skuldgevoelens en op- standigheid te praat. Afgesien daar van dat dit ook kenmerkend van die treursmart is, kan hulle ook gewys word op die vergifnis wat daar in Christus is. Volgens 1 Joh. 1:9, sal die Here wat getrou en regverdig is jou sondes vergewe en jou van alle ongeregtigheid reinig as jy jou sonde bely. Hulle moet dit in die geloof aanvaar, en hulself ook ver gewe. • Nie-veroordelende luister en on- voorwaardelike aanvaarding sodat die treurendes uiting aan hulle emo- sies kan gee sonder die vrees van verwerping. Dit is baie belangrik aangesien baie van die emosies sosi- aal onaanvaarbaar is en dit ook skuldgevoelens by die treurendes opwek — daarom is dit noodsaaklik dat uiting daaraan gegee moet word. Daar sal dan eerder van die tera peutiese verhouding en gesprek ge bruik gemaak word as ’n middel om die treurende persone te help om uiting aan hulle gevoelens te gee en hulp te verleen in die hantering daarvan. Daar moet egter op gewys word dat die gebruik van medikasie in patalogiese rousmart geregverdig is. Basiese riglyne vir verdere hulp verlening • Die begeleiers moet fisies sowel as emosioneel teenwoordig wees om onderskraging en sekuriteit aan die treurendes te verleen. Veral ge- durende die vroeë periode van skok en desoriëntering sal dit waardevol wees indien iemand sommige van die roetinetake en verantwoordelik- hede kan oorneem soos die voorsie- ning van maaltye. Dit is ook tera- peuties indien rigting vir die treu rende familie gegee kan word oor sekere besluite en optredes. Hulle is soms nie in staat om dit self te doen nie. Jackson (1975) skryf in sy boek, Understanding Grief, dat die doel van die begeleier se hulp en onder- steuning daarop gerig m oet wees om die treurende mens te help in die ontkoppeling van die emosio nele bindinge met die ontslape dierbare ten spyte van die hartseer en treursm art wat hulle belewe en om uiteindelik die tipe van interak sie wat verlore is te verplaas. Indivi- due moet ook oorreed word om die D ie h u lp v erlen ers/b eg eleiers moet onthou dat die treurende fa milie ’n breë spektrum van uiteen- lopende gedrag toon. Sommige sal uitbarstings van vyandigheid, woede en intense uiting van hart seer in die vorm van ween toon; ander weer sal nie hulle emosies op 43 CURATIONIS VOL8 NO3 ons dood. Indien hulle nie uiting gee aan al hierdie gevoelens en emosies nie, kan dit vir jare duur. Vanweë die verwaarlosing van die treurende familie in ons gesond- heidsdienssisteem kry ons talle mense met reste van ondeurge- werkte rousmart. voorvereistes vir die rouwerk effek- tief te voltooi. Om te ween en te praat oor alles wat gebeur het en hulle geliefde persoon, dra by tot die besef dat hy werklik dood is. By die herhaling van die gebeure ver- loor die treurproses ook ’n bietjie van sy die emosionele komponent. ’n intense wyse tot uitdrukking laat kom nie, maar op ’n stille wyse treur en ween. Daar moet onthou word dat elke mens ’n individuele krisishanteringstyl het. Moenie die persoon wat stilweg treur probeer forseer om op ’n meer intense wyse aan sy hartseer-emosies uiting te gee nie, of andersom, die persoon wat vryelik uiting gee probeer be- dwing nie — vir hulle is dit normaal. • Laat ons egte besorgdheid en versorging en deernis tog sigbaar wees. Basiese riglyne vir verdere hulp verlening Aangesien die dood van ’n dierbare so ’n ongelooflike gebeure is, en die familie dit nie kan glo nie, wil sommige van hulle die liggaam van die ontslapene in die lykshuis of by die begrafnisondernemers gaan sien — want om te sien is te glo. Indien dit hulle wens is moet hulle nie teëgehou word nie. Hierdie finale afskeid dra ook by tot die realisering van die dood. Die teen- deel is ook waar. Geen persoon moet forseer word om na die ontsla pene te gaan indien dit nie hul be geerte is nie. Hulle het ander ma- niere van aanvaarding en integre- ring van die afsterwe. • Moedig die treurende familie aan om nie ongeduldig met hulself te wees nie, en om nie aanvanklik teveel van hulself te verwag nie. Wys hulle egter op hul verantwoor- delikheid om te besluit oor wat hulle met hul rouproses gaan maak. Hulle kan dit op ’n gesonde of on- gesonde manier verwerk, hulle kan toelaat dat hulle in verbitterde en ontnugterde mense ontwikkel of hulle kan in hul geloof in ons He- melse Vader versterk, vernuwe en gesuiwer word tot groter diensbaar- heid aan hulle God en medemens — ’n mens wat innerlik deur ons Meester genees is. Die hulpverleners se houding teenoor die dood, rou, die uitdruk king van gevoelens in die algemeen, en hulle gevoelens om trent die treu rende individu se partikuliere styl van uiting en hantering van sy treursmart kan ’n belangrike rol in die bevordering of remming van die rouproses speel. Sommige kan on- gemaklik voel met die intense uiting van gevoelens, sommige begeleiers sal hulle nie net onttrek nie maar selfs probeer om hierdie belangrike gevoelens te onderdruk in ’n poging om die ongemak wat dit veroorsaak te voorkom. Onverdraagsaamheid teenoor die spesifieke treursmart hanteringstyle kan die familie se neiging om ’n sekere aanpassings- modus te vermy versterk en so- doende die rouproses erg rem. • Moedig die treurendes aan om oor die afgestorwe dierbare te praat, dit is van groot belang dat hulle oor die negatiewe aspekte sowel as die positiewe sal praat, asook oor hulle wedersydse verhou ding. In die normale gang van sake word al die negatiewe aspekte op die agtergrond gestoot en al die goeie aspekte van hul dierbare word verabsoluteer. g • Die gebruik van medikasie in die hantering van die rouproses bly nog steeds ’n debateerbare punt. Basiese riglyne vir verdere hulp verlening Dit sluit egter nog nie die bege- leidende aspekte uit nie (Barton, ibid: 119-121). Die treurende familie het ’n be hoefte om te praat en te praat, te ween en te ween, om ’n terugblik te werp op die oorledene en alles in verband daarmee, en om alles in hersiening te neem. Hierdie pro sesse is van kardinale belang om die treurendes in staat te stel om die Dit is ook belangrik dat hulle oor hulle opstandigheid sal praat, op- standigheid teenoor die ontslapene of teenoor hulle God, want na alles is Hy die beskikker oor ons lewe en (Vervolg op p 56) 44 CURATIONIS SEPTEMBER 1985
https://openalex.org/W4285445379	http://jurnal.umsrappang.ac.id/mallomo/article/download/391/307	Indonesian	null	Inovasi Kemasan Kopi Robusta Kekinian Desa Sukamaju Berbasis Kearifan Lokal	MALLOMO	2,021	cc-by	3,245	MALLOMO: Journal of Community Service https://jurnal.umsrappang.ac.id/mallomo/index Vol 1, No 2, Juni 2021, pp 64-72 MALLOMO: Journal of Community Service https://jurnal.umsrappang.ac.id/mallomo/index Vol 1, No 2, Juni 2021, pp 64-72 MALLOMO: Journal of Community Service https://jurnal.umsrappang.ac.id/mallomo/index Vol 1, No 2, Juni 2021, pp 64-72 Dini Yuliani1, Irfan Nursetiawan2, Otong Husni Taufiq3 1,2, 3 Ilmu Pemerintahan, Universitas Galuh Email: diniasyari16@gmail.com Abstract. Poverty is still the main topic as a problem that must be resolved and an indicator of the existence of a pre-prosperous society, and this cannot be separated from the factor of economic inequality and the inability of the community to meet their basic needs. Poverty can also mean a lack of social needs and economic inability. As for the objectives of this activity, namely: (1) Socializing contemporary coffee packaging as an adaptive and novelty form of packaging in the modern era; (2) Providing training on the creativity of coffee farmers in coffee making and providing great opportunities in promoting superior products of coffee producers; and (3) Providing business assistance for coffee producers. The method used in this service activity, namely: Focus Group Discussion (FGD) to determine the potential of the coffee- producing community and using the Waterfall method. The results of these activities, namely (1) Coffee packaging innovations that have been carried out have received positive responses from partners and are useful for increasing the number of sales of coffee commodity products; (2) Coffee packaging innovations consist of information on Brand Coffee, Single Origin and Espresso Blend, region, varietal, roast level, cupping notes, height, weight of coffee, roast date, beans, and level of fineness; (3) Community service is carried out through socialization of coffee packaging innovations, packaging design 64 Inovasi Kemasa Kopi Robusta … (Dini Yuliandini) \| 65 PENDAHULUAN Kemiskinan merupakan salah satu permasalahan sosial bagi Bangsa Indonesia dan hal tersebut seperti pandemic yang terus meluas, serta memerlukan penanganan yang serius khususnya oleh Pemerintah Pusat dan Pemerintah Daerah. Kabupaten Ciamis sebagai salah satu bagian dari kabupaten/kota yang berada di wilayah provinsi Jawa Barat tidak terlepas dari permasalahan kemiskinan. Di Kabupaten Ciamis pada tahun 2017 jumlah penduduk miskin berjumlah 96,76 ribu jiwa, sedangkan di tahun 2018 berjumlah 85,72 ribu jiwa dan di tahun 2019 berjumlah 75,41 ribu jiwa (Badan Pusat Statistik, 2019). Walaupun jumlah penduduk miskin mengalami penurunan dari tahun ke tahun, tetapi hal ini tetap menjadi permasalahan yang harus diselesaikan. Salah satu desa yang masyarakatnya masih ada yang tergolong prasejahtera, yakni Desa Sukamaju Kecamatan Cihaurbeuti Kabupaten Ciamis. Desa Sukamaju Kecamatan Cihaurbeuti merupakan desa yang berada di lereng atau kaki bukit Gunung Sawal yang termasuk di daerah kawasan Ciamis Utara dimana mata pencaharian penduduknya dari dulu hingga sekarang mayoritas bercocok tanam. Dengan kondisi tersebut, maka dominasi mata pencaharian masyarakat Desa Sukamaju, yakni petani dan buruh tani. Potensi yang dimiliki oleh Desa Sukamaju, yakni produk kopi yang berasal dari para petani kopi yang dibudidayakan di wilayah kaki Gunung Sawal. Produksi kopi tersebut diinisiasi oleh BUMDEs Maju Mandiri Desa Sukamaju. Konsep pemberdayaan masyarakat, khususnya bagi petani kopi di Desa Sukamaju merupakan salah satu bentuk solusi untuk menurunkan tingkat kemiskinan yang ada di desa tersebut. Produksi kopi tersebut telah memberdayakan para petani kopi dan telah berproduksi untuk kopi serbuk dengan jenis kopi robusta. Namun dalam proses produksi kopi tersebut terdapat kendala, salah satunya dalam hal kemasan produk. Kemasan produk kopi yang diproduksi masih bersifat konvensional, sehingga dirasa kurang menarik untuk pasar saat ini. Dengan bahan baku yang cukup melimpah memang tidak menjadi kendala utama dalam proses produksi, tetapi dalam pengemasan produk menjadi kendala yang mendominasi karena belum semua market menerima produk tersebut. Isu lingkungan dan gaya kekinian menjadi isu sentral dalam era perubahan yang menuntut sebuah inovasi. Menurut Noviadji, B. R. (2014) menyatakan Kemasan adalah wadah atau pembungkus, bagi produk pangan, kemasan mempunyai peranan penting dalam upaya mempertahankan mutu dan keamanan pangan serta meningkatkan daya tarik produk. Agar bahan pangan yang akan dikonsumsi bisa sampai kepada yang membutuhkannya dengan baik dan menarik, maka diperlukan pengemasan yang tepat. Berdasarkan hal tersebut, maka dalam membantu masyarakat melalui kemitraan dengan BUMDes Maju Mandiri khususnya masyarakat petani kopi dapat memahami pentingnya inovasi kemasan kopi untuk peningkatan penjualan produk kopi tersebut. 66 \| MALLOMO: Journal of Community Service 66 Inovasi Kemasa Kopi Robusta … (Dini Yuliandini) training, and business assistance; and (4) Business assistance with partners includes training in partner organization management, assistance in program development, as well as monitoring and evaluation of activities. Abstrak. Kemiskinan masih menjadi topik utama sebagai masalah yang harus diselesaikan dan menjadi indikator dari keberadaan masyarakat pra-sejahtera, serta hal tersebut tidak lepas dari faktor ketimpangan ekonomi dan ketidakmampuan masyarakat dalam memenuhi kebutuhan dasarnya. Kemiskinan dapat berarti pula sebagai kekurangan kebutuhan sosial dan ketidakmampuan secara ekonomi. Adapun yang menjadi tujuan dari diadakannya kegiatan ini, yakni: (1) Mengadakan sosialisasi kemasan kopi kekinian sebagai bentuk adaptif dan kebaruan kemasan di era modern; (2) Memberikan pelatihan kreatifitas petani kopi dalam pembuatan kopi dan memberikan peluang besar dalam mempromosikan produk unggulan produsen kopi; dan (3) Melakukan pendampingan usaha bagi produsen kopi. Metode yang digunakan dalam kegiatan pengabdian ini, yakni: Focus Group Discussion (FGD) untuk mengetahui potensi yang dimiliki masyarakat produsen kopi dan menggunakan metode Waterfall. Adapun hasil kegiatan tersebut, yaitu (1) Inovasi kemasan kopi yang telah dilakukan mendapatkan respon yang positif bagi mitra dan bermanfaat untuk meningkatkan jumlah penjualan produk komoditas kopi; (2) Inovasi kemasan kopi terdiri dari informasi Brand Coffee, Single Origin dan Espresso Blend, region, varietal, level sangrai, cupping notes, ketinggian, berat kopi, tanggal sangrai, biji, serta tingkat kehalusan; (3) Pelaksanaan pengabdian kepada masyarakat dilakukan melalui sosialisasi inovasi kemasan kopi, pelatihan desain kemasan dan pendampingan usaha; dan (4) Pendampingan usaha dengan mitra meliputi pelatihan manajemen organisasi mitra, pendampingan pembuatan program, serta monitoring dan evaluasi kegiatan. Keywords: Innovation, Packaging, Community Empowerment, Coffee. artikel dengan akses terbuka di bawah lisensi CC BY -4.0 Kata Kunci: Inovasi, Kemasan, Pemberdayaan Masyarakat, Kopi. Inovasi Kemasa Kopi Robusta … (Dini Yuliandini) \| 6 Inovasi Kemasa Kopi Robusta … (Dini Yuliandini) \| 6 Kabupaten Ciamis. Kolaborasi BUMDes Maju Mandiri dan petani kopi Desa Sukamaju bersinergi untuk meningkatkan kesejahteraan, serta dari akademisi melalui pengabdian ini mengakselarasi tujuan bersama dari khalayak ramai berkaitan dengan kesejahteraan. PENDAHULUAN Inovasi merupakan salah satu modal untuk melahirkan sebuah pembaharuan dalam setiap hal dan bertujuan untuk kemajuan yang berkelanjutan. Menurut Stephen Robbins (dalam Sukmadi, 2016:31) menyatakan, Inovasi adalah suatu gagasan baru yang diterapkan untuk memprakarsai atau memperbaiki suatu produk atau proses dan jasa. Oleh karena itu, dalam pengabdian ini merupakan sebuah langkah untuk perbaikan, khususnya dalam kemasan kopi yang berasal dari produksi masyarakat Desa Sukamaju Kecamatan Cihaurbeuti 67 1. Kerangka Pemecahan Masalah Modernisasi telah mengubah mindset masyarakat menjadi lebih apatis terhadap tingkat kesejahteraan mereka sendiri. Paradigma yang berkembang, yakni mereka hanya menunggu dari pihak lain untuk membantu mereka dalam meningkatkan kesejahteraan. Seharunya hal tersebut harus segera ditinggalkan, karena berdampak pada laju perekonomian yang cenderung stagnan. Adapun kerangka dalam pemecahan masalah dalam kegiatan pengabdian masyarakat ini dilakukan dengan bertumpu pada penyadaran diri masyarakat. Untuk prinsip-prinsip yang diterapkan dalam pengabdian ini, sejalan dengan konsep Karsidi (2007), yakni: a. Belajar Dari Masyarakat Prinsip yang paling mendasar adalah prinsip bahwa untuk melakukan pemberdayaan masyarakat adalah dari, oleh, dan untuk masyarakat. Ini berarti, dibangun pada pengakuan serta kepercayaan akan nilai dan relevansi pengetahuan tradisional masyarakat serta kemampuan masyarakat untuk memecahkan masalah-masalah sendiri. b. Pendamping sebagai Fasilitator Masyarakat sebagai Pelaku Konsekuensi dari prinsip pertama adalah perlunya pendamping menyadari perannya sebagai fasilitator dan bukannya sebagai pelaku atau guru. Untuk itu perlu sikap rendah hati serta kesediaan belajar dari masyarakat dan menempatkan warga masyarakat sebagai narasumber utama dalam memahami keadaan masyarakat itu sendiri. c. Saling Belajar, Saling Berbagi Pengalaman Salah satu prinsip pendampingan untuk pemberdayaan masyarakat adalah pengakuan akan pengalaman dan pengetahuan lokal masyarakat. Salah satu prinsip pendampingan untuk pemberdayaan masyarakat adalah pengakuan akan pengalaman dan pengetahuan lokal masyarakat. Dengan demikian, kerangka dalam pemecahan masalah untuk khalayak sasaran berorientasi pada partisipasi masyarakat. Pelibatan masyarakat dalam pola pemberdayaan yang akan diterapkan diharapkan menjadi stimulus agar masyarakat mempunyai motivasi untuk terus maju dan berkembang, serta mencapai tingkat kesejahteraan yang diinginkan. Analisis kebutuhan dari mitra atau khalayak sasaran menjadi langkah awal untuk realisasi pemberdayaan dan dari pihak tim pengabdi sebagai pendamping atau fasilitator. Konsep partisipatif yang disisipkan menjadi landasan untuk sebuah kemajuan masyarakat. Menurut Hikam (dalam Karsidi, R., 2001), strategi penguatan civil society, sebaliknya ditujukan ke arah pembentukan gradual suatu masyarakat politik (political-society) yang demokratis- partisipatoris dan reflektif. Sehingga dalam upaya pemberdayaan diupayakan agar melibatkan secara penuh potensi aspiratif dalam masyarakat, sekaligus melakukan kritik secara terus menerus terhadap ketimpangan-ketimpangan yang terjadi. Hal ini akan menjadikan masyarakat menjadi lebih mempunyai peran dan diharapkan pemberdayaan dapat dilakukan. 2. Realisasi Pemecahan Masalah Realisasi pemecahan masalah dalam kegiatan pengabdian ini disajikan dalam tabel 1, sebagai b ik t Realisasi pemecahan masalah dalam kegiatan pengabdian ini disajikan dalam tabel 1, sebagai berikut: 68 \| MALLOMO: Journal of Community Service 68 \| MALLOMO: Journal of Community Service Tabel 1 Kerangka Pemecahan Masalah Kegiatan Indikator Kinerja Luaran Baseline Target Evaluasi awal untuk mengetahui potensi yang dimiliki mitra dalam membuat desain kemasan kopi Belum diketahuinya potensi petani kopi robusta dalam menciptakan sebuah kemasan yang efektif (10%) Diketahuinya potensi petani kopi robusta dalam menciptakan sebuah kemasan yang efektif (90%) Sosialisasi implementasi inovasi kemasan kopi yang kekinian Tingkat pemahaman mitra tentang inovasi kemasan kopi yang kekinian (5%) Tingkat pemahaman mitra tentang inovasi kemasan kopi yang kekinian (80%) Desain kemasan kopi Pelatihan kreativitas pembuatan desain kemasan kopi Tingkat kreativitas desain kemasan kopi (0%) Tingkat kreativitas pembuatan desain kemasan kopi (90%) Terciptanya desain kemasan kopi yang kekinian Sumber: Data Pengabdian, 2020. Tabel 1 Kerangka Pemecahan Masalah Sumber: Data Pengabdian, 2020. 3. Khalayak Sasaran Khalayak sasaran atau mitra dalam kegiatan ini, yakni para petani kopi robusta yang berada di wilayah Desa Sukamaju Kecamatan Cihaurbeuti Kabupaten Ciamis. Mitra tersebut dipilih berdasarkan keinginan yang sama untuk berwirausaha dan memiliki motivasi untuk meningkatkan taraf hidup. Adapun peta lokasinya disajikan ke dalam bentuk gambar, sebagai berikut: Gambar. 1 Peta Lokasi Pengabdian Sumber: Pemerintah Desa Sukamaju, 2019 Gambar. 1 Peta Lokasi Pengabdian Sumber: Pemerintah Desa Sukamaju, 2019 Gambar. 1 Peta Lokasi Pengabdian Sumber: Pemerintah Desa Sukamaju, 2019 Sumber: Pemerintah Desa Sukamaju, 2019 y g g Langkah-langkah yang ditempuh dalam kegiatan pengabdian ini yang dilaksanakan oleh tim pelaksana pengabdian kepada masyarakat, sebagai berikut: a. Evaluasi Awal Inovasi Kemasa Kopi Robusta … (Dini Yuliandini) \| Inovasi Kemasa Kopi Robusta … (Dini Yuliandini) Inovasi Kemasa Kopi Robusta … (Dini Yuliandini) Pada tahap ini dilakukan metode Focus Group Discussion (FGD) untuk mengetahui potensi ang dimiliki masyarakat petani kopi robusta, sehingga dapat ditentukan desain kemasan produk yang sesuai dengan perkembangan zaman. FGD tersebut melibatkan masyarakat sasaran, pemerintah desa dan pihak terkait lainnya. b. Sosialisasi Tahap sosialisasi dengan menggunakan metode ceramah untuk memberikan pengetahuan mendalam mengenai konsep atau desain kemasan yang kekinian. Tahap sosialisasi dengan menggunakan metode ceramah untuk memberikan pengetahuan mendalam mengenai konsep atau desain kemasan yang kekinian. Tahap sosialisasi dengan menggunakan metode ceramah untuk memberikan pengetahuan mendalam mengenai konsep atau desain kemasan yang kekinian. c. Membentuk Kelompok Tani Pada tahap ini dilakukan beberapa sub kegiatan diantaranya, sebagai berikut: 1) Melakukan evaluasi awal dengan menggunakan metode Waterfall, yaitu: a) Communication, pada tahap ini, proses yang dilakukan adalah melakukan pengumpula data tentang petani kopi robusta yang berada di Desa Sukamaju; b) Planning, proses ini merupakan lanjutan dari proses Communication (analisis kebutuhan). Pada proses planning (perencanaan) akan diperoleh dokumen berisikan data-data kebutuhan petani kopi yang ingin ditampilkan pada kemasan kopi robusta; dan c) Modelling, tahap ini merupakan tahap penerjemahan kebutuhan petani kopi kedalam perancangan terhadap desain kemasan yang akan direalisasikan. 2) Pembentukan kelompok. Pada tahap ini dilakukan pembentukan kelompok kepada masyarakat sasaran yang telah ditentukan pada saat evaluasi awal dengan melakukan pemilihan ketua, sekretaris, bendahara dan para anggotanya secara demokratis. d. Pelatihan Keterampilan Mitra Untuk meningkatkan keterampilan mitra, dilakukan dengan metode demonstrasi dan praktik, sebagai berikut: Untuk meningkatkan keterampilan mitra, dilakukan dengan metode demonstrasi dan praktik, sebagai berikut: 1) Demonstrasi yang dilakukan untuk memberikan contoh kepada peserta mengenai pembuatan desain kemasan. 2) Praktik tentang semua tahapan pembuatan kemasan. e. Pendampingan Usaha Tahap ini dilakukan dengan metode pendampingan, yakni pendampingan dalam usaha meningkatkan pemasaran dan penjualan petani kopi. f. Evaluasi Akhir Pada tahap ini dilakukan evaluasi untuk mengetahui keberlanjutan usaha dari petani kopi. f. Evaluasi Akhir Pada tahap ini dilakukan evaluasi untuk mengetahui keberlanjutan usaha dari petani kopi. P t Kh g. Pemantauan Khusus Pada tahap ini dilakukan untuk mengetahui tingkat motivasi mitra untuk terus berwirausaha dan meningkatkan jumlah penjualan barang atau produk yang ditawarkan kepada konsumen atau pelanggan. 4. Metode yang Digunakan 4. Metode yang Digunakan Langkah-langkah yang ditempuh dalam kegiatan pengabdian ini yang dilaksanakan oleh tim pelaksana pengabdian kepada masyarakat, sebagai berikut: E l i A l . e ode ya g gu a a Langkah-langkah yang ditempuh dalam kegiatan pengabdian ini yang dilaksanakan oleh tim pelaksana pengabdian kepada masyarakat, sebagai berikut: Langkah-langkah yang ditempuh dalam kegiatan peng pelaksana pengabdian kepada masyarakat, sebagai berikut: a. Evaluasi Awal 69 Hasil Dan Pembahasan Desa Sukamaju merupakan salah satu desa yang terletak di Kecamatan Cihaubeuti Kabupaten Ciamis yang dipimpin oleh seorang Kepala Desa yang bernama Dede Engkuh dengan masa jabatan dari tahun 2016 sampai dengan 2022 mempunyai beragam potensi yang dapat dimanfaatkan menjadi sumber ekonomi. Alamat kantor desa berada di Jalan Gunung Sawal No. 13 Dusun Sukamaju RT. 01 RW. 01 Kecamatan Cihaurbeuti Kabupaten Ciamis dan disana merupakan salah satu jantung geliat perekonomian masyarakat Desa Sukamaju. Potensi ekonomi perdesaan banyak ditemukan di desa tersebut. Adapun hasil observasi yang telah dilakukan, khususnya berkaitan dengan ekonomi perdesaan, diantaranya : (1) Usaha ekonomi mikro masyarakat Desa Sukamaju; dan (2) Potensi Wilayah Desa Berbasis Lahan Kering dan Pegunungan. Mata pencaharian masyarakat Desa Sukamaju 70 \| MALLOMO: Journal of Community Service 70 Inovasi Kemasa Kopi Robusta … (Dini Yuliandini) \| 71 Selanjutnya dilakukan pelatihan dan pendampingan pembuatan kemasan. Pelatihan dalam pembuatan desain kopi kepada mitra dilakukan melalui 3 (tiga) tahap, yaitu: (1) pengenalan; (2) pemrosesan (process); dan (3) Output. Pengenalan dilakukan dengan melakukan orientasi pada software yang digunakan. Perangkat lunak yang digunakan, yakni dengan menggunakan Corel Draw X7. Kemudian pemrosesan dilakukan dengan melakukan desain sederhana kemasan kopi. Adapun tampilan, software tersebut sebagai berikut: Gambar. 4 Tampilan Software Corel Draw X7 Sumber: Dok. Pribadi, 2020. Gambar. 4 Tampilan Software Corel Draw X7 Sumber: Dok. Pribadi, 2020. Pelatihan yang telah dilakukan, bersifat sederhana karena menggunakan perangkat lunak yang mudah digunakan. Hal ini bertujuan agar kreativitas dari mitra dapat dikembangkan lebih lanjut. Sudut pandang kebermanfaatan juga masih menjadi prioritas dalam pelaksanaan pelatihan tersebut. Antusiasme peserta pelatihan tampak ketika orientasi awal pengenalan software tersebut. Tahapan terakhir dalam proses pelatihan tersebut, mitra telah menguasai prinsip dasar dalam pembuatan desain kemasan. Dalam pelaksanaan kegiatan pengabdian ini, setelah melakukan inovasi kemasan dan mengadakan pelatihan pembuatan desain kemasan kopi, maka langkah terakhir yakni pendambingan usaha. Pendampingan usaha ini bertujuan untuk meningkatkan penjualan produk. Selain itu, proses pendampingan usaha berorientasi pada menyadarkan masyarakat tentang struktur dan strategi perubahan sosial. Dalam pendampingan usaha ini ada beberapa pihak yang terkait, yaitu masyarakat Desa Sukamaju, Badan Usaha Milik Desa (BUMDes) Maju Mandiri dan Pemerintah Desa Sukamaju. Sinergitas antara para stakeholder, serta kelompok petani kopi akan membantu dalam pendampingan usaha. Inovasi masih akan terus dilakukan sesuai dengan kebutuhan pasar akan komoditas kopi. Adapun proses pendampingan yang dilakukan, meliputi: (1) Pelatihan manajemen organisasi mitra; (2) Pendampingan pembuatan program; serta (3) Monitoring dan evaluasi kegiatan. Dari semua kegiatan tersebut diharapkan akan terbentuk perubahan sosial ekonomi masyarakat, khususnya masyarakat di perdesaan. 70 \| MALLOMO: Journal of Community Service sebagian besar menjadi petani. Dengan demikian, maka diperlukan pemberdayaan para petani di wilayah tersebut. Para petani di wilayah tersebut masih termasuk ke dalam masyarakat prasejahtera. Diperlukan bimbingan dan arahan agar masyarakat di desa tersebut menjadi lebih sejahtera. Kegiatan pengabdian ini dimulai dengan tahap sosialisasi. Sosialiasi merupakan langkah awal yang dilakukan dalam kegiatan pengabdian ini. Orientasi pada kegiatan ini dilakukan dengan tujuan agar mitra dapat mengetahui secara lugas berkaitan dengan tujuan dalam kegiatan pengabdian ini. Produk kopi yang ada di Desa Sukamaju Kecamatan Cihaurbeuti Kabupaten Ciamis telah dikemas dengan baik. Namun untuk desain kemasan masih kurang menarik. Berdasarkan hal tersebut, maka inovasi kemasan kopi sangat diperlukan guna menciptakan desain yang dapat diterima pasar. Selain itu, kearifan lokal berkaitan dengan kopi perlu dilestarikan. Hal ini yang akan menjadikan ciri khas dari produk unggulan dan tentunya akan menari dari konsumen nantinya. Inovasi sebagai bagian dari produk kreativitas akan bermanfaat jika diimplementasikan dengan baik. Adapun dokumentasi sosialiasi, sebagai berikut: Gambar.2 Sosialisasi terhadap Mitra Sumber: Dok. Pribadi, 2020 Gambar.2 Sosialisasi terhadap Mitra Sumber: Dok. Pribadi, 2020 Produk kopi yang telah ada, kemudian kemasan kopi tersebut dimodifikasi agar memenuhi beberapa ketentuan kemasan. Adapun kemasan yang dijadikan pembaharu untuk kemasan kopi tersebut, terdiri dari informasi: (1) Brand Coffee; (2) Single Origin dan Espresso Blend; (3) Region; (4) Varietal; (5) Level Sangrai; (6) Testing Notes atau Cupping Notes; (7) Ketinggian; (8) Berat Kopi; (9) Tanggal Sangrai; (10) Biji dan Tingkat Kehalusan. Adapun untuk bentuk dan kemasan yang telah dilakukan inovasi oleh tim pengabdi, sebagai berikut: Gambar. 3 Desain Kemasan Kopi Sumber: Dok. Pribadi, 2020. Gambar. 3 Desain Kemasan Kopi Sumber: Dok. Pribadi, 2020. 71 Inovasi Kemasa Kopi Robusta … (Dini Yuliandini) Simpulan Dan Saran Dalam kegiatan pengabdian kepada masyarakat dapat disimpulkan beberapa hal, sebagai berikut: berikut: 1. Inovasi kemasan kopi yang telah dilakukan mendapatkan respon yang positif bagi mitra dan bermanfaat untuk meningkatkan jumlah penjualan produk komoditas kopi. 1. Inovasi kemasan kopi yang telah dilakukan mendapatkan respon yang positif bagi mitra dan bermanfaat untuk meningkatkan jumlah penjualan produk komoditas kopi. 72 \| MALLOMO: Journal of Community Service 2. Inovasi kemasan kopi terdiri dari informasi Brand Coffee, Single Origin dan Espresso Blend, region, varietal, level sangrai, cupping notes, ketinggian, berat kopi, tanggal sangrai, biji, serta tingkat kehalusan. 3. Pelaksanaan pengabdian kepada masyarakat dilakukan melalui sosialisasi inovasi kemasan kopi, pelatihan desain kemasan dan pendampingan usaha. 3. Pelaksanaan pengabdian kepada masyarakat dilakukan melalui sosialisasi inovasi kemasan kopi, pelatihan desain kemasan dan pendampingan usaha. 4. Pendampingan usaha dengan mitra meliputi pelatihan manajemen organisasi mitra, pendampingan pembuatan program, serta monitoring dan evaluasi kegiatan. Adapun saran ataupun rekomendasi setelah dilakukan kegiatan pengabdian kepada masyarakat ini, sebagai berikut: 1. Pemerintah Desa harus meningkatkan aksesibilitas internet, sehingga akses internet dapat dengan mudah didapatkan oleh masyarakat di perdesaan. 2. Pemerintah Daerah Kabupaten/Kota harus mendukung Usaha Mikro Kecil dan Menengah (UMKM) yang ada di perdesaan. 3. Peningkatan kualitas Sumber Daya Manusia (SDM) perlu ditingkatkan melalui sosialisasi dan edukasi pemanfaatan, serta inovasi teknologi informasi oleh semua pihak. 4. Inovasi kemasan produk komoditas kopi harus disesuaikan dengan kebutuhan pasar. Daftar Rujukan Badan Pusat Statistik. (2019). Jumlah Penduduk Miskin Menurut Kabupaten/Kota di Jawa Barat, 2019. (Online) Tersedia: https://jabar.bps.go.id/dynamictable/2020/01/29/190/jumlah-penduduk- miskin-menurut-kabupaten-kota-di-jawa-barat-ribu-orang-2019.html [05 Januari 2020]. Badan Pusat Statistik. (2019). Jumlah Penduduk Miskin Menurut Kabupaten/Kota di Jawa Barat, 2019. (Online) Tersedia: https://jabar.bps.go.id/dynamictable/2020/01/29/190/jumlah-penduduk- miskin-menurut-kabupaten-kota-di-jawa-barat-ribu-orang-2019.html [05 Januari 2020]. Badan Pusat Statistik. (2019). Jumlah Penduduk Miskin Menurut Kabupaten/Kota di Jawa Barat, 2019. (Online) Tersedia: https://jabar.bps.go.id/dynamictable/2020/01/29/190/jumlah-penduduk- miskin-menurut-kabupaten-kota-di-jawa-barat-ribu-orang-2019.html [05 Januari 2020]. Fajarini, U. (2014). Peranan kearifan lokal dalam pendidikan karakter. SOSIO-DIDAKTIKA: Social Science Education Journal, 1(2), 123-130. Fajarini, U. (2014). Peranan kearifan lokal dalam pendidikan karakter. SOSIO-DIDAKTIKA: Social Science Education Journal, 1(2), 123-130. Noviadji, B. R. (2014). Desain Kemasan Tradisional Dalam Konteks Kekinian. Artika, 1(1), 10-21 Karsidi, R. (2001). Paradigma Baru Penyuluhan Pembangunan dalam Pemberdayaan Masyarakat. Mediator: Jurnal Komunikasi, 2(1), 115-125. Karsidi, R. (2007). Pemberdayaan masyarakat untuk usaha kecil dan mikro (Pengalaman Empiris di Wilayah Surakarta Jawa tengah). Jurnal Penyuluhan, 3(2). Kurniawati, D. P. (2013). Pemberdayaan masyarakat di bidang usaha ekonomi (studi pada Badan Pemberdayaan Masyarakat kota Mojokerto). Jurnal Administrasi Publik, 1(4), 9-14. Noviadji, B.R (2014). Desain Kemasan Tradisional dalam Konteks Kekinian. Artika, 1(1), 10-21. Sukmadi. (2016). Inovasi dan Kewirausahaan (Edisi Paradigma Baru Kewirausahaan). Bandung: Humaniora Utama Press. Suwarno, Y. (2008). Inovasi di sektor publik. STIA-LAN, Jakarta. Winarno dan Herimanto (2014). Ilmu Sosial dan Budaya Dasar. Bandung: Bumi.
https://openalex.org/W3154951122	https://www.mdpi.com/1422-0067/22/8/4013/pdf?version=1618318186	English	null	In Vitro and Vivo Identification, Metabolism and Action of Xenoestrogens: An Overview	International journal of molecular sciences	2,021	cc-by	20,642	Review In Vitro and Vivo Identiﬁcation, Metabolism and Action of Xenoestrogens: An Overview Sci. 2021, 22, 4013. https:// doi.org/10.3390/ijms22084013 Academic Editor: Barbora Szotáková Received: 14 March 2021 Accepted: 12 April 2021 Published: 13 April 2021 Citation: Wang, L.-H.; Chen, L.-R.; Chen, K.-H. In Vitro and Vivo Identiﬁcation, Metabolism and Action of Xenoestrogens: An Overview. Int. J. Mol. Sci. 2021, 22, 4013. https:// doi.org/10.3390/ijms22084013 Keywords: xenoestrogen; bisphenol; polychlorinated biphenyls; phytoestrogen Received: 14 March 2021 Accepted: 12 April 2021 Published: 13 April 2021 Received: 14 March 2021 Accepted: 12 April 2021 Published: 13 April 2021 International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences Review In Vitro and Vivo Identiﬁcation, Metabolism and Action of Xenoestrogens: An Overview Li-Hsuan Wang 1, Li-Ru Chen 2,3 and Kuo-Hu Chen 1,4,* 1 Department of Obstetrics and Gynecology, Taipei Tzu-Chi Hospital, The Buddhist Tzu-Chi Medical Foundation, Taipei 231, Taiwan; 100311023@gms.tcu.edu.tw 2 Department of Physical Medicine and Rehabilitation, Mackay Memorial Hospital, Taipei 10049, Taiwan; gracealex168@gmail.com 3 Department of Mechanical Engineering, National Yang-Ming Chiao-Tung University, Hsinchu 30010, Taiwan 4 School of Medicine, Tzu-Chi University, Hualien 970, Taiwan * Correspondence: alexgfctw@yahoo.com.tw; Tel.: +886-2-6628-9779 Department of Obstetrics and Gynecology, Taipei Tzu-Chi Hospital, The Buddhist Tzu-Chi Medical Foundation, Taipei 231, Taiwan; 100311023@gms.tcu.edu.tw 2 Department of Physical Medicine and Rehabilitation, Mackay Memorial Hospital, Taipei 10049, Taiwan; gracealex168@gmail.com 3 Department of Mechanical Engineering, National Yang-Ming Chiao-Tung University, Hsinchu 30010, Taiwan 4 School of Medicine, Tzu-Chi University, Hualien 970, Taiwan * Correspondence: alexgfctw@yahoo.com.tw; Tel.: +886-2-6628-9779 * Correspondence: alexgfctw@yahoo.com.tw; Tel.: +886-2-6628-9779 Abstract: Xenoestrogens (XEs) are substances that imitate endogenous estrogens to affect the physio- logic functions of humans or other animals. As endocrine disruptors, they can be either synthetic or natural chemical compounds derived from diet, pesticides, cosmetics, plastics, plants, industrial byproducts, metals, and medications. By mimicking the chemical structure that is naturally occurring estrogen compounds, synthetic XEs, such as polychlorinated biphenyls (PCBs), bisphenol A (BPA), and diethylstilbestrol (DES), are considered the focus of a group of exogenous chemical. On the other hand, nature phytoestrogens in soybeans can also serve as XEs to exert estrogenic activities. In contrast, some XEs are not similar to estrogens in structure and can affect the physiologic functions in ways other than ER-ERE ligand routes. Studies have conﬁrmed that even the weakly active compounds could interfere with the hormonal balance with persistency or high concentrations of XEs, thus possibly being associated with the occurrence of the reproductive tract or neuroendocrine disorders and congenital malformations. However, XEs are most likely to exert tissue-speciﬁc and non-genomic actions when estrogen concentrations are relatively low. Current research has reported that there is not only one factor affected by XEs, but opposite directions are also found on several occasions, or even different components stem from the identical endocrine pathway; thus, it is more challenging and unpredictable of the physical health. This review provides a summary of the identiﬁcation, detection, metabolism, and action of XEs. However, many details of the underlying mechanisms remain unknown and warrant further investigation. Citation: Wang, L.-H.; Chen, L.-R.; Chen, K.-H. In Vitro and Vivo Identiﬁcation, Metabolism and Action of Xenoestrogens: An Overview. Int. J. Mol. 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. As endocrine disruptors, xenoestrogens (XEs) are substances that imitate endogenous estrogens to affect the physiologic functions of humans or other animals. In the 1950s, some research demonstrated the hormone-like effects, mostly associated with environmen- tal chemicals in wildlife. Either estrogenic xenobiotics or XEs are considered the major disruptors of endocrine, with a diverse chemical structure [1]. Exposure to not only nature, which phytoestrogens and mycoestrogens are the pertinent examples, but also industrial chemicals, estrogenic, and anti-estrogenic activity have been exhibited [2]. Through the imitations and obstacles in responses via the non-genomic and/or genomic signaling pathway, endogenous estrogens are largely disrupted by XEs [3]. Individuals are exposed to a complex mix of chemicals during their lifetime. XEs, which can be found in every aspect ranging from the environment, food, cosmetics, and other substances, have pros and cons to the human body [4,5]. By mimicking the chemical structure that is naturally occurring estrogen compounds, some XEs are considered the focus of a group of exogenous chemical, which can be derived from the sources as follows: diet, pesticides, cosmetics, Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 4013. https://doi.org/10.3390/ijms22084013 Int. J. Mol. Sci. 2021, 22, 4013 nt. J. Mol. Sci. 2021, 22, x FOR PE 2 of 27 o some plastics, plants, fumes, industrial byproducts, metals and medications (such as oral contraceptives) [4,6–8]. The importance of new chemicals’ endocrine-disrupting potential cannot be ignored [1]. y g g p g p f exogenous chemical, which can be derived from the sources as follows: diet, pesticides, osmetics, some plastics, plants, fumes, industrial byproducts, metals and medications such as oral contraceptives) [4,6–8]. The importance of new chemicals’ endocrine-disrupt- The estrogenic or antiestrogenic activity of chemicals is attributed to the interactions between estrogen receptor (ER) and other compounds. In fact, ER, as a ligand-inducible transcription factor, plays a vital role in development and neoplasia using regulating genes involved in cell proliferation and differentiation. p ) [ , ] p p ng potential cannot be ignored [1]. 1. Introduction The estrogenic or antiestrogenic activity of chemicals is attributed to the interactions etween estrogen receptor (ER) and other compounds. In fact, ER, as a ligand-inducible ranscription factor, plays a vital role in development and neoplasia using regulating Muellery has clearly deﬁned XEs in the review article, which described the mecha- nisms of action and detection methods, stressing the points of molecular cell biological mechanism to estrogen receptor-mediated hormone actions. Figure 1 has displays how the estrogen or XEs work in cells through several consecutive steps [9–11]. p , p y p p g g g enes involved in cell proliferation and differentiation. Muellery has clearly defined XEs in the review article, which described the mecha- nisms of action and detection methods, stressing the points of molecular cell biological mechanism to estrogen receptor-mediated hormone actions. Figure 1 has displays how (1.) Estradiol, a type of estrogen (E), is trapped over by carrier protein in the serum. After the estrogen is released from the blood by the carrier protein, it can pass through the cell membrane without disturbance and enter the cell; g p g p y he estrogen or XEs work in cells through several consecutive steps [9–11]. 1.) Estradiol, a type of estrogen (E), is trapped over by carrier protein in the serum. After the es- trogen is released from the blood by the carrier protein, it can pass through the cell membrane (2.) Located within the nucleus, before activation, the ERs will be bound to various receptor-associated proteins, such as heat-shock proteins (Hsp90); without disturbance and enter the cell; 2.) Located within the nucleus, before activation, the ERs will be bound to various receptor-asso- i t d t i h h t h k t i (H 90) (3.) Estrogen will be bound to the ERs, replacing the receptor-associated proteins; ciated proteins, such as heat-shock proteins (Hsp90); 3 ) Estrogen will be bound to the ERs replacing the receptor associated proteins; (4.) ER dimer is bound to its corresponding DNA-binding domains, and the sequence is named estrogen-responsive element (ERE); 3.) Estrogen will be bound to the ERs, replacing the receptor-associated proteins; 4.) ER dimer is bound to its corresponding DNA-binding domains, and the sequence is named estrogen-responsive element (ERE); (5.) Under the cooperation of assembled substances, including multiple transcription factors (TF), the RNA polymerase (RNA Pol) and other proteins, transcription can be started. 1. Introduction The relevant mRNA sequence emerges as ER target genes transcription is in the process; estrogen-responsive element (ERE); 5.) Under the cooperation of assembled substances, including multiple transcription factors (TF), the RNA polymerase (RNA Pol) and other proteins, transcription can be started. The relevant mRNA sequence emerges as ER target genes transcription is in the process; 6.) Co-activators, including CBP/p300 and SRC-1, are all the linking brackets to ER dimer and p (6.) Co-activators, including CBP/p300 and SRC-1, are all the linking brackets to ER dimer and could play an effect on DNA transcription; ) , g p , g could play an effect on DNA transcription; 7.) After DNA is transcribed into mRNA, RNA should be translated to produce protein to com- p y p (7.) After DNA is transcribed into mRNA, RNA should be translated to produce protein to complete gene expression [10]. ) p p plete gene expression [10]. Figure 1. The molecular mechanisms for the actions of estrogen receptors [9–11]. Like the ligand actions of endogenous hormones, the ER α and β shall be bound and activated before XEs bring their effects fully into play. There is not only one single factor affected by XEs, but opposite directions are also found on several occasions, or even dif- ferent components stem from the identical endocrine pathway; thus, it is more challenging Figure 1. The molecular mechanisms for the actions of estrogen receptors [9–11]. Like the ligand actions of endogenous hormones, the ER α and β shall be bound and activated before XEs bring their effects fully into play. There is not only one single factor affected by XEs, but opposite directions are also found on several occasions, or even different components stem from the identical endocrine pathway; thus, it is more challenging and unpredictable in terms of physical health. Interestingly, XEs are most likely to exert tissue-speciﬁc and non-genomic actions when the concentrations are relatively igure 1 The molecular mechanisms for the actions of estrogen receptors [9 11] Figure 1. The molecular mechanisms for the actions of estrogen receptors [9–11]. u e 1 The ole ula e ha i fo the a tio of e t o e e e to [9 11] Figure 1. The molecular mechanisms for the actions of estrogen receptors [9–11]. igure 1. The molecular mechanisms for the actions of estrogen receptors [9 11]. 1. Introduction The daily sperm production and the number of sperm in the epididymis were reduced. This research conﬁrmed that exposure to BPA and its analogs for a chronic duration could induce struc- tural and functional changes in testicular tissue and endocrine alterations in the male rat reproductive system [14]. To investigate the effects of XEs on skeletal programming, Pelch et al. have compared the skeletal effects of low-dose BPA exposure to mice 9 days prenatal and 12 days postnatal in 2012 [15]. The skeletal health of these mice was assessed during adulthood when they had reached peak bone mass. The study revealed that exposure to 10 µg/kg/day BPA signiﬁcantly increased the femoral length in the male mice but decreased the biomechanical strength in the female mice [15]. g Research across the 1960s to 1970s demonstrated the estrogenicity of a couple of in- dustrial compounds and pesticides, o,p-DDT, kepone, methoxychlor, phenolic derivatives, and polychlorinated biphenyls (PCBs). Several environmental chemicals have been cate- gorized into the list of XEs, including the pesticide toxaphene, dieldrin, and endosulfan, and some different compounds used in the food industry, which are antioxidants, such as t-butylhydroxyanisole. The BBP and 4-OH-alkylphenols are components of plasticizers. The substance BPA was applied in dental restorations [1,5]. pp In Korach’s studies, it is indicated that even weakly active compounds could interfere with the hormonal balance with persistency or high concentrations of XEs. Congenital malformation of wildlife and humans born with birth defects are negatively affected by endocrine-disrupting chemicals XEs. The development of the urinary tract and ner- vous system is particularly sensitive to hormonal disruption over periods of in utero and early postnatal life [5,16]. On top of that, a birth defect can be considered perma- nent damage, whereas the structural changes of the body parts are less affected after reaching adulthood [12,17]. During the 1990s, male reproductive problems ranging from the decline in semen quality, testicular cancer, hypospadias, and cryptorchidism seem to be increasing health issues found in Belgium, Denmark, France, and Great Britain. In 1996, studies suggested that the supernormal levels of estrogens, speciﬁcally diethylstilbestrol (DES), were the leading cause of male fetus reproductive defects. Regarding common XEs stemming from environmental contaminants and chemicals, their adverse effects on male reproductive health are exceptionally crucial. The stages of fetus and childhood are more vulnerable than that of mature adulthood. 1. Introduction Like the ligand actions of endogenous hormones, the ER α and β shall be bound and ctivated before XEs bring their effects fully into play. There is not only one single factor ffected by XEs, but opposite directions are also found on several occasions, or even dif- erent components stem from the identical endocrine pathway; thus, it is more challenging Like the ligand actions of endogenous hormones, the ER α and β shall be bound and activated before XEs bring their effects fully into play. There is not only one single factor affected by XEs, but opposite directions are also found on several occasions, or even different components stem from the identical endocrine pathway; thus, it is more challenging and unpredictable in terms of physical health. Interestingly, XEs are most likely to exert tissue-speciﬁc and non-genomic actions when the concentrations are relatively Int. J. Mol. Sci. 2021, 22, 4013 3 of 27 3 of 27 low. The debate on the risks that humans are exposed to remains a controversy, and a clear-cut relationship between XEs exposure and human health so far has only a little evidence. However, due to the complexity of their mechanisms and potential for adverse effects, how XEs affect normal estrogen signaling remains an open question and is worth investigating [12]. g g The estrogenic environmental compounds, including bisphenol A (BPA) and butyl benzyl phthalate (BBP), may trigger the adverse effects of endocrine-disrupting chemicals on organisms. As the natural estrogen, 17β-estradiol is the major factor in forming breast cancer and further to the progression. An in vitro- in vivo model has been developed to demonstrate the carcinogenicity of natural estrogen 17β-estradiol and xenoestrogenic substances in human breast epithelial cells MCF-10F. In this model, hypermethylation of NRG1, STXBP6, BMP6, SS3, SPRY1, and SNIP were found at different progression phases. Whether BPA and BBP are relevant to breast cancer initiation can be demonstrated using this unique model. The evidence mentioned earlier suggests that natural estrogen 17β-estradiol and xenoestrogenic substances, such as BPA, are considered to trigger a neoplastic transformation in human breast epithelial cells [13]. p p Ullah et al. have conducted an in vivo study and investigated whether chronic ex- posure to low doses of BPA and its analogs affects spermatogenesis with outcomes on oxidative stress and the male reproductive system of 22-day-old rats [14]. Oxidative stress in the testis was signiﬁcantly elevated, while sperm motility was impaired. 1. Introduction An extensive study should explore the underlying problem further and strategically determine the intervention and potential treatment [18]. Int. J. Mol. Sci. 2021, 22, 4013 4 of 27 Estrogens can act on signaling pathways with pertinent examples in diseases, such as cancer, cardiovascular, metabolic, or immune system disorders. Many XEs gathered by natural and synthetic compounds can behave as estrogens and have a close interaction with ERs. These compounds are capable of bonding to the ER in the cell, leading to bioaccumulation as a whole [3,19–21]. In contrast, some XEs are not similar to estrogens in structure and can affect the physiologic functions in ways other than ER-ERE ligand routes. In fact, the high prevalence of XEs has brought tremendous interest in in-depth research concerning hormone-dependent cancers, including breast, ovarian, endometrial, prostate, thyroid, and cervical. y Between 1940 and 1971, nearly 2 to 4.8 million human offspring were exposed to DES, a strong synthetic estrogen. Research has concluded that prenatal women exposed to DES were subject to the growth of adenocarcinomas of the cervix and reproductive disorders [22–24]. Interestingly, DES has been applied in therapeutic hormone replacement for women, extensively using prostate and breast cancer treatment through having cancer- induced factors. On the other hand, DES also functions well to suppress the androgen action and induce apoptosis toward both androgen-dependent tumors and independent by interfering with the cell cycle. To sum up, XEs have their pros and cons, contributing differently to human beings and creatures [22,25,26]. Since XEs can act as endocrine disruptors to affect functions of humans and animals, how they can be detected and how they can act in the endocrine system are important to further studies. This overview provides a summary of the identiﬁcation, detection, metabolism, and action of XEs. 2. Identiﬁcation and Detection of Xenoestrogens The literature revealed that XEs, as active endocrine substances, can interact with estrogen receptors, androgen receptors, or peroxisome proliferative receptors [12]. By inhibiting or activating these nuclear receptors, XEs can affect circulating hormone levels and disrupt normal endocrine function, thus possibly resulting in metabolic syndrome, reproductive dysfunction, and the occurrence of cancers [3,4,27–29]. The exposed concen- tration, dose, and time of XEs, as well as the age of contact persons, have different impacts on affected individuals [30]. In the past, there were various in vivo and in vitro assays developed to measure the estrogenic-like activity of XEs, as well as the concentration and potency of the estrogenic and antiestrogenic compounds. For a better understanding of morphological, histologi- cal, biochemical, and molecular actions, researchers have completed different studies to analyze the estrogenic properties of XEs. Although the in vivo real actions of XEs are unpredictable, many in vitro tests have been continually used to explore the estrogenic potency of XEs [9,31–34]. p y Studies regarding the identiﬁcation and detection of XEs, and investigating the molec- ular mechanisms of interactions between XEs and ERs were solicited from the literature. They described in detail the characteristics of the estrogenic or antiestrogenic potency of different XEs. A summary of the results of these studies is shown in Table 1. Mueller et al. [9] reported a review article in 2004, focusing on several in vitro tests for screening estrogenicity and antiestrogenicity of XEs. These assays were all powerful tools elucidating and describing the effects and mechanisms for XEs actions. While they had discussed in the essay, some mechanism-based assays are inappropriate to high-throughput screening for potent estrogenic and antiestrogenic XEs. Instead, other research [9,35], including high-throughput screening methods published recently, was presented. In vitro assays for identifying and detecting estrogenicity and antiestrogenicity toward different XEs were all summarized in Table 1. 5 of 27 Int. J. Mol. Sci. 2021, 22, 4013 Table 1. A summary of in vitro assays for the measurement of estrogenic and antiestrogenic compounds, partially according to Ref [35]. 2. Identiﬁcation and Detection of Xenoestrogens In Vitro Assay Endpoint of Measurement Advantages Limitations Reference E-Screen ERα (+) cell proliferation Measures physiological endpoint of estrogen action, measures estrogens and antiestrogens No deﬁned ER expression, no mechanistic data [33] Ligand-binding (EDSTAC) a ERα- or ERβ-binding afﬁnity Simple, high-throughput method Does not measure ER activation does not measure physiological response [36,37] ER-binding to ERE ERα- or ERβ-binding afﬁnity to ERE High-throughput method, various EREs can be used Does not measure ER activation, low sensitivity, does not measure physiological response [38,39] GST pull-down/FRET/ two-hybrid assay ERα- or ERβ-ligand-dependent association with coactivators Analysis of molecular interaction, deﬁned ER subtype or ER domain as well as coactivators can be used, measures estrogens and antiestrogens Does not measure direct ER activation, low throughput, does not measure physiological response [40–42] Analysis of gene expression ER-regulated gene expression Analysis of physiological response, versatile, measures estrogens and antiestrogens Low throughput [43,44] Analysis of enzyme activity ER-regulated enzyme activity Analysis of physiological response measures estrogens and antiestrogens Cell lines or primary cell cultures with active marker enzymes suitable only [45,46] Analysis of steroidogenesis (EDSTAC)a Induction and inhibition of estrogen biosynthesis Analysis of physiological response measures ER-independent pathways Cells with active steroidogenesis suitable only [47–51] The ﬂuorescence-based multianalyte chip platform(a ﬂuorescence-based multiplexed protein microarray) The proliferative effect in hormone-sensitive cancer cell line MCF-7 was measured with a resazurin assay. Quantiﬁcation of 10 proteins from MCF-7 cells, representing endpoints of estrogen-and antiestrogen action High throughput screening. Multiparameter panels, fast and highly speciﬁc diagnosis binding afﬁnities, different concentrations and different periods will be necessary to reﬁne the speciﬁc secretion patterns [52–55] Transcriptional activation assays Luciferase activity Rapid screening, identiﬁcation, and characterization of EDCs, the ﬁrst human ovarian cell bioassay of this kind for detecting estrogens Response observed in cell lines does not necessarily reﬂect the toxic or biological potential of a compound in vivo. [56] Triple functional small-molecule–protein conjugate mediated optical biosensor Dye-labeled estradiol (E2) -streptavidin conjugate (estrogenic activity) Easy-to-use and efﬁcient with the high reusable capability Expensive [57] Rapid screening, identiﬁcation, and characterization of EDCs, the ﬁrst human ovarian cell bioassay of this kind for detecting estrogens Transcriptional activation assays Transcriptional activation assays Luciferase activity Triple functional small-molecule–protein conjugate mediated optical biosensor Dye-labeled estradiol (E2) -streptavidin conjugate (estrogenic activity) 6 of 27 Int. J. Mol. Sci. 2021, 22, 4013 Table 1. Cont. 2.2. Ligand-Binding In 1998, Randall Bolger et al. reported a novel technique for detecting XEs named the ﬂuorescence polarization (FP) method. Under room air, the method could measure the capacity of XEs to displace a high-afﬁnity ﬂuorescent ligand from puriﬁed, recombinant hu- man ERα. The ERs in mice have two subtypes, including ERα and ERβ, which are different in the structures of the C-terminal ligand-binding domain and N-terminal transactivation domain. In the mouse model, Kuiper et al. observed ERα- and ERβ-mediated messenger RNA expression via ligand binding and compared the speciﬁcity and afﬁnity of ligand binding to ERα and ERβ via RT–PCR. They also found differences in the distribution and expression of ERα and ERβ in tissues and organs of mice. There were more distribution and expression of ERα in the uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal, while there were more distribution and expression of ERβ in the prostate, lung, bladder, and brain [36,37]. 2. Identiﬁcation and Detection of Xenoestrogens In Vitro Assay Endpoint of Measurement Advantages Limitations Reference Estrogen receptor recombinant yeastscreening (YES)assay Estrogenic activity The simplicity The product of the reporter gene is secreted in the medium, and no cell lysis is required. The presence of yeast cell wall and active transport mechanisms that may differ from those found in mammalian cells and may affect the activity of some test compounds [58] a Recommended for the screening of xenoestrogens by EDSTAC/OECD [46,47] ER estrogen receptor; ERE estrogen-responsive element; GST glutathione-S-transferase; FRET ﬂuorescence resonance energy transfer. Table 1. Cont. The simplicity The product of the reporter gene is secreted in the medium, and no cell lysis is required. Estrogen receptor recombinant yeastscreening (YES)assay a Recommended for the screening of xenoestrogens by EDSTAC/OECD [46,47] ER estrogen receptor; ERE estrogen-responsive element; GST glutathione-S-transferase; FRET ﬂuorescence resonance energy transfer. a Recommended for the screening of xenoestrogens by EDSTAC/OECD [46,47] ER estrogen receptor; ERE estrogen-responsive element; GST glutathione-S-transferase; FRET ﬂuorescence resonance energy transfer. 2.1. E-SCREEN Soto et al. [33] used the E-SCREEN assay to investigate the effects of XEs and 17β-estradiol on cells. Employing human MCF-7 (breast cancer cells) as a target in the assay, XEs were observed competing with E2 for binding to the ER and increased the levels of progesterone receptor (PR) and pS2 in MCF-7 cells. The E-SCREEN assay results have conﬁrmed the estrogenic materials, including alkylphenols, phthalates, PCB congeners, and hydroxylated PCBs. The insecticides dieldrin, endosulfan, and toxaphene are also detected in the assay. 2.3. ER-Binding to ERE Nikov et al. observed differences in afﬁnity of phytoestrogens when combined with ERα or ERβ, respectively. They also mentioned the combinations and interactions of ligand– ER complexes and estrogen response element (ERE) sequences. In their experiments, the FP methods were employed to measure binding afﬁnities of various phytoestrogens, such as genistein, coumestrol, daidzein, glyceollin, and zearalenone to human cells. This article revealed a higher afﬁnity of the phytoestrogens for ERβ than for ERα. The effect of these phytoestrogens on the ability of ERα and ERβ to associate with speciﬁc DNA sequences (EREs) was also investigated. In 2000, Boyer et al. performed a similar method on human ERα for probe of its molecular way on functional speciﬁcity. The implemented binding assays to study the interaction of the receptor with a palindromic estrogen response element derived from the vitellogenin ERE [38,39]. 2.4. Mammalian Two-Hybrid Assays DES is a well-known carcinogen [37]. As a mixture of indenestrol A (IA) S and R enantiomers [39], IA is a metabolic derivative of DES and has a high afﬁnity to bind ER but a weaker biological activity [38]. Mueller et al. found that the estrogenic properties of the S and R enantiomers of IA, IA-S, and IA-R, respectively, had different afﬁnities Int. J. Mol. Sci. 2021, 22, 4013 7 of 27 with ERα and ERβ in cells. Using human endometrial (Ishikawa) and breast MDA cell lines, which stably express either ERα or ERβ, IA-S was found more potent to activate cell transcription through ERα compared to IA-R. However, IA-R had more potency to stimulate ERβ rather than ERα in MDA cells, but this was not the case in endometrial cells. Although IA-R could effectively activate ERβ in vivo, it had a low afﬁnity to bind both ERα and ERβ in vitro. These results showed that IA-R was cell-selective when bound to ERβ. In addition, there existed a single residue within the ligand-binding domains to determine the stereoselectivity of both ERα and ERβ [40–42]. 2.6. Analysis of Enzyme Activity Holinka et al. [46] described alkaline phosphatase as a convenient endpoint to examine mechanisms of hormonal actions. Alkaline phosphatase activity in human endometrial cancer cells of the estrogen-responsive Ishikawa line was markedly stimulated by estrogens, 5a-dihydrotestosterone (DHT), and dehydroepiandrosterone (DHEA). In the previous re- port, E2 and several other estrogens were noticed to greatly enhance the activity of alkaline phosphatase, an enzyme known to be regulated by ovarian hormones in the nonpregnant and pregnant rodent and monkey uterus. The results suggested that estrogen and androgen receptors mediate the stimulation of alkaline phosphatase by the C19 steroids [45,46]. 2.7. Analysis of Steroido-Genesis In H295R cells, various pesticides that had been suspected of interfering with the functions of steroid hormones were examined for their effects on the mRNA expression and catalytic activity of aromatase. Sanderson et al. focused mainly on interactions of XEs with sex hormone receptors, particularly the estrogen receptor. After a period of time, other mechanisms of interference with endocrine functions have gained attention, including the effects of pesticides on the enzymes that are involved in the synthesis and metabolism of steroid hormones. As a key role in producing many endogenous steroid hormones of high potency, the enzymes cytochrome P450 (CYP) that are responsible for the reactions in the biosynthetic pathways of steroids is a research focus [47]. H295R cell line was used to screen several pesticides known or suspected to interfere with steroid hormone function for potential effects on the catalytic activity and mRNA expression of aromatase [47,48]. 2.5. Analysis of Gene Expression Jorgensen et al. demonstrated that estrogenic activity could be evaluated by assaying induction or repression of endogenous estrogen-regulated “marker genes” in human breast cancer MCF-7 cells. The authors mentioned that a cell-based endogenous gene expression assay is very sensitive to what could be used to assay the estrogenicity of different putative estrogenic chemicals. They performed an assay in human MCF-7 that was estrogen-dependent and used a method of polymerase chain reaction (PCR) to observe the changes in gene expression. After differential display using reverse-transcribed (DDRT) PCR technology, the levels of expression could be quantiﬁed by phosphor-imaging [43,44]. 2.8. The Fluorescence-Based Multi-Analyte Chip Platform Using a parallel-proliferation measurement, the ﬂuorescence-based multiplexed pro- tein microarray (Figure 2) presents the bidirectional estrogenic and anti-estrogenic actions of XEs in human MCF-7 cells [52–55]. As a new in vitro tool for screening environmental samples, the ﬂuorescence-based chip platform can analyze multiple targets to detect the estrogenic and anti-estrogenic actions of XEs by observing the expression of proteins. It can quantify 10 proteins secreted by MCF-7 cells, which represent different physiological and pathological endpoints of endocrine actions. Its potential is demonstrated by distinct protein secretion patterns of the cancer cell line after exposure to known ER agonists ß-estradiol, BPA, genistein, and nonylphenol as well as antagonists fulvestrant and tamox- ifen. In parallel, the proliferating effect of endocrine-disrupting substances in MCF-7 cells can be assessed in a proliferation assay based on resazurin. Unlike the detection tools Int. J. Mol. Sci. 2021, 22, 4013 8 of 27 8 of 27 developed earlier, the chip has two advantages, including high-output screening for the endocrine effects of environmental disruptors on human cells and providing messages of the complicated cellular effects on a molecular level. Compared with single marker detection, this multiplexed protein microarray in the chip is more convenient and arrives at better accuracy [52]. REVIEW 8 of 27 developed earlier, the chip has two advantages, including high-output screening for the endocrine effects of environmental disruptors on human cells and providing messages of the complicated cellular effects on a molecular level. Compared with single marker detection, this multiplexed protein microarray in the chip is more convenient and arrives at better accuracy [52]. REVIEW 8 of 27 Figure 2. The fluorescence-based multianalyte chip platform. This equipment contains MCF-7 cells, which are exposed to estrogen receptor agonists and antagonists. Using a resazurin assay, the proliferative effect in hormone-sensitive cancer cell line MCF-7 can be measured. Using a mul- tiplexed protein microarray with fluorescence detection, biomarkers can be quantified in the su- pernatant. By employing immobilized antibodies, the on-chip sandwich immunoassay captures the biomarker and a fluorescently labeled detection antibody for confirmation [52]. Figure 2. The ﬂuorescence-based multianalyte chip platform. This equipment contains MCF-7 cells, which are exposed to estrogen receptor agonists and antagonists. Using a resazurin assay, the proliferative effect in hormone-sensitive cancer cell line MCF-7 can be measured. Using a multiplexed protein microarray with ﬂuorescence detection, biomarkers can be quantiﬁed in the supernatant. 2.8. The Fluorescence-Based Multi-Analyte Chip Platform By employing immobilized antibodies, the on-chip sandwich immunoassay captures the biomarker and a ﬂuorescently labeled detection antibody for conﬁrmation [52]. Figure 2. The fluorescence-based multianalyte chip platform. This equipment contains MCF-7 ells, which are exposed to estrogen receptor agonists and antagonists. Using a resazurin assay, he proliferative effect in hormone-sensitive cancer cell line MCF-7 can be measured. Using a mul- iplexed protein microarray with fluorescence detection, biomarkers can be quantified in the su- pernatant. By employing immobilized antibodies, the on-chip sandwich immunoassay captures he biomarker and a fluorescently labeled detection antibody for confirmation [52] Figure 2. The ﬂuorescence-based multianalyte chip platform. This equipment contains MCF-7 cells, which are exposed to estrogen receptor agonists and antagonists. Using a resazurin assay, the proliferative effect in hormone-sensitive cancer cell line MCF-7 can be measured. Using a multiplexed protein microarray with ﬂuorescence detection, biomarkers can be quantiﬁed in the supernatant. By employing immobilized antibodies, the on-chip sandwich immunoassay captures the biomarker and a ﬂuorescently labeled detection antibody for conﬁrmation [52]. y 9 T i ti l A ti ti A 2.9. Transcriptional Activation Assays 2.9. Transcriptional Activation Assays A plasmid vector contains firefly luciferase genes that are controlled by DNA en- hancers, which can react with androgens, estrogens or retinoic acids. The transfection of a plasmid vector into corresponding receptor-containing cells reflects its ability to respond o respective hormones under luciferase induction. An estrogen-responsive luciferase re- porter plasmid has steadily transfected the recombinant ovarian carcinoma (BG-1) lines n humans. The subsequent recombination of cell lines (BG1Luc4E(2)) can respond to 17β- estradiol of a low concentration (≤1 pM). As a screening system for environmental hor- mones, the detectivity of BG1Luc4E(2)-cell bioassay was identified by the individual re- sponse to common XEs, and also by two novel estrogenic chemicals of PCBs (2,3′,4,4,′- etrachlorobiphenyl and 2,2′,3,5′,6-pentachlorobiphenyl). These cell bioassay systems have applications for rapid screening, identification, and characterization of endocrine- disrupting chemicals. Transcriptional activation assays allow rapid identification of com- A plasmid vector contains ﬁreﬂy luciferase genes that are controlled by DNA en- hancers, which can react with androgens, estrogens or retinoic acids. The transfection of a plasmid vector into corresponding receptor-containing cells reﬂects its ability to respond to respective hormones under luciferase induction. An estrogen-responsive luciferase reporter plasmid has steadily transfected the recombinant ovarian carcinoma (BG-1) lines in humans. The subsequent recombination of cell lines (BG1Luc4E(2)) can respond to 17β-estradiol of a low concentration (≤1 pM). As a screening system for environmental hormones, the detec- tivity of BG1Luc4E(2)-cell bioassay was identiﬁed by the individual response to common XEs, and also by two novel estrogenic chemicals of PCBs (2,3′,4,4,′-tetrachlorobiphenyl and 2,2′,3,5′,6-pentachlorobiphenyl). These cell bioassay systems have applications for rapid screening, identiﬁcation, and characterization of endocrine-disrupting chemicals. Tran- scriptional activation assays allow rapid identiﬁcation of compounds with the potential to affect the ER signaling pathway directly or indirectly [56]. 2.11. Estrogen Receptor Recombinant Yeast Screening (YES) Assay Described by Professor Sumpter, this estrogen receptor recombinant yeast screening (YES) assay can be used to assess the estrogenic activity of chemicals or their metabolites, such as the surfactants and their major degradation products. In principle, the DNA se- quence of the human estrogen receptor (hER) is integrated into the yeast (Saccharomyces cerevisiae) genome, which also contains expression plasmids carrying estrogen-responsive sequences (ERE) that control the expression of the reporter gene lac-Z (encoding the en- zyme β-galactosidase). Thus, in the presence of estrogens, β-galactosidase is subsequently synthesized via the formation of ER-ligand and secreted into the medium, where it causes a colorful change from yellow to red [58]. This recombinant yeast is used to determine whether chemicals or their metabolites, such as the surfactants and their principal degra- dation products, possess estrogenic activity. The results are compared to the effects of the main natural estrogen 17β-estradiol [58]. Using the YES assay, the study revealed none of the parent surfactants tested possessed estrogenic activity. However, one class of surfactants, the alkylphenol polyethoxylates, degrade to persistent metabolites that were weakly estrogenic. Another group of degradation products, the sulfophenyl carboxy- lates, which were derived from the biodegradation of linear alkylbenzene sulfonates, did not appear to possess estrogenic activity [58]. Hence, the estrogen receptor recombinant YES assay can evaluate the estrogenic stimulation provided by estrogens, XEs and other endocrinal disruptors. pounds with the potential to affect the ER signaling pathway directly or indirectly 2.10. Triple Functional Small-Molecule–Protein Conjugate-Mediated Optical Biosensor 2.10. Triple Functional Small-Molecule–Protein Conjugate-Mediated Optical Biosensor It is a challenge to create biosensors for a comprehensive mapping of potential estro- genic chemicals. Using triple functional small-molecule–protein conjugates as probes, flu- orescent ER-based-wave biosensors were reported in 2019 to detect estrogenic activities n water samples [57]. The probe containing a Cy5.5-labeled streptavidin (STV) part and a 17β-estradiol part can act as signal conversion and recognition. When XEs are competing with the E2 part of the probe in binding to ERs, the unbound conjugates will be released. The STV parts then bind with desthiobiotin (DTB) modified with the optical fiber through an STV-DTB affinity interaction. The detection with signal probes is completed by fluo- i i i d d b d di fi ld hi h l t ith t i ti i It is a challenge to create biosensors for a comprehensive mapping of potential es- trogenic chemicals. Using triple functional small-molecule–protein conjugates as probes, ﬂuorescent ER-based-wave biosensors were reported in 2019 to detect estrogenic activities in water samples [57]. The probe containing a Cy5.5-labeled streptavidin (STV) part and a 17β-estradiol part can act as signal conversion and recognition. When XEs are competing with the E2 part of the probe in binding to ERs, the unbound conjugates will be released. The STV parts then bind with desthiobiotin (DTB) modiﬁed with the optical ﬁber through an STV-DTB afﬁnity interaction. The detection with signal probes is completed by ﬂuores- cence emission induced by a descending ﬁeld, which correlates with estrogenic activities in the samples. Int. J. Mol. Sci. 2021, 22, 4013 9 of 27 9 of 27 A facile method for quantifying estrogenic activities is developed by using a triple functional small-molecule–protein conjugate as a sensing element. Following optimization of detection, exposure to environmental estrogens can result in the release of the ﬂuorescein- labeled conjugate in the supernatant. It will increase the amount of ﬂuorescein that is attached to the ﬁber surface, which can be observed by increases in the ﬂuorescent signals. By means of the approach, samples’ estrogenic activities can be measured at a limit of detection (LOD) of 1.05 µg/L, using E2 as a reference. The biosensors provide a reliable application for the detection of estrogenic activities in real samples of wastewater [57]. 3. Metabolism and Action of Xenoestrogens 3.1. The Metabolism of Xenoestrogens There were published data regarding the distribution of relative qualitative tissue expression of ER α and ER β on cells and tissues of humans and mice [37,72–75]. Estrogen contributes largely to cells in various aspects, including development, pro- liferation, migration, and survival [67,76]. The action can be divided into two, namely genomic and non-genomic pathways [76,77]. Receptors, such as ER α, ER β, or even GPER, function well through these pathways [11,78,79]. The binding of estrogens to ERs in the cytoplasm contributes to the genomic pathway. The estrogen/receptor complex then swift to the nucleus; thus, changes are made to gene expression [80]. Additionally, non-genomic pathways, as inﬂuential as they could be, are via the binding to receptors (GPER) in cell membranes to activate secondary cellular messengers without incorporating gene expression in the nucleus. While both pathways are essential to further actions, non-genomic pathways, with their unique feature, have made the process- ing time faster from seconds to minutes; in contrast, genomic pathways are considered less efﬁcient as it could take from an hour up to a few days for the transmission of messages in the organism [76,77]. The ER is located within the nucleus and dimerizes on ligand binding, and then the ER dimer is bound to the estrogen response element (ERE), which is in the sequence of the promoter of the estrogen target gene, being called the ER transcription complex. Dimers, including ER, ER α or ER β homodimer and ER α /ER β heterodimers are capable of binding to the corresponding ERE, which in turn prompts gene expression. Unlike ER β homodimers, ER α/ER β heterodimers seem to be more intensive in all ER activities. Additionally, ER α is discouraged by the existence of ER β activity [81,82]. The factors that accelerate gene transactivation are known as coactivators [79,83]; however, DNA is suppressed by the corepressors [84,85]. Both coactivators and corepressors are deﬁned as the provider of the ER action. p The most common classical mechanism is displayed in Figure 1. Nevertheless, chan- nels that will affect ER would not only be considered a single classical pathway but also the multiple interveners [11]. Moreover, then, the Kinase cascades or ER phosphorylation may be signaling information toward the ligand-independent activation. 3. Metabolism and Action of Xenoestrogens 3.1. The Metabolism of Xenoestrogens The estrogenic or antiestrogenic activity of chemicals is attributed to the interactions between ER and other compounds. In fact, ER, as a ligand-inducible transcription fac- tor, plays a vital role in development and neoplasia, regulating genes involved in cell proliferation and differentiation [9]. As a role of the major transcription factor in cell proliferation and differentiation, ER is sensitive to any disruption of the signaling pathways, leading to infertility, developmental abnormalities, or endocrine cancer discovered in both human beings and wildlife. The damage to health may be originated from exposure to the estrogenic or antiestrogenic activ- ities of chemicals [59]. Endocrine-active compounds may also interfere with other signaling systems, most signiﬁcantly the androgen and thyroid hormone system, steroidogenesis [60], and part of the aryl hydrocarbon (Ah) receptor [61]. The ER, as the nuclear receptor superfamily, is a factor of ligand-inducible transcrip- tion. Hence, far, two subtypes of the ER are ER α [62–65] and ER β [64], respectively, and both receptors have unique tissue distribution, playing a signiﬁcant role in physiol- ogy [66,67]. In 1999, Korach suggested that most aberrant phenotypes are proved to be linked to ER α from the ER knockout mice study. (e.g., hypotrophy of the uterus, infertility, and rudimentary mammary gland development) [67]. While the reproductive system is signiﬁcantly affected by the ER α, another impact upon non-classical estrogen target tissues, such as brain, skeletal, immune, cardiovascular system, adipose tissue, and the male reproductive tract, is also crucial [66,68]. As opposed to ER α, with diverse inﬂuences on multiple systems, ER β impacts are more speciﬁc to the female ovarian function [69,70]. Therefore, more attention has been paid to the XEs in Int. J. Mol. Sci. 2021, 22, 4013 10 of 27 10 of 27 which the dominance of ER α is much more than ER β; however, the interesting ﬁndings indicate that than ER α, ER β has correctly shown higher afﬁnity [10,71]. which the dominance of ER α is much more than ER β; however, the interesting ﬁndings indicate that than ER α, ER β has correctly shown higher afﬁnity [10,71]. Researchers have made attempts to reach the conclusions of the distribution and locations of the ER through the study of mice or even human body tissues. In 2001, Mueller and Korach et al. demonstrated the RNA and protein expression of ER in the human body [10,70]. 3. Metabolism and Action of Xenoestrogens 3.1. The Metabolism of Xenoestrogens To be more speciﬁc, the estrogen ligand is connected to the ERE in which the signal of E2-target genes can be clearly expressed, such as the ER-DNA interaction classical pathway [12]. However, not all of the ERE would be found in the E2-sensitive gene promoter sequence, implying that the ER modes of action are diverse rather than a simple pathway. p p y Almeida et al. reported that ERα in osteoblast progenitors expressing Osterix1 (Osx1) potentiates Wnt/β-catenin signaling, thereby increasing proliferation and differentiation of periosteal cells [86]. Further, this signaling pathway was required for optimal cortical bone accrual at the periosteum in mice. Notably, this function did not require estrogens. The osteoblast progenitor ERα mediated a protective effect of estrogens against endocortical but not cancellous bone resorption. ERα in mature osteoblasts or osteocytes did not inﬂuence cancellous or cortical bone mass. Hence, the ERα in both osteoblast progenitors and osteoclasts functions to optimize bone mass, but at distinct bone compartments and in response to different cues [86]. p In another study, it was hypothesized that ERα in osteocytes was important for trabecular bone in male mice and for cortical bone in both males and females [87]. Dmp1- Cre mice were crossed with ERα(ﬂox/ﬂox) mice to generate mice lacking ERα protein expression speciﬁcally in osteocytes (Dmp1-ERα (−/−)). Male Dmp1-ERα (−/−) mice displayed a substantial reduction in trabecular bone volume (−20%, p < 0.01) compared with controls. ERα in osteocytes regulates trabecular bone formation and thereby trabecular bone volume in male mice, but it is dispensable for the trabecular bone in female mice and Int. J. Mol. Sci. 2021, 22, 4013 11 of 27 11 of 27 the cortical bone in both genders. The authors proposed that the physiological trabecular bone-sparing effect of estrogen is mediated via ERα in osteocytes in males but via ERα in osteoclasts in females [87]. Kapara et al. have adopted a nondestructive approach for detecting and localizing ERα expression at the single-cell level using surface-enhanced Raman spectroscopy (SERS) combined with functionalized gold nanoparticles (AuNPs) [88]. The author developed an approach based on the percentage area of SERS response to qualitatively measure expression level in ERα-positive (ERα+) breast cancer cells. Speciﬁcally, the calculation of relative SERS response demonstrated that MCF-7 cells (ERα+) exhibited higher nanotag accumulation resulting in a 4.2-times increase in SERS signal area compared to SKBR-3 cells (ERα-) [88]. 3. Metabolism and Action of Xenoestrogens 3.1. The Metabolism of Xenoestrogens The result of this article conﬁrmed the strong targeting effect of ERα- AuNPs towards the ERα receptor. It opened up the possibilities of using SERS as a tool for the estimation of ERα expression levels without the requirement of destructive and time-consuming techniques. Therefore, the potential of using SERS as a rapid and sensitive method to understand the activity of SERDs in breast cancer is demonstrated [88]. ER β has been suggested to possess antiproliferative and antitumor effects in breast and prostate cancer cells in some previous articles, but other studies have indicated its tumor-promoting effects [89]. The author studied the effects of ERB-041-treated colon cancer cells in a zebraﬁsh xenograft model and found signiﬁcantly less distant metastasis of ERB-041-treated cells compared to vehicle-treated cells. These results further support ERβ’s antitumor role in colorectal cancer and the possible use of its agonist in colorectal cancer patients [89]. Estrogen-related receptor β (ERRβ) is a nuclear receptor critical for many biological processes. Despite the biological and pharmaceutical importance of ERRβ, deciphering the structure of ERRβ has been hampered by the difﬁculties in obtaining a pure and stable protein for structural studies [90]. In fact, the ERRβ ligand-binding domain remains the last unsolved ERR structure and also one of only a few unknown nuclear receptor structures. The authors conﬁrmed a critical single-residue mutation resulted in robust solubility and stability of an active ERRβ ligand-binding domain, thereby providing a protein tool enabling the ﬁrst probe into the biochemical and structural studies of this important receptor [90]. The promoter context and estrogenic ligands are the joint dependents upon the ex- pression of ER-target genes and ER-mediated cellular functions. In the previous discussion regarding the classic pathway, estrogenic ligands are highlighted as the trigger of ER con- formational changes, and with further interactions with other coregulators and subsequent transcriptional activity moving forward [91,92]. It is indicated that the XEs work in different functions, even if they are identical. The tissue speciﬁcity marks its uniqueness, and XEs could also turn to other tissues with complexity. As ERα and ERβ have different functions and effects, the physiological functions and the organs and tissues are also distributed differently in the body parts; thus, identical XEs could lead to different consequences and effects on the body. Generally, XEs are either partial agonists or partial antagonists, which indicate that they are possibly less effective than estrogen in the human body [9]. 3. Metabolism and Action of Xenoestrogens 3.1. The Metabolism of Xenoestrogens As mentioned above, the complexity of mechanisms that affect XEs in the human body includes the genomic pathway, non-genomic pathway, and various transcription factors. Hereafter, coactivators, corepressors, or even the interactions between signaling cascades and other receptors will lead to estrogenic effects. Therefore, the mechanisms mentioned above should be considered in the experiment and evaluation. The diversiﬁcation of ER action is the key to be understood [93]. In recent decades, people became aware of the exogenous compounds that work on the signaling pathways of endogenous hormones in the human body or organisms, including synthesis, storage, metabolism, transport, and elimination in organisms [94,95]. g y g p g As endocrine-disrupting chemicals (EDCs), these XEs may lead to biological and pathological changes or even accumulated damage to future generations (e.g., bioaccu- Int. J. Mol. Sci. 2021, 22, 4013 12 of 27 12 of 27 mulation). There are various sources and types of XEs in the environment, and many of these XEs have the similarity of structures with estrogens [19,96]. The chemical structural similarity between these XEs and naturally occurring estrogen compounds can cause the human body and organism simulations (e.g., 17β-estradiol). EDCs behave like natural (estrone (E1), E2, estriol (E3)) and synthetic estrogens DES, [4,7]. Among all estrogens, including E1, E2 and E3, E2 is the most potent and serves as the major estrogen that exerts endocrinal functions in human bodies. The mechanisms of action of XEs could be divided into critical factors as follows: Endogenously occurring estrogens simulation, an antagonist of endogenous estrogens, and the intervention of metabolism and biosynthesis production of estrogen [8]. Additionally, EDCs have been mentioned to act through complex tissue-selective modulation on ERs and other signaling pathways in vivo [19]. As mentioned above, ER α and ER β act as an intermediary in various tissues and those with distinct biological effects, including mammary glands, bone, brain, and vas- cular system in both genders. Due to the partial different tissue distribution and distinct physiological functions, XEs could show agonist or antagonist activity at the time of development [94]. The relationship between ERs and coactivators/corepressors is critical to the regulation of DNA and RNA, which will also impact the expression of ER-target genes. In fact, the characteristic of tissues speciﬁc to different organs includes expression of speciﬁc cofactors, the ER α/ER β ratio, and the level of expression of speciﬁc intracellular kinases (including cytoplasmic tyrosine kinases). 3. Metabolism and Action of Xenoestrogens 3.1. The Metabolism of Xenoestrogens For example, the afﬁnity of the phytoestrogen genistein to ER β is much greater than that of ER α. There is even more evidence to show that genistein impacts the proliferation or antiproliferation in cancer cells [97]. 3.2. The Actions of Xenoestrogens 3.2.3. DDT [2,2-Bis(p-chlorophenyl)-1,1,1-trichloroethan] and Its Metabolite DDE Another example of XEs is DDE, a metabolite of DDT and a component of pesticides. DDE is seen as more effective due to its afﬁnity and lipophilicity, and it is unlikely to be catabolized by organisms. Both DDT and DDE are XEs with estrogenicity, which could inﬂuence the reproduction function of organisms in the environment. For instance, in the alligators of Lake Apopka, biologists have discovered that male crocodiles have a micropenis and various abnormalities of the testes due to their exposure to DDT and DDE substances [108]. Fujisaki et al. have reviewed past data records from the early 1980s that the American alligator (Alligator mississippiensis) population decreased in Lake Apopka after DDT and DDE exposure. Thus, the US government has conducted the extensive restoration of the swamp of Lake Apopka and proposed environmental-related restrictions on the lake in response to such a decrease. According to monitoring by the Florida Fish and Wildlife Conservation Commission, the adult alligator population gradually began to increase in the early 1990s after such efforts were made [109]. y The consequences of DDT and DDE have also been found in human bodies; this was in Wolff et al.′s ﬁndings in the organochlorine article in 1993, including DDT, PCBs and other substances in relation to the risk of breast cancer. The correlation between breast cancer and DDT or DDE was mentioned [12,110]. Due to the inefﬁciency of metabolism and their solubility in lipids, these agents have been found in human tissue that causes lifelong sequestration in adipose tissue. Wolff et al. have concluded that DDE in serum is the most signiﬁcant trigger of breast cancer development instead of associating with PCBs. This research suggests that environmental chemical contamination with organochlorine residues may be a critical factor in breast cancer [110]. In addition to the impacts of breast cancer, there is also evidence that shows methoxy- chlor (pesticide) and DDT have the potential to cause uterine proliferation and weakening of normal follicle development in the female reproductive system [19]. 3.2.1. Selective Estrogen Receptor Modulators (SERMs) and Aromatase Interferer 3.2.1. Selective Estrogen Receptor Modulators (SERMs) and Aromatase Interfere Due to the characteristics mentioned above, tamoxifen and raloxifene have been applied to the current treatments, and the process is so-called selective ER modulators (SERMs). These kinds of XEs sometimes could have impacts on the ER nongenomic path- ways that increase endocrine disruption. There were examples showing XEs in different structures, with high concentrations, BPA, and DES, all capable of activating ERs, thus increasing the risk of developing breast cancer [98]. SERMs were found to demonstrate selectivity toward ERs in the bone, thereby re- ducing side effects. However, they lack the efﬁcacy of traditional estrogen. SERMs are generally inﬂuenced by their binding afﬁnity for ER α and ER β and the effect of the bound ligand on the ER structure. However, the precise mode of action of each SERM remains unknown. One endogenous compound—27-hydroxycholesterol (27HC)—has been found to bind to and modulate the activity of ERs in vivo and to behave like a SERM. In mice, 27HC behaves as an ER antagonist and reduces the protective effects of estradiol. However, in cellular models of ER-positive breast cancer, 27HC acts as a partial ER agonist. In ovariectomized mice with elevated 27HC levels, a dramatic loss of bone was observed. Further research on 27HC could lead to developing new drugs. Aromatase inherently existed in the human body to convert androgens to estrogens. However, a type of XE, such as tributyltins, has an aromatase suppressing effect, leading to an imbalance between androgens and estrogen. Tributyltins is a kind of coating, which often exists in the hull of ﬁshing boats or plastic products, and the potential health effects on organisms are as follows: teratogen; teratogenicity; diabetes mellitus; hyperlipidemia; metabolic syndrome; increase in fat depot size; obesity; hepatic steatosis; hypertrichosis; osteoporosis; decreased sperm production; breast cancer; endometrial cancer [99,100]; polycystic ovary syndrome (PCOS) [101–104]. Int. J. Mol. Sci. 2021, 22, 4013 13 of 27 13 of 27 3.2.2. Polycyclic Aromatic Hydrocarbons (PAHs) 3.2.2. Polycyclic Aromatic Hydrocarbons (PAHs) Some XEs are combined with sex hormone-binding globulin, which decreases E2 plasma transport in the cell. An example is an interaction between polycyclic aromatic hydrocarbons (PAHs) and ERE-dependent E2-target gene transcription. PAHs are a col- lective term for more than a hundred different chemical substances, which are formed when coal, fuel oil, gas, trash, or other organic substances are incompletely burnt, and some PAHs are artiﬁcially manufactured. PAHs are found in coal tar, crude oil, and a few are applied in medicines or manufacturing dyes, plastics, and pesticides [105–107]. On the other hand, some metabolites of PAHs could be combined with ERs to recruit other coregulators, and the enhancing performance of E2 target genes could be achieved. Dioxin is one of the metabolites of PAHs. In the case of dioxin, it could be bound with aryl hydrocarbon receptor (AhR) that leads to heterodimerization along with aryl hydrocarbon nuclear translocator (Arnt). If so, the interaction between ER and ERE could facilitate the expression of the E2 target gene through such a complex [94]. 3.2.5. The Relationship between EDCs and Diseases It has been realized that estrogenic or antiestrogenic effects of different EDCs involve environmental pollution and affect human hormonal discrepancies. According to the historical data, breast cancer incidence and prevalence have been increasing since the 1940s [12,115–117]. The risk of breast cancer accelerates with increased cumulative estrogen exposure or the rise of XEs in the environment. Many researchers are dedicated to identify- ing related risk factors, DDT and its metabolite (metabolite) DDE [110]. As PAHs, dioxin, PCBs and DES were mentioned earlier, and all have been reviewed in the literature for the associated carcinogenic effects (carcinogenic effects, carcinogenic elements) or toxicity to the mammary gland (toxicant). 2,3,7,8-chlorodibenzo-p-dioxin has been proved to be toxic to the mammary glands of mice. Dioxin delays the proliferation and differentiation of the mammary glands of breast development. This ﬁnding has an identical conclusion obtained in the human body [118]. Another study reported that 200 young Belgian girls had delayed pubertal development related to their blood doubling of serum dioxin levels [119]. Similarly, PAHs have also affected a signiﬁcant increase in postmenopausal women’s breast cancer development. Additionally, DES exposure during human pregnancy can trigger oncogenesis in the vagina and breast [120–122]. Moreover, Hoover proposed in 2011 that mothers who have been exposed to diethylstilbestrol will inﬂuence their next generation in utero, with the incidence and progress of future breast cancer development. In fact, the growth may become more signiﬁcant due to the age of these DES daughters [123]. In 2016, Ellinon Axiomaticon reviewed studies that investigated the relationship be- tween endometriosis and endocrine disruptors. Hormonal inﬂuences may be the key factor that affects the extrauterine growth and proliferation of endometrial cells [12]. Other re- ports demonstrated the association of membranous ectopic diseases of rodents or primates with the common environmental pollutants, including TCDD (2,3,7,8-tetrachlorodibenzo- p-dioxin), dioxin, BPA, and DES [124–128]. For the pathophysiology in relation to en- dometriosis that TCDD may cause, changes in the relative levels of ERβ and ERα in endometrial tissue determine the performance of the estradiol-regulated progesterone receptor (PR). The reduction of the ERα-to-ERβ ratio may result in the expression of PR being suppressed [129,130]. If the mother is exposed to speciﬁc-XEs, such as DES or BPA during pregnancy, increased endometriosis will be passed into the next generation, either in the human body or mice [125,127,131]. 3.2.5. The Relationship between EDCs and Diseases Similarly, endometrial cancer has the same trigger for developing ovarian cancer, which is considered estrogen and XEs [132,133]. For instance, methoxychlor [MXC; 1,1,1- trichlor-2,2-bis(4-methoxyphenyl) ethane] is an organochlorine pesticide used in agriculture since DDT was banned. The metabolite of methoxychlor is 2,2-bis(p-hydroxyphenyl)-1,1,1- trichloroethane (HPTE), activating ER in ovarian cancer cells and mitogenic activities in ovarian tissues. Moreover, triclosan is often used as a common ingredient in soaps, deodor- ants, toothpaste, and other hygiene products. Methoxychlor and triclosan are substances that contain organochlorine, regulating cell cycles and apoptosis-related genes by combin- ing with ERs, thereby stimulating the growth of ovarian cancer cells and the consequences of cancer reaction [134]. In addition, genistein, which is a type of phytoestrogen, is similar in structure to estrogen, thus mimicking E2 and stimulating cell proliferation activity [135]. 3.2.4. Polychlorinated Biphenyls (PCBs) Although Wolff et al. in 1993 reported that breast cancer was less associated with PCBs level in serum, other studies have suggested that PCBs work the same way in humans as scientists have long observed the effects of PCBs on other organisms. Bergeron noted the relationship between turtle sex determination and environmental contamination and the EDCs, such as PCB isomers, can alter sex ratios in turtles [12,111]. In the early 1970s, the ﬁndings in the experiments regarding PCBs in mice showed that exposure at birth reduced the reproductive ability of male rats [112] because of the alternation of steroid hormone-metabolizing enzymes [113]. Although Wolff et al. con- cluded that breast cancer was not associated with PCBs in serum in the early days, there Int. J. Mol. Sci. 2021, 22, 4013 14 of 27 have been many reports about PCBs and breast cancer progression in the future. Exposure to PCB174 has been conﬁrmed to be associated with an increase in breast cancer mortality, and it is still positively related to breast cancer-speciﬁc mortality after 5 or even 15 years of follow-up after being diagnosed. Moreover, there are various reports, which made similar conclusions [93,114]. 3.2.6. Bisphenol A (BPA) Industrial compounds, such as BPA and 4-nonylphenol (NPH), have residues in food (e.g., in canned vegetables) or dental materials. BPA is a chemical compound used in Int. J. Mol. Sci. 2021, 22, 4013 15 of 27 15 of 27 food or plastic containers. The coating on metal cans could protect food and beverages. Potentially, the residual compounds may all be detected [4,136–138]. If the mother mouse is exposed to the environment with BPA before delivery, the number of precancerous lesions will increase after the next generations turn into adulthood, and such correlation can be considered dose-related. However, high dosage BPA and 4-NPH can induce the occurrence of breast cancer cells in situ [137,139,140]. The possibility of XEs increase the risk of carcinogenesis in different vertebrate reproductive systems has been discussed. Moreover, current scientiﬁc and medical research has found that the impact of these environmental pollutants has been subjected to cancer-related and metabolic diseases, such as obesity, diabetes mellitus, hypertension, and cardiovascular disease [141,142]. Low levels of estrogen are related to developing glucose intolerance and insulin resistance [143]. As is, BPA can increase or decrease insulin production in the body by mimicking the effect of the body’s endogenous estrogen, which mechanism is similar to insulin regulation by 17β-estradiol. Recently, different research has proved the effects of BPA on insulin resistance, both for children and adults. BPA, as an environmental hormone, plays an important role in the pathophysiology of diabetes mellitus [144–146]. In addition, BPA can also reduce glycogen synthesis, thereby reducing glucose oxidation and reducing the use of glucose by muscle cells, and the sensitivity of muscle cells to insulin will be decreased [147]. BPA Exposure can also contribute to weight gain in mice, especially in female mice; the para-physiological mechanism may result from interference with the neurotransmitter signaling pathway, which causes the change in energy metabolism [142,148]. A study researching children’s obesity has conﬁrmed that compared with normal weight, over- weight and obesity are signiﬁcantly associated with the urinary BPA levels of the children. Moreover, BPA may trigger insulin resistance in children and increase the risk of diabetes mellitus, especially for obese children [149]. In 2017, Lidia Caporossi conducted a literature review to analyze the effects of BPA on different metabolic diseases. Most of the reports are cross-sectional studies [141]. Fénichel pointed out that the prevalence of type-2 diabetes in the world has increased dramatically in the last few decades. 3.2.6. Bisphenol A (BPA) There is also increasing evidence that shows these EDCs may also play a key role in the occurrence of metabolic diseases. In the observations of rodents, it was found that BPA stimulated the production and secretion of pancreatic β cells, interfering with insulin signals, which cause insulin resistance and β cell destruction/damages [150]. g In addition to the risks regarding BPA that may result in various types of reproductive organ cancers and metabolic diseases, recent studies have also suggested that BPA can affect healthy bones. Except for the characteristics of estrogenicity and antiandrogenicity, it has been hypothesized that BPA can bind to the ER and exert its antiandrogenic, inﬂammatory, and oxidative properties [151]. Because bones will transform in response to the stimulation of hormones, inﬂammatory and oxidative status, BPA exposure can impair bone health. In 2018, Chin et al. reviewed the evidence of the effects of BPA and its derivatives on skeletal health in humans and animals and reported that BPA would decrease the proliferation of osteoblast and osteoclast precursor cells and induce apoptosis [151]. In various (in vivo and in vitro) animal models, BPA and its derivatives have different positive and negative actions. While BPA increased femoral bone mineral content in male rats, it decreased femoral mechanical strength in female rats. In estrogen-deﬁciency models, BPA improved bone mineral density and microstructures in aromatase-knockout mice; however, it lowered the trabecular density in ovariectomized rats. The major limitations of current evidence are the small sample size and the cross-sectional rather than longitudinal study design. In conclusion, BPA can affect the skeletal health of vertebrate animals, and its impacts depend on the types and sex of animals. However, the effects of BPA on bone mineral density and bone health of humans warrant further investigation [151]. Another study conducted by Kim et al. had attempted to analyze the relationships be- tween serum BPA concentration, bone mineral density (BMD) and biochemical bone mark- Int. J. Mol. Sci. 2021, 22, 4013 16 of 27 16 of 27 ers in postmenopausal women with osteoporosis [152]. The relationship between BPA and clinical variables was analyzed by the Pearson’s correlation test and the Kruskal–Wallis test. Serum BPA concentration was measured by enzyme-linked immunosorbent assay (ELISA). The mean BPA concentration of 51 postmenopausal women was 1.44 ± 0.52 ng/mL. The results showed no statistically signiﬁcant correlation between BPA concentration and clini- cal variables. 3.2.7. Heavy Metals: Cadmium (Cd) and Arsenic (As) Heavy metals can act as XEs and inﬂuence female reproduction systems. One example is the chemical Cd, which can be found in cigarettes, paints, plastics, batteries, and foods. As toxins to humans, inorganic Cd and As are cytotoxic at a high concentration level [4]; however, they can simulate estrogen and exert XEs-like actions at a low concentration level. Although the literature has conﬁrmed the estrogenicity of inorganic Cd in tumor cell lines, the underlying mechanisms remain unclear and need more research. Both inorganic Cd and As stimulate cell proliferation in the pituitary gland and uterus by increasing the expression of proliferation markers, thus affecting hormone-dependent tumor progression. The anterior pituitary gland is responsible for hormone synthesis and secretion (such as prolactin and luteinizing hormone). Both inorganic Cd and As can increase prolactin synthesis. In 2016, Ronchetti et al. found that low doses of Cd can exert strong xenoestrogenic effects on the anterior pituitary gland [161]. 3.2.6. Bisphenol A (BPA) The author concluded that there was no statistically signiﬁcant correlation between serum BPA and clinical variables related to bone metabolism [152]. Upson et al. have conducted a population-based case-control study to investigate the association of BPA exposure with the risk of endometriosis [153]. The authors mea- sured and analyzed urinary BPA concentrations for more than 400 cases. For cases and controls, the median creatinine-uncorrected total BPA concentrations (µg/L) were 1.02 (IQR: 0.43–2.12) and 0.86 (IQR: 0.36–2.01), and the median creatinine-corrected total BPA concentrations (µg/g) were 1.32 (IQR: 0.79–2.21) and 1.24 (IQR: 0.65–2.54), respectively. The result showed statistically signiﬁcant positive associations when evaluating total urinary BPA concentrations was only in relation to non-ovarian pelvic endometriosis, but not in relation to ovarian endometriosis [153]. Except for the association with female reproductive diseases, including breast cancer, ovarian cancer, vaginal cancer and endometriosis, literature has conﬁrmed the effects of XEs on the male genital system. Increasing evidence has shown the connection between ex- posure and EDCs and impairment of male reproductive function. The impact can originate from the interference of hormone, cell signaling pathway and metabolism, and will be aug- mented, especially if the exposure to XEs occurs during early growth development [154]. Recent research has highlighted the possible relationship between unexplained male infer- tility and exposure to low-dose EDCs in the fetal testis and adult endocrine system [155]. Exposure to EDC may result in the impairment of testis functions in different spermatogen- esis stages, depending on the time point of exposure. BPA induced meiotic abnormalities in the reproductive system of adult male mice [156] and signiﬁcantly reduced the number of sperms in juvenile male mice as a result of interruption of meiotic progression [157,158]. Dibutyl phthalate (DBP) and methoxychlor (MXC) can signiﬁcantly lower the weight of the testes as a consequence of a reduction in the number of spermatogenic elements and spermatozoa [159,160]. The use of DBP can block spermatogenesis, thus prohibiting the production of sperms and even leading to necrosis of the seminiferous tubules [159,160]. 3.2.7. Heavy Metals: Cadmium (Cd) and Arsenic (As) 3.2.8. Phytoestrogens Phytoestrogens are another type of XEs, including isoﬂavonoids, lignans, coumestans, and pisatin [24]. Some plants, such as soybeans, contain phytoestrogens with active ingredients of genistein and daidzein [4,162]. Phytoestrogens can combine and activate estrogen receptors in the brain to inﬂuence functions of the brain, thus possibly resulting in neurobehavioral disruptions. On the other hand, other studies indicated that pregnant women might beneﬁt from the intake of soybean foods that contained phytoestrogens [163]. Int. J. Mol. Sci. 2021, 22, 4013 17 of 27 17 of 27 In vitro or in vivo studies to analyze the ﬁnal effects of phytoestrogens may be quite different. For instance, at low doses (from 10 nM to 1 µM), genistein showed mitogenic effects on breast cancer cell growth, whereas, at higher concentrations (>10 µM), it showed antiproliferative effects [164]. Some of these effects are explained by their interactions with ER subtypes. As mentioned above, the ratios and the expressions of ERα and ERβ are different in various tissues depending on the period of life. Moreover, the abilities of ER subtypes to recruit cofactors, regulate gene expression and stimulate or inhibit cell growth are slightly different. Therefore, in vivo, phytoestrogens may have a complex role, acting as weak estrogens and antiestrogens depending on the tissue. Furthermore, it is believed that the signaling pathways induced by phytoestrogens are not completely identical to those induced by estrogens [164]. Regarding the preventive effects on diseases or cancers, a higher intake of phytoe- strogens, such as isoﬂavones, is associated with a moderately lower risk of developing coronary heart disease. It may also reduce the risks of breast and colorectal cancer as well as the incidence of breast cancer recurrence. Consumption of phytoestrogens or soy foods is associated with reduced risks of endometrial and bladder cancer [165,166]. Regarding the therapeutic effects on menopausal syndrome or other diseases, phytoestrogens have been found to alleviate vasomotor syndromes even after considering placebo effects, reduce bone loss in the spine and ameliorate hypertension and in vitro glycemic control. They may also alleviate depressive symptoms during pregnancy. On the other hand, phytoe- strogens have not shown deﬁnitive effects regarding improving cognition and urogenital symptoms [165,166]. Because of lacking standardization in the study designs, such as the ingredients and doses of phytoestrogens and the durations and outcomes of trials, it cur- rently remains difﬁcult to draw overall conclusions for all aspects of phytoestrogens. 3.2.8. Phytoestrogens These limitations warrant further investigations of the use of phytoestrogens for women’s health. 4. Discussion On the molecular level, many XEs are known to be structurally related to the steroid hormones produced by the human body (structural similarity to the natural estrogens). Therefore, they have estrogenicity or antiestroginecity and can be bound with the receptors of the organism, manipulating differentiation and modulation of cell proliferation, apopto- sis, cytokine production, and cell cycle progression, which should have been controlled over by the endogenous 17β-estradiol [3,6]. On the other hand, some substances, such as Cd, PCB and dioxin, can function as potential endocrine disruptors despite no structural similarity to the natural estrogens. However, the exact mechanism by which this metal Cd may interfere with the reproductive system has not been fully elucidated. The interrup- tion in the steroidogenic pathway by Cd toxic action may be explained in a few different ways [175]. The changes in E2 and progesterone levels may result from the impairment of steroidogenic enzymatic activities by Cd. Under the actions of Cd, conversion of cholesterol to pregnenolone is supposed to be the cause of abnormalities in the metabolism of sex hormones. Thus, endocrine disruptors, including Cd, PCB and dioxin, can exert effects via various pathways other than classical ER-signaling. Evidence shows the possible impacts of environmental XEs on developing humans and animal groups’ evolution in recent years. The issue could be considered crucial in the long run instead of a short-term (maldevelopment) effect. Some accumulated effects of XEs or EDCs may only occur until the individual matures, reaching adulthood, rather than being effective in a short period [12]. Xenoestrogen-related endometriosis, which damages the reproductive system, precancerous lesions, and proliferation of cancer cells are pertinent examples. For example, cervical cancer or prostate cancer is reported to be relevant environmental estrogens cases. An article reporting how XEs or EDCs affect the female reproductive system, particularly the endometrium, was published by Karoutsou et al. in 2016 [12], in which it includes the idea of the term window. Explaining from a period in a development perspective, the term window of susceptibility means that the developing organisms can be altered by environmental factors, which results in structural, functional, and/or cellular changes. The occurrence of such alterations during these windows could only be identiﬁed until the late stages [12]. The use of certain pesticides, which refers to the XEs in relation to the biological damage and adverse events to the reproductive system, was initially documented back in the 20th-century [27–29]. 3.2.9. Diethylstilbestrol (DES) and 17-α-ethinylestradiol DES is one type of synthetic estrogen with strong potency. During 1940–1971, several million people were exposed to DES [21,22,167]. DES can inhibit the actions of adrenal androgens and possibly interfere with cell cycles to induce apoptosis of prostate cancer cell lines [22,25,26]. A study from Egypt pointed out a higher incidence of uterine, cervical, and ovarian cancer in urban areas than in rural areas. The incidence of uterine cancer is 6-fold in urban areas than in rural areas. Correspondingly, there was a higher exposure to XEs (e.g., DES) for females living in the urban areas of Egypt than those living in rural areas [136]. Such a difference in exposure to XEs between urban and rural areas could be noted in other countries [136,168–171]. Voisin et al. exposed mangrove rivulus (Kryptolebias marmoratus) for ﬁrst post-hatching 28 days to 4 and 120 ng/L 17-α-ethinylestradiol, as a model of XE environment [172]. The results showed the effects of 17-α-ethinylestradiol were tissue- and dose-dependent. A total of 31, 51 and 18 proteins were differentially abundant at 4 ng/L in the brain, liver and ovotestis, compared to 20, 25 and 39 proteins at 120 ng/L, respectively. This study demonstrated the long-term effects of early-life endocrine disruption at the proteomic level in diverse estrogen-responsive pathways 5 months after the exposure. The lowest tested and environmentally relevant concentration of 4 ng/L 17-α-ethinylestradiol had the highest impact on the proteome in the brain and liver, highlighting the potency of endocrine disruptors at low concentrations [172]. Hill Jr et al. have researched the concentration-dependent effects of a weak estrogen receptor agonist, 4-NPH and a potent estrogen receptor agonist, 17α-ethinylestradiol (EE) on sex ratios, gonad morphology, vitellogenin (VTG) induction and breeding success in zebraﬁsh (Danio rerio) [173]. Fish were exposed from 2 to 60 days post-hatch (dph) to NPH (10, 30, or 100 microg/L nominal), EE (1, 10, or 100 ng/L nominal), or solvent control (acetone; 0.2% v/v) in a static-renewal system with replacement every 48 h. The percentage of males at 60 dph changed from 45% in solvent controls to 0% at 10 ng/L EE and 10% at 100 microg/L NPH. In the EE exposure group, a concentration-dependent Int. J. Mol. Sci. 2021, 22, 4013 18 of 27 18 of 27 increase in the number of ﬁsh with undeveloped gonads at 60 dph was observed. 3.2.9. Diethylstilbestrol (DES) and 17-α-ethinylestradiol However, the sex ratios of adults determined at 160 dph revealed no signiﬁcant departure from 1 male: 1 female, suggesting that exposure of zebraﬁsh to estrogenic chemicals during sexual differentiation and early gametogenesis did not irreversibly alter phenotypic sex. These results suggest that functional reproductive capacity (breeding success) may be more sensitive than gross morphological endpoints (condition, ovo-somatic index, sex ratio) in adult zebraﬁsh exposed to XEs during sexual differentiation and early gametogenesis [173]. Liao et al. have compared the sensitivities of rare minnows during different life stages to 17β-estradiol (E(2)) at environmentally relevant (5, 25, and 100 ng L−1 and high (1000 ng L−1) concentrations by using VTG and gonad development as biomarkers under semistatic conditions [174]. After 21 days of exposure, VTG concentrations in whole-body homogenates were analyzed. The results indicated that the lowest observed effective concentration for VTG induction was 25 ng L−1 E(2) in the adult stage but 100 ng L−1 E(2) in the larval and juvenile stages. After exposure in the early life stage, the larval and juvenile ﬁsh were transferred to clean water until gonad maturation. No signiﬁcant difference in VTG induction was found between the exposure and control groups in the adults, but a markedly increased proportion of females and appearance of hermaphrodism in the juvenile-stage group exposed to 25 ng L−1 E(2). These results showed that VTG induction in the adult stage is more sensitive than in larval and juvenile stages following exposure to E(2). The juvenile stage may be the critical period of gonad development [174]. 4. Discussion Nowadays, the indirect evidence shows that endocrine disruptors Int. J. Mol. Sci. 2021, 22, 4013 19 of 27 19 of 27 are related to various diseases, and their relationship can also be observed in the studies, including metabolic syndrome (especially hypertension and diabetes, obesity), asthma, and various reproductive system-related cancers [6]. Such diseases are not subject, partic- ularly to males or females. Evidence can also be found in breast, cervical, prostate, and vaginal cancer [4]. Previous studies have shown indirect evidence regarding the diseases or harmful organisms triggered by XEs. As mentioned before, these reports focus on the more common XEs, such as DDT and its metabolites DDE, PCBs, DES, 2,3,7,8-TCDD (dioxin), and BPA. As mentioned previously, DDE has a higher afﬁnity and lipophilicity and is not easily catabolized by organisms. Lake Apopka in Florida is seriously affected by DDT and its metabolites. It is discovered that male crocodiles have a micropenis, various abnormalities of the testes, and the cause may be with the exposure of DDT and DDE to crocodiles [108]. Moreover, metallic endocrine disruptors, such as Cd and phytoestrogens, are other ex- amples. Notably, the damages that isoﬂavones, coumestrol, and clover do to the human body will reﬂect on the reproductive system and possibly position as the trigger of cancer development. They will also affect other systems, such as the nervous, skeletal, human brains, and even the entire body’s metabolism. However, it can be more beneﬁcial to apply natural and synthetic XEs as they can be considered medications to improve human health, treat and prevent diseases if used appropriately. The current review has some inherent limitations. The ﬁrst limitation lies in the focus of the research XEs per se. Because of considering the moral hazard in conducting studies regarding the harmful effects of XEs on humans, many of the studies were observational and animal, thus limiting the application of the review results to humans. Second, studies were conducted with different research designs and subjects (human; animals), and differ- ent results were reported with researchers of different skills and training. Therefore, it is somewhat difﬁcult to make comparisons and inferences in the analysis. Moreover, there are fewer randomized controlled trials found on the topic of XEs after selection. Thus, the evidence available in XEs is not as strong as that in other topics. 4. Discussion Furthermore, there are many intermediating factors, which affect the outcomes, so the direct causal relationship of XEs effects is hard to be proved. Finally, many of the beneﬁcial and harmful effects of XEs, which have been accumulated in organisms, are dose-dependent and time-dependent, which may not be observed during a shorter period. Maybe a better investigation for the action of XEs can be achieved by a longer and closer follow-up of the research subjects. Currently, more experiments are required to study the substances in the environment. This is due to the great spectrum of toxins that organisms in the world are exposed to. The worst case of malignant tumors can develop over the years, and many factors also determine the complex process of carcinogenesis development. It is understandable to review the documentary of environmental estrogens and their inﬂuences on individual development, as well as to conduct more studies for further clariﬁcation. 5. Conclusions As endocrine disruptors, XEs can be either synthetic or natural chemical compounds derived from sources, including diet, pesticides, cosmetics, plastics, plants, industrial byproducts, metals, and medications. By mimicking the chemical structure that is naturally occurring estrogen compounds, even the weakly active compounds could interfere with the hormonal balance with persistency or high concentrations of XEs, thus possibly being associated with the occurrence of the reproductive tract or neuroendocrine disorders and congenital malformations. In contrast, some XEs are not similar to estrogens in structure and can affect the physiologic functions in ways other than ER-ERE ligand routes. In addition to the classical ER-signaling pathway, some endocrine disruptors can exert effects via various pathways other than classical ER-signaling. The dose-related and time-dependent effects of XEs on organisms should be consid- ered. XEs are most likely to exert tissue-speciﬁc and non-genomic actions when estrogen concentrations are relatively low. Current research reported that there is not only a single Int. J. Mol. Sci. 2021, 22, 4013 20 of 27 20 of 27 factor affected by XEs, but opposite directions are also found on several occasions, or even different components stem from the identical endocrine pathway. Thus, the roles of XEs are more challenging and unpredictable in terms of physical health. Although there are numerous studies of XEs or endocrinal disruptors in the literature, many of them are observational and animal, thus limiting the application of the studies to humans. In addition, there are fewer randomized controlled trials of this topic found in the literature. Furthermore, many intermediating factors, which affect the outcomes are difﬁcult to be controlled in these studies, so the direct causal relationship of XEs effects is hard to be proved. Moreover, many of the beneﬁcial and harmful effects of accumulated XEs are dose-dependent and time-dependent, which may not be observed during a shorter period, and may need a longer and closer follow-up of the research subjects. This review provides a current summary of the identiﬁcation, detection, metabolism, and action of XEs. How- ever, many details of the underlying mechanisms remain unknown and warrant further investigation. Author Contributions: L.-H.W. and K.-H.C. conceived and designed the study; L.-H.W., L.-R.C. and K.-H.C. performed the data collection; L.-H.W., L.-R.C. and K.-H.C. analyzed the data; L.-H.W. and K.-H.C. wrote the paper. All authors have read and agreed to the published version of the manuscript. 5. Conclusions Funding: This work and APC were funded by a grant from Taipei Tzu-Chi Hospital, Taiwan (TCRD-TPE-110-06) for KH Chen. 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https://openalex.org/W2291389152	https://boa.unimib.it/bitstream/10281/103884/2/10281-103884.pdf	English	null	A TMS investigation on the role of the cerebellum in pitch and timbre discrimination	Cerebellum & ataxias	2,016	cc-by	6,153	© 2016 Lega et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. A TMS investigation on the role of the cerebellum in pitch and timbre discrimination Carlotta Lega1, Tomaso Vecchi2,3, Egidio D’Angelo2,3 and Zaira Cattaneo1,3* Abstract Background: Growing neuroimaging and clinical evidence suggests that the cerebellum plays a critical role in perception. In the auditory domain, the cerebellum seems to be important in different aspects of music and sound processing. Here we investigated the possible causal role of the cerebellum in two auditory tasks, a pitch discrimination and a timbre discrimination task. Specifically, participants performed a pitch and a timbre discrimination task prior and after receiving offline low frequency transcranical magnetic stimulation (TMS) over their (right) cerebellum. Results: Suppressing activity in the right cerebellum by means of inhibitory 1 Hz TMS affected participants’ ability to discriminate pitch but not timbre. Conclusion: These findings point to a causal role of the cerebellum in at least certain aspects of sound processing and are important in a clinical perspective helping understanding the impact of cerebellar lesions on sensory functions. Keywords: Pitch, Timbre, Transcranial magnetic stimulation (TMS), Auditory discrimination * Correspondence: zaira.cattaneo@unimib.it 1Department of Psychology, University of Milano-Bicocca, Milan, Italy 3Brain Connectivity Center, C. Mondino National Neurological Institute, Pavia, Italy Full list of author information is available at the end of the article Lega et al. Cerebellum & Ataxias (2016) 3:6 DOI 10.1186/s40673-016-0044-4 Lega et al. Cerebellum & Ataxias (2016) 3:6 DOI 10.1186/s40673-016-0044-4 Procedure The aim of this study was to analyse the role of the cere- bellum in pitch and timbre processing using transcranial magnetic stimulation (TMS). While neuroimaging tech- niques provide correlational evidence regarding the activation in a specific brain region during an ongoing cognitive process, TMS allows establishing the causal role of specific cortical areas in a given task [37, 38]. Moreover, participants in TMS experiments act as their own controls overcoming some of the limitations intrinsic in patients’ studies, such as potential differences in pre- morbid ability, and variability depending on high hetero- geneity of lesions’ sizes and gravity. Specifically, in this study we applied off line low frequency repetitive TMS to induce transient suppression of cerebellar activity [39, 40] before participants’ performance in a pitch and timbre dis- crimination tasks. If the cerebellum is causally involved in pitch and timbre processing, participants should perform worse following real than sham (faked) stimulation. Figure 1 shows the experimental paradigm (Fig. 1a) and the timeline of an experimental trial (Fig. 1b). Partici- pants seated comfortably in a dimly lit room and stimuli were binaurally delivered through professional head- phone (Sennheiser HD 280 Pro headphone). Each sub- ject took part in two different sessions (Real and Sham) that were separated by an average of 6 days (range 5–7). In each session, participants performed both the pitch discrimination task and the timbre discrimination task twice: once before, and once after receiving 15 min of off-line 1Hz rTMS over the right cerebellum. During TMS, no task was performed and participants were instructed to minimise movements and be silent. The post-stimulation task started immediately after the end of the stimulation. Both the pitch and timbre discrimin- ation tasks required participants to indicate by left/right key pressing using their dominant hand whether two consecutively presented sounds (separated by 1 sec of silence interval) were identical or different. Participants were instructed to respond as fast and as accurately as possible. Intertrial interval was 2 sec. In each task, 42 sounds were presented: in half of the trials the two sounds to be compared were identical, in the other half they were different. In the different trials of the pitch task, the second pure tone presented could be 20, 30 or 40 Hz higher (ascending trials) or lower (descending tri- als) compared to the first one. The number of descend- ing and ascending trials was counterbalanced. Background via brain stimulation has been found to impact on visual processing [16, 17]. The cerebellum is traditionally considered a “motor con- troller” and its role in the acquisition of motor skills is well established [1, 2]. Nevertheless, accumulating evi- dence suggests that the cerebellum may play a critical role in non-motor functions, contributing to cognitive and emotional processing [3–6]. In particular, a cerebel- lar involvement has been found in high-order cognitive processes such as language [7], working memory [8], and spatial processing [9, 10]. Furthermore, the cerebel- lum seems to play a role in the generation of sensory predictions [11, 12], optimising perception [13]. Accord- ingly, patients with cerebellar lesions are often impaired in processing visual information, such as in deciding speed and direction of moving stimuli [14]. Neuroimaging evi- dence also shows that the cerebellum is significantly active in neurologically unimpaired individuals during sensory discrimination, such as visual (and auditory) motion dis- crimination [15]. Also, interfering with cerebellar activity The cerebellum is also involved in auditory processing, where it plays a critical role in timing [18–21]. Cerebellar activations have also been observed in healthy subjects during passive listening of both music [22, 23] and speech [24, 25]. Moreover, pitch discrimination and melody dis- crimination [26–28], as well as sound intensity and dur- ation discrimination [29], activate cerebellar regions. For instance, Petacchi and colleagues [23] showed that cere- bellar activity significantly increased during a pitch dis- crimination task compared to passive listening, with the cerebellum responding more when the difficulty of the discrimination to be performed increased. Importantly, cerebellar activity during auditory discrimination tasks has been consistently observed even in the absence of any motor or cognitive component [30]. Patients’ findings sup- port evidence obtained in healthy individuals: cerebellar disorders are often associated to deficits in melody recog- nition [31], in discriminating small differences in sound duration [32] and in pitch discrimination [33]. Although there is considerable evidence that the cere- bellum contributes to auditory perception [30], the pre- cise role of the cerebellum in different aspects of sound Lega et al. Cerebellum & Ataxias (2016) 3:6 Page 2 of 7 Page 2 of 7 were presented at a level of 75 dB SPL. Stimuli used in the Timbre discrimination task consisted of 21 complex tones of 200 msec duration. Background Sound files in the timbre task were created from digitized samples of real musical instruments, with all instruments be- longing to the wind or string family. Sound files used in the timbre task were taken from the University of Iowa Musical Instrument Samples (Lawrence Fritts, http://theremin.music.uiowa.edu/MIS.html). processing is not completely clear. In particular, whilst neuroimaging [23, 30] and patients’ [33] studies converge in indicating a role of the cerebellum in pitch processing, whether the cerebellum also contributes to other sound features such as timbre (i.e., the property of a sound which allows a person to distinguish musical instrument when pitch, loudness and duration remain identical), is less clear. Indeed, whereas some neuroimaging studies re- ported significant cerebellar responses to sound timbre [34, 35], in other studies investigating timbre processing cerebellar activations were not considered [36]. Participants Fourteen participants (9 F; mean age = 21.93 ys; SD = 1.86) took part in the experiment. All participants were right- handed [41] and had less than 3 years of formal musical training, as revealed by a self-reported history of musical experience. Prior to the experiment, each participant filled in a questionnaire (translated from Rossi et al. [42]) to evaluate compatibility with TMS. None of the volunteers reported neurological problems, familiarity for seizures nor was taking any medication that could interfere with neuronal excitability. Written informed consent was obtained from all participants before the experiment. The protocol was approved by the local ethical committee. Participants’ treatment was conducted in accordance with the Declaration of Helsinki. Procedure In the timbre task, the two sounds to be compared in each trial were identical in terms of frequency and intensity, but they had different timbre. In particular, two different string sounds may be presented, or two different wind sounds (wind and string sounds were never presented in the same trial to avoid ceiling effects in recognition). Task order (pitch and timbre discrimination), TMS con- dition order (Real vs. Sham), and the response key assignment for same/different response were counterba- lanced across participants. The software E-prime 2.0 (Psychology Software Tools, Pittsburgh, PA) was used for stimuli presentation, data collection and TMS trig- gering. Pre and post-stimulation task sessions lasted approximately 10 min (5 min for each task). Stimuli Stimuli used in the pitch discrimination task consisted of 21 pure tones (i.e., tones with a sinusoidal waveform, where the wave consists of a single frequency) of 200 ms generated through the software Audacity (http://audacity.sourceforge.net/). All tones had a fre- quency comprised between 1000 and 1200 Hz, and Lega et al. Cerebellum & Ataxias (2016) 3:6 Page 3 of 7 Fig. 1 a The experimental paradigm: participants underwent two experimental sessions, one with real TMS, and the other with sham TMS (order of sessions counterbalanced). In each session, participants performed the task twice, once before and once after receiving 1 Hz repetitive 15 min TMS over the right cerebellum. b The timeline of an experimental trial. In the pitch discrimination task the two sounds were pure tones only differing for pitch. In the timbre discrimination task the two sounds were tones of the same instrumental family (wind vs. string) presented at the same high frequency and differing only in their timbre characteristic Fig. 1 a The experimental paradigm: participants underwent two experimental sessions, one with real TMS, and the other with sham TMS (order of sessions counterbalanced). In each session, participants performed the task twice, once before and once after receiving 1 Hz repetitive 15 min TMS over the right cerebellum. b The timeline of an experimental trial. In the pitch discrimination task the two sounds were pure tones only differing for pitch. In the timbre discrimination task the two sounds were tones of the same instrumental family (wind vs. string) presented at the same high frequency and differing only in their timbre characteristic Transcranial magnetic stimulation g TMS was administered over the right cerebellum by means of a Magstim Rapid2 machine (Magstim Co Ltd, Whitland, UK) with a 70 mm butterfly coil. An air- cooled coil was used, in order to avoid coil overheating. A fixed intensity of 45 % of the maximum stimulator output was used, in line with prior studies [43]. The right cerebellar hemisphere was targeted in light of pre- vious evidence pointing to a right lateralized cerebellar activation during timbre processing [35], whereas pitch discrimination seems to induce bilateral cerebellar acti- vations [23]. The right cerebellum was localised in each participant as the region located 1 cm under and 3 cm lateral to the inion as in prior studies [44, 45]. Prior studies using neuronavigated TMS have demonstrated that this point lies over the cerebellar hemisphere [17, 46]. The coil was placed over the right cerebellum with the handle point- ing upward, parallel to the inion-nasion line [44, 47]. Previ- ous studies have shown that rTMS at 1 Hz temporarily reduces the excitability of the stimulated cortex for a time window that outlasts the period of stimulation [39, 40]. Sham stimulation was conducted with the coil held at a 90° position in order to ensure that the magnetic field did not stimulate the target area. The stimulation paradigm in the sham condition was the same as that of real rTMS stimulation. Analyses were performed on mean accuracy scores and on mean reaction times (RT) for correct responses. Prior to analyses, reaction times 3 s.d.’s above or below the partici- pants’ mean were removed (this corresponded to 1.33 % and 1.99 % of the trials in the pitch and timbre discrimin- ation task, respectively). A repeated-measures analysis of variance (ANOVA) with TMS condition (real vs. sham) and Session (pre-stimulation vs. post-stimulation) as within- subjects factors was performed separately for the pitch and the timbre discrimination task on accuracy scores and cor- rect RT. Bonferroni-Holmes correction was applied to post-hoc comparisons. Pitch discrimination task Mean accuracy was above 73 % (SD = 9 %) in all the experimental conditions. Analysis on accuracy scores re- vealed no significant main effects of TMS, F(1,13) = 3.75, p = .08, ηp 2 = .22, and of Session, F(1,13) = 1.47, p = .25, ηp 2 = .10. The interaction TMS by Session was not signifi- cant, F(1,13) < 1, p = .92, ηp 2 = .00. Mean correct RT are shown in Fig. 2. The ANOVA on correct RT showed no sig- nificant main effect of either TMS, F(1,13) = 1.30, p = .27, ηp 2 = .09, or Session, F(1,13) = 2.86, p = .11, ηp 2 = .18. The interaction TMS by Session was significant, F(1,13) = 8.08, Lega et al. Cerebellum & Ataxias (2016) 3:6 Page 4 of 7 Fig. 2 Mean response latencies for correct responses as a function of TMS (Real vs. Sham) and Session (Pre vs. Post stimulation) in the Pitch discrimination task. Participants were significantly faster (as indicated by the asterisk) in the post compared to the pre session when sham TMS was delivered, reflecting learning effects. RT were comparable in the pre and post real TMS sessions, suggesting that real TMS affected learning effects. Error bars represent ±1 SEM Fig. 2 Mean response latencies for correct responses as a function of TMS (Real vs. Sham) and Session (Pre vs. Post stimulation) in the Pitch discrimination task. Participants were significantly faster (as indicated by the asterisk) in the post compared to the pre session when sham TMS was delivered, reflecting learning effects. RT were comparable in the pre and post real TMS sessions, suggesting that real TMS affected learning effects. Error bars represent ±1 SEM p = .01, ηp 2 = .38. Post-hoc t-tests revealed that participants were significantly faster in responding in the post-sham stimulation session compared to the pre-sham stimulation session, t(13) = 3.52, p = .016, reflecting learning effects. In turn, RT were comparable between pre-real and post-real stimulation sessions, t(13) = .63, p = .54, suggesting that real TMS interfered with learning. Moreover, whilst RT were comparable in the sham and real pre-sessions, t(13) = 1.24, p = .24, indicating a similar level of baseline performance, participants tended to be slower following real, t(13) = 2.31, p = .09 (p = .03 uncorrected), than sham TMS. Timbre discrimination task Mean accuracy was above 82 % (SD = 9 %) in all the ex- perimental conditions. The ANOVA on accuracy scores revealed no significant main effect of either TMS, F(1,13) = .50, p = .49, ηp 2 = .04, or Session, F(1,13) = .16, p = .22, ηp 2 = .12. The interaction TMS by Session was not significant, F(1,13) < 1, p = .99, ηp 2 = .00. Figure 3 shows mean participants’ correct RT. The ANOVA re- vealed a significant main effect of Session, F(1,13) = 4.95, p = .04, ηp 2 = .28: participants were overall faster in the post-stimulation session (irrespective of stimulation being real or sham), reflecting learning effects. Neither the main effect of TMS, F(1,13) = .03, p = .87, ηp 2 = .00, nor the inter- action TMS by Session, F(1,13) = .57, p = .46, ηp 2 = .04, reached significance. Pitch discrimination task of prior neuroimaging and patients’ data suggesting a cerebellar involvement in processing of music and single sound features [31–33, 48]. We found that interfering with cerebellar excitability via offline low frequency TMS significantly affected pitch discrimination, whereas it had no effect on timbre discrimination. In particular, in the pitch discrimination task real TMS counteracted learning effects that emerged in the other experimental conditions as faster responses in the post compared to the pre stimulation sessions. Our findings are consistent with prior neuroimaging and neuropsychological evidence suggesting that the cerebellum is involved in perceptual tasks [8, 49, 50], possibly monitoring the incoming sensory events to optimize perception [13, 51]. More specifically, our data add to previous studies that showed that discrimination of pitch and melody elicit activation of cerebellar regions [26, 28, 33], pointing to a causal role for the (right) cere- bellum in processing pitch. Accordingly, resting state fMRI has shown functional connectivity between bilat- eral anterior cerebellum and the auditory cortex in the temporal lobes [52]. In line with this, the cerebellum and the lateral anterior temporal lobe appeared to be bidirectionally interconnected during a rhyming judg- ment task [53]. More in general, consistent evidence suggests that the cerebellum projects not only to motor, but also to somatosensory areas [6]. Still, it is important to consider that the type of stimulation we used may have only affected posterior cerebellar regions, given the deep position of the cerebellum [54]. Indeed, coil geom- etry seems to be an important factor in determining effective stimulation of deep cerebellar regions, with the Discussion figure of eight coil likely being suboptimal when target- ing motor areas [54]. Nonetheless, cerebellar stimulation parameters similar to ours significantly affected percep- tual [16] and cognitive (for instance, linguistic) functions in prior studies (e.g. [55, 56]), suggesting that the stimu- lation we used was able to interfere with neural activity in the cerebellar-cortical network subtending discrimin- ation of sound features. Although prior neuroimaging evidence also suggested a possible role for the cerebellum in timbre processing [34, 57], we did not find evidence for this in our study. On the one hand, the lack of TMS effect in the timbre discrimination task ensures that the effects we reported in the pitch discrimination task were not due to unspe- cific effects of TMS slowing down responses regardless of the specific task at play. On the other hand, it is pos- sible that real TMS affects auditory discrimination task only when the task has a certain level of complexity. Per- formance accuracy was indeed overall higher in the timbre than in the pitch task. This is in line with prior literature showing that non-musicians are more sensitive in sound categorization to changes in timbre than to changes in pitch [58]. Interestingly, previous studies demonstrated a positive correlation between cerebellar activation and task difficulty [15, 23, 28, 59]. Moreover, the right cerebellum may be more important than the left in difficult auditory discrimination [57]. Discussion In this study we aimed to shed light on the possible causal role of the cerebellum in auditory processing, and in particular in pitch and timbre discrimination, in light Lega et al. Cerebellum & Ataxias (2016) 3:6 Page 5 of 7 Fig. 3 Mean response latencies for correct responses as a function of TMS (Real vs. Sham) and Session (Pre and Post stimulation) in the Timbre discrimination task. Participants were overall faster in the post compared to the pre session, reflecting learning effects. The type of stimulation (Real vs. Sham) did not affect performance. Error bars represent ±1 SEM Fig. 3 Mean response latencies for correct responses as a function of TMS (Real vs. Sham) and Session (Pre and Post stimulation) in the Timbre discrimination task. Participants were overall faster in the post compared to the pre session, reflecting learning effects. The type of stimulation (Real vs. Sham) did not affect performance. Error bars represent ±1 SEM figure of eight coil likely being suboptimal when target- ing motor areas [54]. Nonetheless, cerebellar stimulation parameters similar to ours significantly affected percep- tual [16] and cognitive (for instance, linguistic) functions in prior studies (e.g. [55, 56]), suggesting that the stimu- lation we used was able to interfere with neural activity in the cerebellar-cortical network subtending discrimin- ation of sound features. right hemispheric specialization in processing spectral sound features, that are critical for discriminating timbre differences [62–64]. In line with these findings, the left cerebellum may be more important than the right in timbre processing (cerebral cortex fibers mainly project- ing to the contralateral cerebellar cortex [65]). However, other studies in infants [66] and in adults [35, 67] re- ported left hemispheric cerebral cortex engagement underlying perception of timbre change. Moreover, Reiterer and colleagues [35] showed a right cerebellar activation during timbre processing, speculating that pre-linguistic sound features (including timbre) may be represented by a complex network that connects Broca’s area and the right cerebellum. In turn, previous neuro- imaging studies mainly indicate bilateral cerebellar acti- vation during pitch processing [23, 26–28, 30, 34], although some degree of lateralization may occur depending on task complexity [29, 57, 68]. Evidences are thus not entirely consistent regarding lateralization of timbre and pitch processing. Future studies may address this issue by comparing the effect of left and right cere- bellar stimulation on auditory discrimination. References 1. Manto M, Bower JM, Conforto AB, Delgado-García JM, Da Guarda SNF, Gerwig M, et al. Consensus paper: Roles of the cerebellum in motor control- the diversity of ideas on cerebellar involvement in movement. Cerebellum. 2012;11(2):457–87. 1. 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Neuroreport. 2000;11(6):1301–6. Acknowledgement f h h d tomography (PET) study. Hear Res. 2011;282(1–2):35–48. None of the authors declare to have any financial or non-financial compet- ing interests. None of the authors declare to have any financial or non-financial compet- ing interests. 24. Callan DE, Tsytsarev V, Hanakawa T, Callan AM, Katsuhara M, Fukuyama H, et al. Song and speech: Brain regions involved with perception and covert production. Neuroimage. 2006;31(3):1327–42. Authors’ contributions d h 21. Teki S, Grube M, Kumar S, Griffiths TD. Distinct neural substrates of duration- based and beat-based auditory timing. J Neurosci. 2011;31(10):3805–12. CL carried out the TMS study, performed the statistical analyses and drafted the manuscript. ZC conceived of the study and drafted the manuscript. TV and ED participated in design of the experiment. All authors read and approved the final manuscript. 22. Brown S, Martinez MJ, Parsons LM. Passive music listening spontaneously engages limbic and paralimbic systems. Neuroreport. 2004;15(13):2033–7. 23. Petacchi A, Kaernbach C, Ratnam R, Bower JM. Increased activation of the human cerebellum during pitch discrimination: A positron emission tomography (PET) study. Hear Res. 2011;282(1–2):35–48. Conclusions In sum, our findings show that the (right) cerebellum plays a causal role in pitch processing. Future research is needed to better clarify the role of cerebellum in other aspects of auditory processing, such as rhythm or com- plex melody recognition and discrimination. Moreover, level of expertise in determining the involvement of the cerebellum in auditory functions deserves consideration. In fact, prior studies showed greater engagement of the In interpreting our data, it is also worth mentioning that pitch and timbre processing may have a different degree of lateralization in the brain. 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https://openalex.org/W2985374215	https://phcfm.org/index.php/phcfm/article/download/2012/3477	English	null	The execution rate of procedures to diagnose extrapulmonary tuberculosis in Botswana	African journal of primary health care & family medicine	2,020	cc-by	5,481	African Journal of Primary Health Care & Family Medicine ISSN: (Online) 2071-2936, (Print) 2071-2928 Original Research Page 1 of 6 Dates: Keywords: extrapulmonary tuberculosis; execution rate of procedures; medical officers; Botswana. How to cite this article: Mudiayi T-K, Tshitenge S, Kgwaadira BT, Nkubito GK. The execution rate of procedures to diagnose extrapulmonary tuberculosis in Botswana. Afr J Prm Health Care Fam Med. 2020;12(1), a2012. https://doi.org/ 10.4102/phcfm.v12i1.2012 Copyright: © 2020. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. The execution rate of procedures to diagnose extrapulmonary tuberculosis in Botswana Read online: Authors: Tantamika-Kabamba Mudiayi1 Stephane Tshitenge2 Botshelo T. Kgwaadira1 Grace K. Nkubito1 Affiliations: 1Botswana National Tuberculosis Programme (BNTP), Ministry of Health and Wellness, Gaborone, Botswana 2Department of Family Medicine and Public Health, Faculty of Medicine, University of Botswana, Gaborone, Botswana Corresponding author: Stephane Tshitenge, stephotshitenge@yahoo.com Dates: Received: 19 Dec. 2018 Accepted: 12 Aug. 2019 Published: 27 Jan. 2020 How to cite this article: Mudiayi T-K, Tshitenge S, Kgwaadira BT, Nkubito GK. The execution rate of procedures to diagnose extrapulmonary tuberculosis in Botswana. Afr J Prm Health Care Fam Med. 2020;12(1), a2012. https://doi.org/ 10.4102/phcfm.v12i1.2012 Copyright: © 2020. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Background: Extra-pulmonary tuberculosis (EPTB) accounts for about 20% of TB cases worldwide. Its diagnosis is challenging. Aim: This study meant to assess the prevalence of EPTB types, procedures to diagnose EPTB and medical officers’ (MOs) views on procedures performed in the diagnosis of EPTB over a 2-year period in Botswana. Setting: The study was conducted in 13 urban and rural facilities of 29 health districts in Botswana. Methods: This was a cross-sectional study that reviewed patients’ TB data and administered a questionnaire to MOs. Methods: This was a cross-sectional study that reviewed patients’ TB data and administered a questionnaire to MOs. Methods: This was a cross-sectional study that reviewed patients’ TB data and administered a questionnaire to MOs. This significantly contribu to TB morbidity, as demonstrated by an high TB and HIV co-infection rate of 55% amongst cases in 2014.2,4,5 Extra-pulmonary TB (EPTB) accounts for about 20% of global TB cases, disproportionately affects individuals with HIV in endemic areas and displays more dive clinical manifestations and a worse outcome than pulmonary TB (PTB).2,6,7,8 It also presents m diagnostic challenges, especially in the developing world, because of scarcity of both diagno tools and procedures performed to obtain and analyse tissues.4,9,10,11,12 In Botswana, annual repo showed a steady rise in EPTB rates from 11% in 1990 and 13% in 2006 to 17% in 2008 and 19% 2013 respectively.2,11 Whilst bacteriological confirmation constitutes an important indicator for adequate managem of PTB and is well documented as demonstrated in annual performance reports, data on qua of diagnosis of EPTB are lacking.2,4,12 With partners’ support, Botswana National Tuberculosis Programme (BNTP) has invested in diagnostic processes, including a high laboratory coverage, the capacity-building of health c workers (HCWs) through training and clinical mentorship programmes and of late, introduction of the Xpert MTB/RIF assay technology that has decreased the time to TB diagno and has improved diagnostic accuracy.13 basic procedures to diagnose EPTB such as pleural tap (p = 0.032) or FNA (p < 0.0001). Conclusion: This study reviewed and evaluated the proportion of EPTB and inquired abou MO’s experience in managing EPTB. Despite MOs’ attendance at Botswana Nation Tuberculosis Programme (BNTP) TB case management (TBCM) training, the emphasis by th BNTP guidelines and availability of logistics, the execution rate of procedures to diagnos EPTB was still low in Botswana. Keywords: extrapulmonary tuberculosis; execution rate of procedures; medical officer Botswana. ne: Scan this QR code with your smart phone or mobile device to read online. Conclusion: This study reviewed and evaluated the proportion of EPTB and inquired about MO’s experience in managing EPTB. Despite MOs’ attendance at Botswana National Tuberculosis Programme (BNTP) TB case management (TBCM) training, the emphasis by the BNTP guidelines and availability of logistics, the execution rate of procedures to diagnose EPTB was still low in Botswana. African Journal of Primary Health Care & Family Medicine ISSN: (Online) 2071-2936, (Print) 2071-2928 Page 1 of 6 Original Research African Journal of Primary Health Care & Family Medicine ISSN: (Online) 2071-2936, (Print) 2071-2928 Page 1 of 6 Original Research African Journal of Primary Health Care & Family Medicine ISSN: (Online) 2071-2936, (Print) 2071-2928 Page 1 of 6 Original Research Methods: This was a cross-sectional study that reviewed patients’ TB data and administered a questionnaire to MOs. Results: About 2 in 10 TB (n = 2996, 22.7%) cases were classified as EPTB. The most common site of EPTB was pleural (n = 1066, 36.7%) followed by lymph node (LN) (n = 546, 18.8%). A pleural tap was performed in 182 (17.0%) cases of pleural TB and a fine needle aspiration (FNA) in one-third (n = 160, 29.6%) of LN TB cases. There were statistical differences in work experience amongst MOs’ responses regarding their self-reported confidence to undertake basic procedures to diagnose EPTB such as pleural tap (p = 0.032) or FNA (p < 0.0001). http://www.phcfm.org Open Access ne: Scan this QR code with your smart phone or mobile device to read online. ding author: Tshitenge, tenge@yahoo.com 19 Dec. 2018 12 Aug. 2019 27 Jan. 2020 e this article: K, Tshitenge S, BT, Nkubito GK. tion rate of s to diagnose onary tuberculosis na. Afr J Prm Health Med. 2020;12(1), t ps://doi.org/ hcfm.v12i1.2012 he Authors. AOSIS. This work under the ommons n License. Introduction Africa accounts for 3 in 10 cases of tuberculosis (TB) worldwide and about 4 in10 cases of mortality globally.1 In 2014, Botswana registered 6542 cases with a death rate of 9% in a populat of 2 million.2,3 Botswana has a high human immunodeficiency virus (HIV) prevalence in gene population (18.5%; Botswana AIDS Impact Survey [BAIS IV] 2013). Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Introduction Africa accounts for 3 in 10 cases of tuberculosis (TB) worldwide and about 4 in10 cases of TB mortality globally.1 In 2014, Botswana registered 6542 cases with a death rate of 9% in a population of 2 million.2,3 Botswana has a high human immunodeficiency virus (HIV) prevalence in general population (18.5%; Botswana AIDS Impact Survey [BAIS IV] 2013). This significantly contributes to TB morbidity, as demonstrated by an high TB and HIV co-infection rate of 55% amongst TB cases in 2014.2,4,5 Extra-pulmonary TB (EPTB) accounts for about 20% of global TB cases, but disproportionately affects individuals with HIV in endemic areas and displays more diverse clinical manifestations and a worse outcome than pulmonary TB (PTB).2,6,7,8 It also presents more diagnostic challenges, especially in the developing world, because of scarcity of both diagnostic tools and procedures performed to obtain and analyse tissues.4,9,10,11,12 In Botswana, annual reports showed a steady rise in EPTB rates from 11% in 1990 and 13% in 2006 to 17% in 2008 and 19% in 2013 respectively.2,11 Copyright: © 2020. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Whilst bacteriological confirmation constitutes an important indicator for adequate management of PTB and is well documented as demonstrated in annual performance reports, data on quality of diagnosis of EPTB are lacking.2,4,12 With partners’ support, Botswana National Tuberculosis Programme (BNTP) has invested in TB diagnostic processes, including a high laboratory coverage, the capacity-building of health care workers (HCWs) through training and clinical mentorship programmes and of late, the introduction of the Xpert MTB/RIF assay technology that has decreased the time to TB diagnosis and has improved diagnostic accuracy.13 http://www.phcfm.org http://www.phcfm.org Page 2 of 6 Page 2 of 6 Original Research Patients’ data were collected manually by TB coordinators and TB focal persons after orientation for consistency by investigators using the ‘record review data abstraction form’ where relevant variables were recorded for easy and rapid data extraction. Information was collected from (1) unit (facility/district) TB registers, (2) patient TB treatment card and (3) Record Review Data Abstraction Form. Variables consisted of the year, district, health facility (HF), patients with TB, EPTB and their site. The majority of medical officers (MOs) in Botswana are trained in an African setting. Introduction As TB is a common disease there, MOs are expected to be familiar with procedures to diagnose EPTB that include basic skills such as fine needle aspiration (FNA), lumbar puncture (LP), abdominal tap, pleural tap and excision biopsy of superficial tissue. Advanced skills to diagnose EPTB, such as pericardial tap, pleural biopsy, abdominal ultrasound and echocardiography, are generally performed by radiographers, radiologists or other specialists in referral hospitals. Besides, TB coordinators and TB focal persons were assigned to disseminate information to MOs on the upcoming study at their sites; consent forms were then distributed. Self- administered questionnaires were issued to MOs willing to participate in the study; they were asked to return questionnaire in 2 weeks’ time in a designated box. Information collected was (1) demographic information (sex, work experience, TB experience, place of practice), (2) whether s/he was confident of TBCM, (3) when was last TB refresher course attended, (4) whether there was available logistics such as TBGs and medical equipment and (5) access to a supervisor or a mentor. This study aimed to auditing the quality of diagnosis of EPTB in Botswana. It meant to assess the prevalence of EPTB types, rates of procedures performed to diagnose it and MOs’ views on procedures performed in the diagnosis of EPTB. Ethical consideration Ethical clearance was granted by Health Research Development Council (HRDC) in the notification of IRB Review Ref. No: PPME-13/18/1 Vol VIII (328), and access to records was granted by the Ministry of Health & Wellness (MoH&W) in a Letter of Support for EPTB Study – Ref: DPH 20/11 XII (22) sent to respective DHMTs. The BNTP guidelines (TBGs) provide guidance for diagnosis of EPTB. Extra-pulmonary tuberculosis was defined as the TB occurring outside the lung.10 Presumed diagnosis of EPTB was made when a patient presented symptoms and/or signs of TB related to an extra-pulmonary site, revealed by procedures (e.g. imaging or laboratory investigations) and followed by a decision taken by an MO to initiate anti-TB therapy. Diagnosis of EPTB is confirmed when the presence of Mycobacterium tuberculosis is demonstrated on specimens taken from affected anatomical sites by a positive culture, smear or Xpert MTB/RIF assay. A miliary TB was considered an EPTB if it signalled a disseminated TB. In Botswana, diagnosis of EPTB is the responsibility of MO. At a facility level, manual TB registers, electronic TB registers and patient TB cards help to capture routine patient information. Patient treatment cards are an important tool for assessing treatment adherence and keeping notes on disease evolution. Methods This was a cross-sectional study that reviewed patients’ TB data from 01 January 2010 to 31 December 2012 and administered a questionnaire to MO during the same period. The BNTP supervises the TB programme. Botswana health services comprise 29 health districts that run TB preventive and curative activities. Laboratory and radiology services are available in all district hospitals, which have secondary care facilities, as well as primary hospitals. The BNTP’s role includes conducting TB training for trainers (TOT), TB case management (TBCM) training, clinical mentorship visits and monitoring and evaluation of the programme. The role of TOT is to implement in-service TB training at local level. We summarised the data using mean value ± standard deviation (s.d.) for normally distributed variables, median ± interquartile range for skewed and frequency in percentage for binomial variables. Chi-square test was used to analyse association between MOs’ responses about their comfortability to perform basic and advanced skills and their work experience. R software, version 3.0.0, with R commander package, version 1.9-6, was used to capture and analyse the data. The level of statistical significance was p = 0.05. We selected 13 of the 29 health districts of Botswana. For this study, Gaborone and Francis town, the two major cities in Botswana, were selected. We also selected 11 districts, and amongst these, eight were rural and three were urban districts. The selected districts were Chobe, Ghanzi, Jwaneng, Kgalagadi North, Kgalagadi South, Kweneng East, Mabutsane, Ngamiland, Okavango, Tutume and Tonota. These districts had the highest TB notifications and accounted for about 60% of TB patients in Botswana.2 Ethical clearance was obtained from the Health Research Development Council (HRDC) in the notification of IRB Review, and access to the records was obtained from the Ministry of Health & Wellness (MoH&W) in the letter of support for EPTB sent to respective District Health Management Teams (DHMTs). Results The TB registers of 13 selected districts had a record of 13, 148 TB cases, of these 2996 (22.7%) cases were classified as EPTB. Amongst the EPTB cases, 1619 (54.0%) were males and 1377 (46.0%) were females. Human immunodeficiency virus test was recorded in 2707 (90.4%) of EPTB cases, and TB/HIV co-infection was observed in 1920 (70.2%) of EPTB cases. Concomitant PTB/ EPTB was found in 874 (29.1%) of EPTB cases, and of these http://www.phcfm.org Open Access Open Access Page 3 of 6 Original Research Page 3 of 6 cases, only 96 (11%) were confirmed bacteriologically. The EPTB case mortality rate was 10.5%. (n = 106, 81%) of MOs responded that they were ‘comfortable’ in the reading of abnormal CXR (Table 2). The majority (n = 105, 80.2%) of MOs felt ‘comfortable’ in conducting a pleural tap whereas more than half (n = 73, 55.7%) of MOs felt ‘comfortable’ in conducting an FNA. There were statistical differences in work experience amongst MOs’ responses regarding their self-reported confidence to undertake basic procedures to diagnose EPTB such as pleural tap (p = 0.032) or FNA (p < 0.0001). cases, only 96 (11%) were confirmed bacteriologically. The EPTB case mortality rate was 10.5%. The most common site of EPTB was pleural (n = 1066, 36.7%) followed by lymph node (LN) (n = 546, 18.8%) (Figure1). Pleural TB was diagnosed using chest X-ray (CXR) in 870 (81.8%) of cases and a pleural tap was performed in 182 (17.0%) cases. To diagnose LN TB, an FNA was accomplished in one-third (n = 160, 29.6%) of the cases whereas close to half (n = 94, 43.5%) of abdominal TB was diagnosed using an abdominal ultrasound alone and/or in combination with abdominal tap (n = 7, 3.2%) and/or biopsy in one case (0.5%). In TB meningitis, an LP was performed in about half (n = 85, 48.6%) of the cases (Figure 2). On the other hand, 2 in 10 MOs felt ‘comfortable’ in performing advanced skills to diagnose EPTB such as pericardial tap (n = 19, 14.5%) or pleural biopsy (n = 20, 15.3%). There was a statistical difference in work experience amongst MOs’ responses regarding their self-reported confidence to undertake advanced procedures to diagnose EPTB such as pericardial tap (p = 0.002) or pleural biopsy (p = 0.003). Of the 141 questionnaires distributed to MOs, 131 (93%) were returned. Results All the returned questionnaires were filled satisfactorily and included in the study. About three- quarters of MOs had either 6–10-year work experience (n = 48, 36.6%) or more than 10-year experience (n = 45, 34.4%) (Table 1). Seventy-nine (60.3%) MOs worked in a hospital setting whereas 50 (38.2%) MOs practised in primary health care (PHC) clinics, and two (1.5%) were from private practice HF. 0.6 1.6 2.2 0 26.6 0.5 3.2 43.5 0.9 18.9 47.2 0.6 48.6 0.8 60 77 0.1 17 81.8 0 10 20 30 40 50 60 70 80 90 Histopathology Biochemistry Cytology All relevant cases (n = 1591) Lymph node biopsy Fine needle aspiraon Tuberculosis adenis (n = 540) Abdominal biopsy Abdominal tap Abdominal ultrasound Abdominal tuberculosis (n = 216) Pericardial tap Echography Chest X ray Tuberculosis pericardias (n = 106) Computerised Tomography Scan Lumbar puncture Tuberculosis meningis (n = 177) Bone biopsy X-ray Tuberculosis of the bone/joint (n = 125) Chest X ray Miliary tuberculosis (n = 187) Pleural biopsy Pleural tap Chest X ray Pleural tuberculosis (n = 1063) Percentage Diagnosc procedures in extra-pulmonary tuberculosis cases FIGURE 2: Performance of diagnostic procedures in extra-pulmonary tuberculosis cases, according to the site of infection in 13 health districts of Botswana, n = 2413, from 01 January 2010 to 31 December 2012. About half (n = 63, 48.1%) of MOs responded that they had attended at least one BNTP’s TBCM training or a ToT course on TBCM (n = 61, 46.5%). Also, 6 in 10 (n = 83, 63%) MOs responded that they never participated in any kind of in- service TB training in their HFs, and 7 out of 10 (n = 85, 64.9%) MOs responded that they had the latest BNTP guidelines. Also, two-thirds (n = 87, 66.4%) of MOs responded that they had previous TB management experience of at least 10 years. Amongst these, 31 (35.6%) responded that they managed TB cases every week, whilst 28 (32.2%) and 25 (28.7%) responded that they managed TB cases every month and on daily basis, respectively. Whilst 105 (80%) MOs indicated that they felt ‘comfortable’ when managing PTB in general, 83 (69%) MOs felt ‘comfortable’ when also managing EPTB. Results The majority 0 5 10 15 20 25 30 35 40 Skin Genitourinal track Pericardial Bone Meninges Miliary Unknown Abdominal Other Lymph nodes Pleural Percentage % Percentage Sites of extra-pulmonary tuberculosis FIGURE 1: Frequency of extra-pulmonary tuberculosis, according to the site of infection, in 13 health districts of Botswana, n = 2996, from 01 January 2010 to 31 December 2012. 0 5 10 15 20 25 30 35 40 Skin Genitourinal track Pericardial Bone Meninges Miliary Unknown Abdominal Other Lymph nodes Pleural Percentage % Percentage Sites of extra-pulmonary tuberculosis FIGURE 2: Performance of diagnostic procedures in extra-pulmonary tuberculosis cases, according to the site of infection in 13 health districts of Botswana, n = 2413, from 01 January 2010 to 31 December 2012. FIGURE 2: Performance of diagnostic procedures in extra-pulmonary tuberculosis cases, according to the site of infection in 13 health districts of Botswana, n = 2413, from 01 January 2010 to 31 December 2012. TABLE 1: Sex distribution and work experience of medical doctors in 13 health districts of Botswana, from 01 January 2010 to 31 December 2012 (n = 131). Work experience Male Female Total % 0–5 years 21 17 38 29.0 6–10 years 40 8 48 36.6 > 10 years 40 5 45 34.4 Total 101 30 131 100 TABLE 1: Sex distribution and work experience of medical doctors in 13 health districts of Botswana, from 01 January 2010 to 31 December 2012 (n = 131). FIGURE 1: Frequency of extra-pulmonary tuberculosis, according to the site of infection, in 13 health districts of Botswana, n = 2996, from 01 January 2010 to 31 December 2012. Open Access http://www.phcfm.org Page 4 of 6 Original Research TABLE 2: Extrapulmonary tuberculosis diagnostic skills and work experience of medical doctors in 13 health districts of Botswana, from 01 January 2010 to 31 December 2012 (n = 131). Discussion This study audited the quality of diagnosis of EPTB in the 13 health districts of Botswana and evaluated the proportion and types of EPTB and diagnostic procedures. The study meant to describe the execution rate of procedures in diagnosing EPTB and assess the level of confidence of MOs to perform basic or advanced procedures in the diagnosis of EPTB. In our study, 60.3% of MOs reported that they practised in a hospital setting and 38.2% reported that they practised in PHC clinics. A pleural tap was performed in about 2 in 10 (17.0%) TB pleural effusion cases, whereas an FNA was executed in only one-third (29.6%) of LN TB cases. Despite attendance to BNTP TBCM trainings (4 in 10 MOs), availability of BNTP guidelines (64.9% of cases) and MOs’ experience of at least 10 years (66.4% of MOs), a basic procedure such as pleural tap was performed only in 17% of pleural TB cases. Also, an LP was performed in only about half of TB meningitis cases. The low execution rate of such basic skills to diagnose EPTB appeared not to be generated because of lack of skills or logistics, as the majority of MOs felt ‘comfortable’ in performing a pleural tap (80.2%) or an FNA (55.7%). In Botswana, public health service is free of charge, and equipment to perform investigations are largely available. The low execution rate of LP could be explained by local cultural belief, as patients’ relatives often decline this procedure fearing death. In this study, 2–3 in 10 TB patients suffered from EPTB, whilst 3 in 10 EPTB patients had both PTB and EPTB. The proportion of EPTB varies from region to region, reflecting HIV prevalence. For instance, in our study, the majority of EPTB patients (70.2%) were HIV positive. Whilst countries such as Benin, with a low HIV prevalence (1%) compared to Botswana, reported an EPTB prevalence of 9%, and in Tanzania EPTB accounted for 15% in one study, and amongst them 58.3% were HIV positive.12,13,14 In this study, 36.6% EPTB cases were of pleural site and 18.8% of LNs site whereas both PTB and EPTB were observed in 29.1% of EPTB cases. African countries display similar patterns of EPTB sites of infection. Results Variable Responses Work experience Total Pearson’s chi-square p-value 0–5 years 6–10 years > 10 years n % n % n % n % Basic EPTB diagnostic procedures Abnormal chest X-ray C 31 81.6 35 72.9 40 88.9 106 81.0 0.153 NR 3 - 10 - 4 - 17 13.7 NC 4 - 3 - 1 - 8 5.3 Spine X-ray C 20 52.6 31 64.6 30 66.7 81 61.8 0.040* NR 8 - 14 - 12 - 34 24.9 NC 10 - 3 - 3 - 16 12.3 Pleural tap C 25 65.8 41 85.4 39 86.7 105 80.2 0.032* NR 6 - 6 - 4 - 16 12.2 NC 7 - 1 - 2 - 10 7.6 LN FNA C 14 36.8 31 64.6 28 62.2 73 55.7 < 0.0001* NR 2 - 11 - 11 - 24 18.7 NC 22 - 6 - 6 - 34 25.9 Abdominal tap C 28 73.7 45 93.8 41 91.1 114 87.0 0. 038* NR 3 - 2 - 2 - 7 5.3 NC 7 - 1 - 2 - 10 7.7 Advanced EPTB diagnostic procedures Pericardial tap C 5 13.2 3 6.3 11 24.4 19 14.5 0.002* NR 3 - 11 - 15 - 29 22.1 NC 30 - 34 - 19 - 83 64.4 Pleural biopsy C 5 13.2 4 8.3 11 24.4 20 15.3 0.006* NR 5 - 12 - 17 - 34 26.0 NC 28 - 32 - 17 - 77 58.7 Total n 38 - 48 - 45 - 131 100 - Percentage (%) 29.0 - 36.6 - 34.4 - 100 - n, number; EPTB, extra pulmonary tuberculosis; C, comfortable; NR, need a refresher course; NC, not comfortable; LN FNA, lymph node fine needle aspiration. p < 0.05 is statistically significant. TABLE 2: Extrapulmonary tuberculosis diagnostic skills and work experience of medical doctors in 13 health districts of Botswana, from 01 2012 ( 131) rculosis diagnostic skills and work experience of medical doctors in 13 health districts of Botswana, from 01 January 2010 to 31 December n, number; EPTB, extra pulmonary tuberculosis; C, comfortable; NR, need a refresher course; NC, not comfortable; LN FNA, lymph node fine needle aspiration p < 0.05 is statistically significant. Pakistan reported a dissimilar pattern, as LN and spine TB were the most diagnosed EPTB types in 60% of EPTB cases.15,16 Competing interests The authors have declared that no competing interests exist. Acknowledgements The authors would like to thank the following institutions and persons: Ministry of Health & Wellness, Botswana, BNTP staff, I-TECH-Botswana and Dr Jenny Ledikwe, DHMTs, Ms Cynthia Cephas, Ms Eugenie Masitha, Titi Tsholofelo, Ms Dorothy Mpho Seretse and Dr Tudu Monagen. They are grateful to Dr Farai Mavhunga, NTP Manager, Namibia and Dr Victor Williams, Technical Advisor at URC, Swaziland, for their remarks. They are deeply indebted to Dr Mookesi Molefi, University of Botswana, for his inputs in data analysis. In this study, there was a statistical difference in work experience amongst MOs’ responses regarding their self- reported confidence for undertaking basic procedures to diagnose EPTB such as pleural tap (p = 0.032) and FNA (p < 0.0001). Also, there was a statistical difference in work experience amongst MOs’ responses regarding their self- reported confidence for undertaking advanced procedures to diagnose EPTB such as pericardial tap (p = 0.002) and pleural biopsy (p = 0.003). Medical practice is grounded not only on scientific knowledge and training but also on clinical experience. With passage of time, MOs gain insights into professionalism and from transfer of skills. Original Research The study did not attempt to assess association between procedures’ performance rate and MOs’ confidence to perform these procedures. Further studies addressing these issues may be required. The study did not attempt to assess association between procedures’ performance rate and MOs’ confidence to perform these procedures. Further studies addressing these issues may be required. essential tests such as histology or biochemistry were underutilised where only 18% of EPTB cases had laboratory- confirmed diagnosis. Also in Cameroun, the overall performance of investigations was 17%, whereas in a study from Pakistan, histology was carried out in 47%, radiology in 23% and microbiology in 9% of EPTB cases.14,15,16 Fear of delaying patient’s treatment initiation could explain the non-performance of procedures. Authors’ contributions All authors contributed equally to this work. Finding out Mycobacterium tuberculosis in pleural fluid or biopsy specimens remains the gold standard for diagnosing pleural TB. However, as MOs are not ‘comfortable’ to perform pleural biopsy and given the unavailability of biopsy kits in primary care, a lymphocytic predominant exudate and a high adenosine deaminase (ADA) level may be considered as diagnostics for diagnosis of pleural TB in a high TB prevalence setting such as Botswana. We recommend the availability of ADA analysis in PHC laboratories.10,19 Botswana TB guideline has recently recommended the use of Xpert MTB/RIF assay in the routine diagnosis of EPTB for a biopsy tissue sample, cerebrospinal fluid and urine, as it could improve the quality of EPTB diagnosis and reduce delay in initiation of treatment.20 Disclaimer The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. Limitations of the study included those inherent to retrospective design such as incomplete patient records, potential for recall bias and inability to assess incidence. This study did not assess reasons for low rate of procedures’ execution to diagnose EPTB by MOs, or MO views on the CME and the support they had from the BNTP. Funding information This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Conclusion This study reviewed and evaluated the proportion and types of EPTB and inquired about MOs’ experience in managing EPTB. About 2 in 10 TB patients suffered from EPTB. Pleural and LN TB are the most commonly encountered types of EPTB. Despite MO attendance to BNTP TBCM training and the availability of TBGs and equipment, the execution rate of procedures to diagnose EPTB was still low in Botswana. We recommend portfolio-based learning for MOs managing EPTB, as it has proven to be an effective method to learn skills in CME. There is a need to review the support system of mentoring of MOs, continuous medical education (CME) and in-service lectures as currently implemented in the TB programmes which are mostly educational outreach visits (EOV). A review study reported that although more effective than educational materials or audit and feedback, EOV had modest effects on the effectiveness of guidelines’ implementation strategies, improving health professional practice and healthcare outcomes.17 Autonomy and self-direction by identifying educational needs and planning to meet individual learning objectives are important principles of adult learning. We recommend portfolio-based learning for MOs managing EPTB as it has proven to be a more effective method to learn skills in CME.18 Data availability statement Data sharing is not applicable to this article as no new data were created or analysed in this study. Discussion As an illustration, a study from Tanzania reported a similar pleural TB prevalence of 44% and a higher LN TB prevalence of 31%.14 In a study from Cameroun, it was reported that concomitant TB and ETPB occurred in 20% of cases whereas a study conducted in The low rate of procedures executed to diagnose EPTB also could be because of poor adherence of guidelines. Studies from various countries indicated inadequate adherence to institutions’ protocol on the procedures to diagnose EPTB. For instance, in a Tanzanian study, although available, http://www.phcfm.org http://www.phcfm.org Open Access Page 5 of 6 Original Research Page 5 of 6 References 1. World Health Organization. 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https://openalex.org/W4367317335	https://journal.ipb.ac.id/index.php/jmagr/article/download/44936/25478	English	null	The Effect of Goverment Policy and Market Failure on Divergence of Cassava Competitiveness in South Lampung	Jurnal Manajemen & Agribisnis/Jurnal Manajemen Agribisnis	2,023	cc-by	7,442	1 Corresponding author: Email: dyaharing@yahoo.com Dwika Mutiara Abriani), Dyah Aring Hepiana Lestari)1, Dwi Haryono) )Department of Master Agribusiness, Faculty of Agriculture, University of Lampung Jl. Soemantri Brojonegoro No. 1, Bandar Lampung 35141, Indonesia Abstract: Indonesia is one of the world's leading cassava producers, and Lampung Province is the main producer. Efforts to increase cassava production must be accompanied by good farming activities that link to a complete agribusiness system. This study aims to analyze the divergence effect of cassava competitiveness as an effect of government policies and market failure in South Lampung. This study was conducted using a survey method. The number of respondents was 64 cassava farmers, 8 cassava collectors, and 1 tapioca industry. Data collection was carried out in August-September 2022. This study uses PAM (Policy Analysis Matrix) to analyze the competitiveness and the divergence effect and market structure analysis to analyze the market failure. The result showed that there is a market failure of cassava because the market structure is not perfectly competitive (oligopsony). Therefore, the divergence effect of cassava did not reach the target because it caused the transfer of inputs prices from traders to farmers due to the fertilizer subsidy and the transfer of income from consumers to farmers due to the determination of cassava minimum price (Rp900/kg). However, cassava farming has a competitiveness with the value of comparative advantage (DRCR = 0.30) which is smaller than the value of competitive advantage (PCR = 0.58). The changes from comparative to competitive advantage require government policies to control market failure and to reduce the high transaction cost economy. Kata kunci: efek divergensi, kegagalan pasar, ubi kayu, oligopsoni, sistem agribisnis Jurnal Manajemen & Agribisnis, Vol. 20 No. 1, March 2023 Permalink/DOI: http://dx.doi.org/10.17358/jma.20.1.130 Jurnal Manajemen & Agribisnis, Vol. 20 No. 1, March 2023 Permalink/DOI: http://dx.doi.org/10.17358/jma.20.1.130 Available online at http://journal.ipb.ac.id/index.php/jmagr http://journal.ipb.ac.id/index.php/jmagr THE EFFECT OF GOVERMENT POLICY AND MARKET FAILURE ON DIVERGENCE OF CASSAVA COMPETITIVENESS IN SOUTH LAMPUNG Dwika Mutiara Abriani), Dyah Aring Hepiana Lestari)1, Dwi Haryono*) INTRODUCTION the situation. This condition caused the processing industry to fulfill its raw material needs by importing semi-finished cassava products in starch, pellets, chips, and coarse flour (Ministry of Trade Republic of Indonesia, 2017 in Sinaga et al. 2021)). In fact, farmer’s income will increase if they can increase domestic cassava production rather than importing it (Saptana et al. 2022). Policies to increasing production can be carried out by facilitating farmers to access production facilities (seed, fertilizers, pesticides), capital assistance, and marketing (Saptana et al. 2022). Cassava is one of the leading commodities in the food crops sub-sector because it can substitute for Indonesia’s main foods (rice and corn), industrial raw materials, feed ingredients, and imports/export commodities. China is Indonesia’s largest cassava export country, with US$ 19.71 million (48.14% of the total value) in 2020. On the import side, most of the cassava in Indonesia is imported from Thailand, for US$ 62.83 million (7.47% of the total value) in 2020. Lampung Province is a cassava production center in Indonesia (from seven other provinces) with a production of 6,819,473 tons (36.41% of the total value) in 2020 (Pusat Data dan Sistem Informasi Pertanian, 2021). According to (Zakaria et al. 2021), agribusiness cassava has several problems. However, the institutional collaboration in sustainable partnerships between farmer group association (Gapoktan), cassava industries (tapioca), and an ICT system could improve the farm, industries, and overall cassava agribusiness performance. Besides that, the government must keep the low prices of cassava for the tapioca industries to grow. Efforts that can be made is to provide a reasonable price or a decent price for farmers and industries. According to (Saptana et al. 2022), the output price policy can be carried out through guaranteed output prices by optimizing the function of the farmers market as a captive market for farmers. Therefore, Lampung Province must increase cassava production and productivity to always contribute in fulfill the demand for cassava in national and international markets. On the other hand, the government provides a policy to support the cassava farmers in order to compete with imported cassava products. Efforts to increase cassava production must be accompanied by good farming activities that link to a complete agribusiness system. Increasing competitiveness can be achieved through the essence of competitiveness itself, namely productivity. Sources of productivity growth are technical change, technical efficiency, and economics of scale through farming activities (Saptana, 2016). Article history: Received 6 January 2023 Revised 23 February 2023 Accepted 27 February 2023 Available online 31 March 2023 Keywords: divergence effect, market failure, cassava, oligopsony, agribusiness system Abstrak: Indonesia merupakan salah satu negara penghasil ubi kayu dunia dan Provinsi Lampung merupakan penghasil utamanya. Upaya peningkatan produksi ubi kayu harus diiringi dengan kegiatan usaha tani yang baik dan mempunyai keterkaitan dalam sistem agribisnis yang utuh. Penelitian ini bertujuan untuk menganalisis efek divergensi daya saing ubi kayu akibat kebijakan pemerintah dan kegagalan pasar di Lampung Selatan. Penelitian ini dilakukan dengan menggunakan metode survei. Jumlah responden sebanyak 64 orang petani, 8 orang pedagang pengumpul, dan 1 pabrik tepung tapioka. Pengumpulan data dilakukan pada bulan Agustus-September 2022. Penelitian ini menggunakan PAM (Policy Analysis Matrix) untuk menganalisis daya saing dan efek divergensi serta menggunakan analisis struktur pasar untuk menganalisis kegagalan pasar. Hasil penelitian menunjukkan bahwa terdapat kegagalan pasar ubi kayu yang diakibatkan oleh struktur pasar yang tidak bersaing sempurna (oligopsoni). Oleh karena itu, efek divergensi ubi kayu tidak mencapai sasaran karena menyebabkan terjadinya transfer harga input dari pedagang ke petani akibat subsidi pupuk dan transfer pendapatan dari konsumen ke petani akibat penetapan harga minimum ubi kayu (Rp 900/kg). Namun, usaha tani ubi kayu tetap berdaya saing dengan nilai keunggulan komparatif (DRCR = 0.30) yang lebih kecil dari nilai keunggulan kompetitifnya (PCR = 0.58). Untuk mewujudkan keunggulan komparatif menjadi kompetitif diperlukan kebijakan pemerintah yang ditujukan untuk menghilangkan adanya kegagalan pasar dan menekan tingginya biaya transaksi. This is an open access article under the CC BY license Kata kunci: efek divergensi, kegagalan pasar, ubi kayu, oligopsoni, sistem agribisnis 130 Copyright © 2023, ISSN: 1693-5853/E-ISSN: 2407-2524 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 METHODS Apart from being caused by government policies, divergence effect is also caused by market failure. Market failure of cassava in this study can be identified through the market structure analysis in the terms of marketing channels. Market structure defined as market characteristics based on the number of producers and consumers, several types and characteristics of the products produced, freedom and barriers of entry and exit of the market, and market price information for the products (Rumallang et al. 2019). This research was conducted in Jati Agung and Tanjung Bintang, South Lampung which was carried out purposively. It is because these two districts have the largest harvest area each of 2.000 ha and 385 ha and the highest cassava production each of 53,224 tons and 10,231.76 tons (BPS Kabupaten Lampung Selatan, 2022). The population of this study was all farmers who farm cassava in Jati Agung (1,000 people) and Tanjung Bintang (100 people). Sampling using a simple random sampling method according to Isaac and Michael in (Ismail, 2018) as follows : The competitiveness of cassava is highly dependent on the use of inputs from cassava farming which are tradable and non-tradable to produce output in the form of cassava tubers which can be distributed according to valid prices at the farmer level. This marketing activity will be resulting revenue which will determine the amount of cassava farming income that also will indicate whether the cassava farming has competitive and comparative competitiveness which can be shown from the value of private profits and social benefits by using PAM method. In addition, PAM can explain government policies based on the divergence effects that has an impact on the input, output, and input and output of cassava farming. The divergence effect can also explain cassava marketing activities by using market structure analysis that aims to increase income which is influenced by farmers’ receipts from the sale of cassava. The size of the farmer’s income is influenced by the efficiency or not of the marketing activities of the cassava itself. The framework can be seen in Figure 1. s = s = Notes: s (Farmers sample); ʎ (Confidence level (90% = 1.645)); N (Farmers population (1,100 people)); P (True chance (0.5)); Q (Wrong chance (0.5)); d (Deviation (10% = 0.1)). Based on these calculations, the sample used was 64 farmers which were divided into 58 farmers from Jati Agung and 6 farmers from Tanjung Bintang. INTRODUCTION The first line represents the private prices, the second line represents the social price, and the third line represents the impact of government policies (divergence) (Lestari et al. 2020) (Table 1). The PAM method explains the divergence effect as an effect of the government policies. As in research (Septarisco and Prihtanti, 2019), divergence effect are divided into government input policies (IT, NPCI, and FT), government output policies (OT and NPCO), and government input-output policies (EPC, NT, PC, and SRP). Calculations from this analysis are in the notes of Table 1. This study used the PAM (Policy Analysis Matrix) method to describe competitiveness which is shows three lines. The first line represents the private prices, the second line represents the social price, and the third line represents the impact of government policies (divergence) (Lestari et al. 2020) (Table 1). The PAM method explains the divergence effect as an effect of the government policies. As in research (Septarisco and Prihtanti, 2019), divergence effect are divided into government input policies (IT, NPCI, and FT), government output policies (OT and NPCO), and government input-output policies (EPC, NT, PC, and SRP). Calculations from this analysis are in the notes of Table 1. not. Therefore, this study will complement the previous research. This study does not only focus on the level of competitiveness of cassava but also analyze the divergence effect of competitiveness as an effect of government policies and market failure as well as what operational policies are to turn comparative advantage into a competitive advantage. This study using PAM (Policy Analysis Matrix) and market structure analysis. PAM can identify policy variables that directly affect the competitiveness and market structure analysis can identify the market failure that occur in the cassava’s market. INTRODUCTION These efforts can assist farmers in increasing productivity as well as comparative and competitive advantages in cassava farming. Problems can occur from tapioca industries side and farmer’s side. Tapioca industries may meet a raw material supply constraints during certain months, resulting in increase of the cost of tapioca production and decrease in profitability or competitiveness. The high costs between farmers and tapioca industries in the cassava transaction process result in weak linkages between them. During the rest of the harvest season, farmers must cover the supply of cassava by using high refraction and low prices of cassava. In other months, the tapioca industries meet a shortage of raw material supplies resulting in a decrease of the revenue and possibly a loss for the year. During the last ten years, there has been no policies from the government for cassava. The government only issued several programs that appeal to cassava industries and held a meeting between farmers and the tapioca industries in Lampung Province in 2021. The results of the meeting were to increase cassava production and productivity, do a transparent cassava transaction with a beneficial partnership, determine a minimum price of cassava (Rp900/kg) with a maximum fraction of 15%, form a forum for tapioca industries in Lampung Province, and conduct the counseling, coaching, and supervision in the development of cassava. There have been many studies on the competitiveness of cassava in Lampung Province. The results of previous studies showed cassava in Central Lampung (Asnawi & Mejaya, 2016; Endaryanto et al. 2022; Zulkarnain et al. 2021; Rosanti et al. 2018), East Lampung (Endaryanto et al. 2022; Rosanti et al. 2018), and North Lampung (Iswara et al. 2021; Rosanti et al. 2018) have a competitive and comparative advantages. However, there is no research has been found whether cassava in South Lampung have a competitiveness or Cassava production produced by Indonesian farmers cannot suffice market demand due to increased demand the development of the animal feed industry (especially large ruminants). In addition, the decrease in the harvested area of Indonesian farmers has exacerbated 131 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 This study used the PAM (Policy Analysis Matrix) method to describe competitiveness which is shows three lines. Cassava Respondents Characteristic The characteristics of respondents in this study were classified into three, that are farmers, collectors, and tapioca industry as shown in the following Table 2. There are 64 farmers in this study, based on age is on evenly distributed because most farmers are in the age group of 38–54 years. The level of education received is the high school level of 59.38% which is classified as a moderate level of education. The largest number of family responsibilities is in the group of 3-5 people with 15–27 years of farming experience. Land area with a range of 0.5–2 hectares is 84.36% which is classified as medium land with 78.12% of farmers having their own land. RESULTS is a tapioca industry named PT. Darma Agrindo that located in Jati Agung. PT. Darma Agrindo applies cassava prices in an integrated manner with all tapioca industries under PT. Bumi Waras with a total of 18 tapioca industries in Lampung Province. The increasing or decreasing cassava prices was influenced by the price of tapioca flour in the market. METHODS Besides farmers, there were 8 cassava collectors and 1 tapioca industry who were taken using the snowball sampling method as (Abriani et al. 2022) research. This study used primary data, was obtained from interviews with respondents (farmers, cassava collectors, and tapioca industry), and secondary data, was obtained from various sources, such as kind of literature, books, journals, and agencies related to this research. Data collection was carried out in August-September 2022 using the survey method. 132 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 P ISSN: 2407 5434 E ISSN: 2407 7321 Accredited by Ministry of RTHE Number 32a/E/KPT/2017 Vol. 20 No.1, March 2023 Table 1. Policy Analysis Matrix (PAM) Description Revenue Cost Profit Tradable Input Non-Tradable Input Private Price A B C D Social Price E F G H Divergence I J K L Source: Monke & Pearson (1995) Notes Cassava Farming: Private Profits D = A – (B+C) Social Profits H = R – (F+G) Output Transfers (OT) I = A – E Input Transfers (IT) J = B – F Factor Transfers (FT) K = C – G Net Transfers (NT) L = D – H Private Cost Ratio (PCR) = C/(A-B) Domestic Resources Cost Ratio (DRCR) = G/(E-F) Nominal Protection Coefficient Output (NPCO) = A/E Nominal Protection Coefficient Input (NPCI) = B/F Effective Protection Coefficient (EPC) = (A-B)/(E-F) Profitability Coefficient (PC) = D/H Subsidy Ratio for Producers (SRP) = L/E Figure 1. Framework of the divergence effect of cassava competitiveness as an effect of government policies and market failures in South Lampung Cassava farming Input Output Input prices Output prices Private cost Social cost Private revenue Social revenue Government policies Market failure Market structure analysis Divergence effect PAM Table 1. Policy Analysis Matrix (PAM) Cassava farming Divergence effect Market failure Government policies Market structure analysis Market structure analysis Figure 1. Framework of the divergence effect of cassava competitiveness as an effect of government policies and market failures in South Lampung 133 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Determination of Input and Output Social Prices of Cassava in South Lampung The social prices of cassava in this study are calculated based on its border prices that consists of CIF (Cost Insurance and Freight) prices and FOB (Free on Board) prices (Table 4). The social price of currency exchange rates is the price of domestic money that is associated with foreign currencies on the money market, so it is obtained the SER (Shadow Exchange Rate) value. The SCF (Standard Conversion Factor) value is obtained from the calculation of the export value minus export taxes and the import value plus import taxes (Nursalam et al. 2018) then resulting the SCF value of 1.00. Furthermore, the SER value obtained by dividing the rupiah exchange rate of Rp14,265 (Bank Indonesia, 2021) with the SCF value of 1.00 so that the SER value is Rp14,265.73/US$. The social price of cassava uses the FOB price of tapioca Bangkok (US$ 483.30/ton) for the period January to December 2021 (Thai Tapioca Starch Association (TTSA), 2022). The price of tapioca is the price of an imported commodity in Indonesia so that the FOB of tapioca added with shipping and insurance costs will produce a CIF price of US$ 52.72/ton (Zulkarnain et al. 2021). These results then are converted into domestic currency and multiplied by the social price of the currency exchange rate to obtain a CIF price in the domestic currency of Rp7,646.72/kg. Furthermore, these results are added to the cost of loading and unloading, shrinkage, etc (3% of FOB) (Zulkarnain et al. 2021) and transportation to the province (Rp100/kg), so that the CIF value in domestic currency is Rp7,976.12/kg. The quality of cassava is equal to the quality of imports so that the conversion process to tapioca is calculated (Rosanti et al. 2018). The conversion value is 25% of the CIF value in domestic currency (SKKNI Bidang Pengolahan Tapioka Kementerian Perindustrian, 2019 in (Zulkarnain et al. 2021)) so that the social export price in a trader is Rp1,994.03/kg. This value is added by distribution from the farmers (Rp100/kg) so that the social price of cassava in farmers is Rp2,094.03/kg. The social price of urea (890 US$/ton) and NPK Phonska (745 US$/ton) is based on FOB prices (The Pink Sheet, 2022). This value is multiplied by the social price of the currency exchange rate then added by loading and unloading, warehouse, depreciation, etc (3% of FOB), transportation (Rp10/kg), and distribution costs to farmers (Rp55/kg) (Zulkarnain et al. Identification of Inputs and Outputs of Cassava Farming in South Lampung Cassava farming uses inputs as tradable inputs and non- tradable inputs. The tradable inputs consist of chemical fertilizers and pesticides. While, the non-tradable inputs consist of land, seeds, manures, agricultural tools and machines (ATM), and labors. The output produced is cassava with have matures between 7–9 months with a different types of seed varieties (Bayeman, Thailand, Garuda, and Cassesart). The different types of seed varieties are not affecting the production and productivity because these are suitable for cultivation in South Lampung climate conditions. Besides farmers, there are 8 cassava collectors who in productive age with an education level is high school of 63.5%. There are 50.00% of cassava collectors have selling cassava experience in a range of 16–25 years so they have sufficient experience. In addition, there Table 2. Characteristics of farmers and cassava collectors Farmers Collectors Variables Categories Number (People) % Categories Number (People) % Age (years) 21 – 37 7 10.94 40 – 50 5 62.50 28 – 54 45 70.31 51 – 55 2 12.50 55 – 71 1 18.75 56 – 65 1 25.00 Education (levels) ES 12 18.75 ES 3 36.50 JHS 13 20.31 SHS 5 63.50 SHS 38 59.38 (-S) 1 1.56 Family Responsibilities (years) 0 – 2 12 18.75 5 – 15 2 25.00 3 – 5 52 81.25 16 – 25 1 50.00 26 – 40 2 25.00 Experience (years) 2 – 14 28 43.75 15 – 27 32 50.00 28 – 40 4 6.25 Land Area (hectares) > 2 2 3.12 0.5 – 2 54 84.36 < 0.5 8 12.25 Land Ownership (Status) Own 50 78.12 Rent 14 21.88 Notes: ES = Elementary School, JHS = Junior High School, SHS = Senior High School, (-S) = Not Studying Table 2. Characteristics of farmers and cassava collectors 134 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 farmers are required to pay Land and Building Taxes (PBB) that social price is the same as the private price (Rp47,768/ha/year). Determination of Input and Output Private Prices of Cassava in South Lampung The private prices used in this study is the price that used at the time the research was conducted (planting season in 2021) (Table 3). The private land prices use the actual price of land rent and land tax in cash per hectare per year. The private seed prices use the actual prices of seed for one cassava seed. The private labor prices use the actual wage paid by farmers to labor in one working day. The private fertilizers, herbicides, and ATM prices use the actual prices of them. The private cassava prices use the prices received by farmers when selling cassava. The social price of seeds is the same as the private price (Rp129/stem) because seeds are obtained from local seeds (Iswara et al. 2021). The social price of the agriculture tools and machines is the same as the depreciation value per year (Rp186,595/year) (Rosanti et al. 2018). The social price of capital is calculated by adding the interest rate value to the average inflation (Alghoziyah et al. 2016). The interest rate working capital for a commercial banks in 2021 is 8.72% and the average inflation rate in 2021 is 1.87% (Bank Indonesia, 2021) so the social price for capital interest is 10.59%. The social price of labor is estimated to be 80% of the private price is Rp63,375/day (Suryana, 1980). This is because labors in the Indonesian agricultural sector is generally uneducated (unskilled) and have low productivity, yet the level of wages provided often exceeds the opportunity cost (Zulkarnain et al. 2010). Thus, the wages that should be paid are lower than the labor wages paid by farmers (Suryana, 1980 in Rosanti et al. 2018). Determination of Input and Output Social Prices of Cassava in South Lampung 2021), so its obtained the urea social price of Rp13,032.39/kg and NPK Phonska of Rp10,901.81/kg. The social price of manures used manures private price (Rp673/kg) because manures is a non-tradable input. The social price of herbicides is based on its private price which is reduced by a 10% import tariff and a 10% value-added tax (Kata et al. 2020) (Rp539,355/liter). The social price of land is the actual land rent value of Rp6,843,750/ha/year) because land can’t be traded internationally (Sukmaya et al. 2017). Meanwhile, 135 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Divergence effect of tradable input occurred due to input policy (fertilizer subsidies) which resulted in transfers of production inputs from input traders to farmers of Rp5,016,016. This situation does not occur in the non-tradable input market because farmers pay the non-tradable input higher than the social prices. These results are compatible with the research (Mubarokah and Tamami, 2019), the cost of non-tradable inputs is higher than the tradable inputs. It happened because the inputs used in cassava farming come from domestic. This means that Indonesian is still able to provide input this cassava farming. Based on the PAM table in Table 5, cassava competitiveness can be determined through PCR and DRCR calculations. The results of PCR and DRCR were 0.58 and 0.30. The PCR value was higher than the DRCR value, which means that without government policies, cassava farming requires fewer domestic factors to produce a unit of added value compared to with government policies. In the other words, fulfilling the demand for cassava is better if it is cultivated domestically rather than importing it. Table 3. Input and output private prices of cassava Descriptions Units Private Prices (Rp/Units) Land rent Ha/year 6.843.750 Land tax Ha/Year 34.040 Seeds Stem 129 Labors HOK 79.219 Herbicides Liter 674.193 Manures Kg 673 Ureas Kg 2.680 NPK Kg 2.795 ATM Year 186.595 Cassava Kg 1.158 Table 4. Input and output social prices of cassava Descriptions Units Private Prices (Rp/Units) SER US$ 14,265.73 Urea Kg 13,032.39 NPK Kg 10,901.80 Manures Kg 672.00 Herbicides Liter 539,355.00 Land rent Ha/years 6,843,750.00 Land tax Ha/years 34,040.00 Seeds Stem 129.00 ATM Year 186,595.00 Capital % 10.59 Labors HOK 63,375.00 Cassava Kg 2,094.03 Competitiveness of Cassava in South Lampung Table 4. Input and output social prices of cassava Descriptions Units Private Prices (Rp/Units) Divergence Effect of Cassava Competitiveness as an Effect of Government Policies in South Lampung The impact of government policies can be seen from three indicators, that are input policies, output policies, and input-output policies (Table 6). The results showed that the IT value has a negative sign (-5.016.016) with the NPCI value < 1 (0.3441), which means that there were no transfers from farmers to tradable input traders due to the fertilizer subsidy policy. These results are compatible with the research of (Fadli et al. 2017) that the impact of input policies is that farmers receive lower prices due to the form of input subsidy policies. Besides input subsidies, government policies affecting tradable inputs and domestic factors may consist of trade policies, interest rate subsidies, regional minimum wages (UMR), and value added tax. Competitiveness of Cassava in South Lampung Competitiveness of Cassava in South Lampung The impact of government policies in inputs and outputs of cassava in South Lampung Input Policies Output Policies Input-Output in the research of (Lestari et al. 2017), the negative NT value caused loss for farmers due to government policies applied to inputs and outputs so that the private profits earned by farmers are lower than social benefits. The PC value is positive (0.39) but less than 1, which means the market failures and government policies have a negative impact because they reduce cassava farmers income by 64.0%. According to (Novia et al. 2017) even though the profits received are lower than they should be because of the value of PC > 0, they still do not run into big losses. The OT value is negative (26.434.045) with the NPCO value < 1 (0.5530), which means there is no transfer from consumers to farmers. It is caused the income received by farmers to be lower than it should be due to the imperfect market structure of the cassava (oligopsony). This condition is the same as the research of (Meliyana et al. 2013) that the incentives provided by the government to farmers have not been able to increase cassava production and competitiveness in North Lampung. Therefore, government must make policies to keep cassava selling prices at a low level is detrimental to producers and profitable to tapioca industries and consumers. The SRP value is negative (0.3913), which means government policies have a negative impact on the production cost structure because the production costs paid by farmers are higher than the profits that should be received. The SRP value is negative because farmers have limited knowledges and abilities for cultivation and technological adaptation so the use of resources and government assistance is not as efficient as in the research of (Halimah et al. 2021). The EPC value is positive of 0,61 but less than 1, which means there are market failure and the government’s policies are not protective the cassava domestic, so the farmers do not get a real benefit due to the low cassava price. Existing government policies should be able to protect farmers and encourage exports. According to (Handayani et al. 2020), the EPC value > 1 indicates that farming activities must be increased to be more competitive. Competitiveness of Cassava in South Lampung Efforts can be made by increasing production, improving production quality through post-harvest technology, and increasing product added value (development of derivative products) so that government policies are more in favor to farmers. Competitiveness of Cassava in South Lampung The results in Table 5 showed that cassava farming in South Lampung is financially and economically profitable in one hectare with the value of private and social profits that are positive of Rp 12.436.706 and Rp35,579,607 respectively. According to Nurfaizah in (Ramadan, 2018), the value of social profit which is higher than the private profit can be caused by the FOB price of cassava on the international market which is higher than the domestic price. Divergence effect of profit has a negative value of (Rp23,142,901), which means that farmers received lower profits than they should. In addition, there is a transfer income to consumers from farmers at Rp26,434,045/hectare. This is due to oligopsony practices that resulted a market failure because the cassava collectors and tapioca industry control the cassava price in the market, while the farmers only act as price takers. Therefore, the government’s policy regarding determining a cassava minimum price of Rp900/kg does not make an effective contribution to farmer’s revenue. The FT value is 1,724,872, which means the costs incurred by farmers to pay for non-tradable inputs are higher than they should be. The result indicates that there are government’s interventions resulting in market distortions observed by variable detrimental to cassava farmers. According to the research of (Rahman et al. 2016), increasing the wage labor market must be done. This is an effort that will enable farmers to get benefits from increased production through wages because labor is the main variable input in the production process of agricultural commodities. 136 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Table 5, Competitiveness of cassava in South Lampung Description Revenue (Rp) Cost (Rp) Profit (Rp) Tradable Input Non-Tradable Input Private Price 32,707,564 2,630,955 17,639,903 12,436,706 Social Price 59,141,609 7,646,971 15,915,032 35,579,607 Divergence -26,434,045 -5,016,016 1,724,872 -23,142,901 Table 5, Competitiveness of cassava in South Lampung Table 6. The impact of government policies in inputs and outputs of cassava in South Lampung Input Policies Output Policies Input-Output Policies Indicators Value Indicators Value Indicators Value Input Transfers (IT) -5,016,016 Output Transfers (OT) -26,434,045 Effective Protection Coefficient (EPC) 0.5841 Nominal Protection Coefficient Input (NPIN) 0.3441 Nominal Protection Coefficient Output (NPON) 0.5530 Net Transfers (NT) -23,142,901 Factor Transfers (FT) 1,724,872 Profitability Coefficient (PC) 0.3495 Subsidy Ratio for Producers (SRP) -0.3913 Table 6. Managerial Implication Divergence effect of cassava competitiveness is caused by government policies on cassava input-output and cassava market failure. The results showed that the government policies did not reach the target because the income received by farmers to be less than it should be. This is supported by the market distortion of the market structure of cassava in the form of oligopsony (there are more farmers than traders) so that farmers only act as price taker. Although cassava farming in South Lampung is already competitive, it still needs to be improved considering that Lampung Province is one of the cassava producers that supports world cassava’s demand. Therefore, the best policy is needed to increase the competitiveness of cassava. This policy can be pursued by increasing cassava production and productivity through a good agribusiness system. Figure 2. The marketing channels of cassava in South Lampung Producer (64 farmers) 2.530.570 kg (100%) Cassava Collectors (8 people) 1.283.750 kg (50,72%) Tapioca Industry II (1.246.820 kg (49,28%)) Figure 2. The marketing channels of cassava in South Lampung The farmers barriers are the low price of cassava that is setted by the tapioca industry followed by the refraction of cassava tonnage. This result is compatible with the research of (Abriani et al. 2022), that farmers can only accept the price offered by traders because farmers do not have power in setting prices. In addition, farmers do not have adequate transportation, so they must carry out a harvesting system with collectors. This condition can be overcome by coordination between agencies that can regulate the availability of the required facilities and infrastructure. Cassava sales by producers occur to information on prices and refraction as well as pressure on household needs. The source of information regarding prices comes from tapioca industry so that this becomes a benchmark for cassava collectors in determining prices to farmers. According to the research (Endaryanto et al. 2022) increasing cassava production can be carried out with strategies, that are improving technology by increasing seed quality, balancing the access of fertilizer subsidies, increasing access to irrigation infrastructure facilities, and developing human resources. Besides that, farmer cooperatives organize farmers and increase their negotiating powers so that can help farmers save money by lowering transaction costs and adding value. Divergence Effect of Cassava Competitiveness as an Effect of Market Failure in South Lampung The traders in this study were cassava collectors, who bought cassava directly from farmers, and tapioca industry, who bought cassava from collectors or farmers, who were both in the same location. The marketing channels in this study can be seen in Figure 2. There are two marketing channels which have many producers than consumers, which means the market The NT value is negative, which means the government policies do not reach the target because of farmers suffer a loss of Rp23,142,901. This condition occurs 137 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 /2017 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 damaged but still has to sell because of household pressure. The weather barrier is not only perceived by farmers, but also collectors and tapioca industry to carry out the harvest will be hampered if the weather is inadequate. During the rainy season, it is difficult for vehicles that transport cassava to enter the land because the roads are muddy. During the dry season, cassava is difficult to harvest because of the hard soil condition. structure is oligopsony. The oligopsony market structure is a market structure that is not perfectly competitive because consists of several consumers who are facing many producers, so the market structure is inefficient. The marketing channels are free to entry and exit the market as seen from the fact that there is no regional division of purchasing and buying cassava by farmers, collectors, and tapioca industry. Although there are no barriers to entry and exit the market, barriers are still occurred in each marketing channel. Conclusions Divergence effect of cassava competitiveness on inputs (fertilizer subsidies) causes farmers pay less for tradable inputs but pay more for non-tradable inputs. f Divergence effect of cassava competitiveness on inputs (fertilizer subsidies) causes farmers pay less for tradable inputs but pay more for non-tradable inputs. Divergence effect of cassava competitiveness on output (cassava minimum price of Rp900/kg) causes the income received by farmers are lower than it should be due to imperfect market structure (oligopsony) and cassava private prices is lower than the social prices, so it caused a transfer income from farmers to consumers. Divergence effect of cassava competitiveness on input- output did not reach the target because of the selling price of cassava is low so it caused losses for farmers due to reduced revenue and the costs paid by farmers higher than the profits earned. However, cassava farming has a competitiveness with the value of comparative advantage (DRCR = 0.30) which smaller than the value of competitive advantage (PCR = 0.58). For the changes from comparative to competitive advantage require government policies to control the market failure and to reduce the high transaction cost economy. Managerial Implication The policy to increase the competitiveness of cassava in Lampung Province that can be done is to increase output by 20% even though the cost of fertilizer increases by 20%. The main effort to increase cassava production can be done by increasing harvested area and productivity. According to Taslim & Rifin (2020) land expansion by utilizing idle land can be done to increase the productivity of cassava. Intensification in increasing production and productivity of cassava can be done by using technology, fertilizers, and superior seeds. The volume of production and productivity from tapioca processing also needs to be improved. Based on Saptana et al. (2021) research, the government can Furthermore, the weather became another barrier perceived by farmers. When it’s rainy season, the cassava rot more quickly and farmers do not want to sell it because the water content is very high, so they are worried that the refraction given will be even bigger. This is compatible with the research of (Zakaria et al. 2019) that cassava has characteristic that is easily 138 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis, Vol. 20 No.1, March 2023 moral and financial assistance. For other researchers is that further research is needed regarding finding the right strategies to increase the competitiveness of cassava in South Lampung. develop the farming activities in production centers with an agricultural area approach to achieve an efficient business scale. Therefore, it is necessary to support agricultural infrastructure, the use of certified seeds, complete and balanced fertilization, organic fertilizers, and using agricultural mechanization with rotary hand tractors. FUNDING STATEMENT: This study does not receive any specific grant from funding institution in the commercial, public, academic, or for – non – profit sector. REFERENCES Abriani DM, Lestari DAH, Rosanti, N. 2022. 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https://openalex.org/W2981287670	https://europepmc.org/articles/pmc6812673?pdf=render	English	null	RNA-seq transcriptome profiling of porcine lung from two pig breeds in response to <i>Mycoplasma hyopneumoniae</i> infection	PeerJ	2,019	cc-by	10,080	RNA-seq transcriptome profiling of porcine lung from two pig breeds in response to Mycoplasma hyopneumoniae infection Ligang Ni1,2, Chengyi Song1, Xinsheng Wu1, Xuting Zhao2, Xiaoyan Wang Bichun Li1 and Yuan Gan3 Ligang Ni1,2, Chengyi Song1, Xinsheng Wu1, Xuting Zhao2, Xiaoyan Wang1, Bichun Li1 and Yuan Gan3 1 College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, China 1 College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, China 2 Department of Animal Science and Technology, Jiangsu Agri-animal Husbandry Vocational College, Taizhou, Jiangsu, China 3 Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu, China How to cite this article Ni L, Song C, Wu X, Zhao X, Wang X, Li B, Gan Y. 2019. RNA-seq transcriptome profiling of porcine lung from two pig breeds in response to Mycoplasma hyopneumoniae infection. PeerJ 7:e7900 http://doi.org/10.7717/peerj.7900 ABSTRACT Background. Mycoplasma hyopneumoniae (Mhp) is the main pathogen causing res- piratory disease in the swine industry. Mhp infection rates differ across pig breeds, with Chinese native pig breeds that exhibit high fecundity (e.g., Jiangquhai, Meishan, Erhualian) more sensitive than Duroc, Landrace, and other imported pig breeds. However, the genetic basis of the immune response to Mhp infection in different pig breeds is largely unknown. However, the genetic basis of the immune response to Mhp infection in different pig breeds is largely unknown. Aims. The aims of this study were to determine the relative Mhp susceptibility of the Chinese native Jiangquhai breed compared to the Duroc breed, and identify molecular mechanisms of differentially expressed genes (DEGs) using an RNA-sequencing (RNA- seq) approach. Methods. Jiangquhai and Duroc pigs were artificially infected with the same Mhp dose. The entire experiment lasted 28 days. Daily weight gain, Mhp-specific antibody levels, and lung lesion scores were measured to evaluate the Mhp infection susceptibility of different breeds. Experimental pigs were slaughtered on the 28th day. Lung tissues were collected for total RNA extraction. RNA-seq was performed to identify DEGs, which were enriched by gene ontology (GO) and the Kyoto Encyclopedia annotation of Genes and Genomes (KEGG) databases. DEGs were validated with real-time quantitative polymerase chain reaction (RT-qPCR). Submitted 24 May 2019 Accepted 16 September 2019 Published 21 October 2019 Corresponding author Xiaoyan Wang, wxyan@yzu.edu.cn Academic editor Chris Vulpe Additional Information and Declarations can be found on page 15 DOI 10.7717/peerj.7900 Copyright 2019 Ni et al Results. Infection with the same Mhp dose produced a more serious condition in Jiangquhai pigs than in Duroc pigs. Jiangquhai pigs showed poorer growth, higher Mhp antibody levels, and more serious lung lesions compared with Duroc pigs. RNA- seq identified 2,250 and 3,526 DEGs in lung tissue from Jiangquhai and Duroc pigs, respectively. The two breeds shared 1,669 DEGs, which were involved in immune- relevant pathways including cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, and chemokine signaling pathway. Compared to Jiangquhai pigs, more chemokines, interferon response factors, and interleukins were specifically activated in Duroc pigs; CXCL10, CCL4, IL6 and IFNG genes were significantly up-regulated, which may help Duroc pigs enhance immune response and reduce Mhp susceptibility. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Conclusion. This study demonstrated differential immune-related DEGs in lung tissue from the two breeds, and revealed an important role of genetics in the immune response to Mhp infection. ABSTRACT The biological functions of these important DEGs should be further confirmed and maybe applied as molecular markers that improve pig health. Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 Experimental design and sample collection Experimental design and sample collection Twenty healthy 50-day-old Jiangquhai pigs were selected from the Jiangquhai Pig Breed Conservation Farm (Taizhou, China), and twenty healthy 50-day-old Duroc pigs were selected from the Xingtai Agriculture and Animal Husbandry Technology Development Company (Yangzhou, China). All pigs were free of all major porcine diseases and confirmed to be negative for Mhp, PRRSV, pseudorabies virus, and classical swine fever virus infection by PCR or reverse-transcription (RT)-PCR. Jiangquhai and Duroc pigs were randomly assigned to the control or infected group during the experiment and raised separately in isolation. Ten Jiangquhai and ten Duroc pigs were inoculated with 5 mL viral suspension of a virulent strain of Mhp (106 colour changing units [CCU]) (Xiong et al., 2014), which was provided by the Veterinary Medicine Institute of Jiangsu Academy of Agricultural Sciences (Nanjing, China). The remaining ten Jiangquhai and ten Duroc pigs were treated with an equivalent volume of aseptic physiological saline, which served as a negative control group. Four groups (i.e., Jiangquhai infection, Jiangquhai control, Duroc infection, Duroc control) were raised in isolation rooms to prevent cross-infection. Approval for the study was provided by the ethics committee of Yangzhou University (SYXK(Su) IACUC 2016-0131). On day 28, all pigs were euthanized by stunning, and lung tissues were collected and stored at −70 ◦C. At this time, pulmonary MPS lesions were confirmed and assessed with the scoring system (Steinmann, Blaha & Meemken, 2014; Lee et al., 2011). From the beginning to the end (day 28) of the study, all experimental pigs were weighed prior to feeding in the morning to compare weight gain between groups. To assess the immune response, blood samples were collected on days 0 and 28 via jugular venipuncture into normal serum tubes without anticoagulant. The serum was separated by centrifugation (1,600× g for 10 min at 4 ◦C), divided into aliquots, and stored at −20 ◦C until analysis. Mhp-specific antibody in peripheral blood was detected. RNA-seq library preparation and sequencing Total RNA was extracted from lung tissue of three infected and three control pigs of each breed (three pigs selected randomly from each group) using TRIzol reagent (Invitrogen, South San Francisco, CA, USA) following the manufacturer’s protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with an RNA Integrity Number (RIN) >7 were subjected to subsequent analysis. The libraries were constructed using the TruSeq Stranded mRNA LTSample Prep kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Then, these libraries were sequenced on the Illumina Hiseq 2500 platform (Shanghai OE Biotech Co., Ltd, Shanghai, China), and 125-bp paired-end reads were generated. INTRODUCTION Mycoplasma hyopneumoniae (Mhp) exists in every country where pigs are raised and is the main pathogen leading to respiratory disease in the swine industry (Maes et al., 2008; Stark, Nicolet & Frey, 1998). The pathogen resides in the respiratory tract, and its secretions can be found in infected pigs for a long time (Maes et al., 1996). The main clinical symptoms of infected pigs are dry cough, as well as dramatically reduced porcine growth and feed conversion rates, which cause great losses to the pig industry (Maes et al., 1996; Sarradell et al., 2003). Production practices on some Chinese pig farms revealed that Chinese local breeds are more sensitive to Mhp than imported breeds such as Duroc and Landrace. The Meishan and Erhualian, which are characterized by high fecundity, show extremely high susceptibility to Mhp infection (Fang et al., 2015; Maingi et al., 2014). This suggests that genetic components contribute to breed susceptibility or resistance to Mhp infection. Recently, it was reported that quantitative trait loci (QTLs) are associated with respiratory disease lesions, and five QTL were detected in Landrace pigs (Okamura et al., 2012). In Chinese Erhualian pigs, QTLs affecting respiratory disease were identified by a genome-wide association study; CXCL6, CXCL8, KIT, and CTBP2 were highlighted as candidates that might associated with resistance or susceptibility to swine enzootic pneumonia-like respiratory disease (Huang et al., 2016). However, the genetic basis for the immune response to Mhp infection among different pig breeds remains largely unknown. Jiangquhai is a Chinese pig breed distributed in the central Jiangsu Province. Similar to the Meishan and Erhualian breeds, Jiangquhai pigs exhibit sensitivity to Mhp infection (Maingi et al., 2014). Vaccines and antibiotics are used to control the occurrence of mycoplasma pneumonia of swine (MPS); however, these methods are not sufficient. It is therefore necessary to study the molecular mechanism of pathogenesis. This knowledge can be applied to carry out disease resistance breeding. In this study, we investigated the immune response of Jiangquhai and Duroc pigs to artificial Mhp infection using an RNA-sequencing (RNA-seq) approach. The goals were to identify genetic components that contribute to Mhp susceptibility or resistance and lay a foundation for genetic breeding that improve pig health. 2/21 Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 MATERIALS & METHODS Experimental design and sample collection RNA-seq data analysis The FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) value of each gene was calculated using cufflinks (Cole et al., 2012), and the read counts of each gene were obtained by HTSeq-Count (Anders, Pyl & Huber, 2015). Differentially expressed genes (DEGs) were identified using the DESeq estimateSizeFactors and nbinomTest functions, corrected p-value of 0.05 and log2 (Fold change) (log2 FC) of 1 were set as the threshold for significantly differential expression. Hierarchical clustering analysis of DEGs was performed to inspect sample relations. The DEGs were annotated by GO functional enrichment and KEGG pathway analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID). RT-qPCR veriﬁcation RNA was extracted from lung tissue of six infected and six control pigs of each breed using the TRIzol reagent following the manufacturer’s protocol. First-strand cDNA synthesis was performed using 5 µg of RNA and the Superscript II cDNA amplification system (Invitrogen, South San Francisco, CA, USA) according to the manufacturer’s protocol. Quantitative PCR was performed using an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) and Power SYBR Green PCR Master Mix (Invitrogen, South San Francisco, CA, USA). The gene for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was included as an endogenous control, and the specific primers used in the RT-qPCR assays are listed in Table S1. Relative expression of target genes was determined by the comparative cycle threshold (CT) method (Livak & Schmittgen, 2001) and the 1CT value was calculated by subtracting the target CT of each sample from its GAPDH CT value. Quality control and mapping Raw data (raw reads) were processed using the NGS QC Toolkit (Ravi, Mukesh & Liu, 2012). Low-quality reads and those containing poly-N were removed to ensure high- quality mapping. Then the clean reads were mapped using the Sus scrofa genome 11.1 Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 3/21 as a reference with bowtie2 (Langmead & Salzberg, 2012) or Tophat software packages (http://tophat.cbcb.umd.edu/) (Kim et al., 2013). Statistical analysis Weight gain and antibody level data are presented as mean ± standard error (SE). Comparison of variables was performed using one-way analysis of variance with SPSS13.0 software (SPSS Inc., Chicago, IL, USA). Eﬀect of Mhp infection on weight gain, antibody production, and lung lesions in Jiangquhai and Duroc pigs The average daily weight gain (ADG) of the Jiangquhai infected pigs was highly significantly lower than the Jiangquhai control pigs (p < 0.01), while the ADG of Duroc infected pigs was not significantly different from Duroc control pigs (p > 0.05) (Table 1). These results demonstrate that Mhp infection had a greater impact on the growth rate of Jiangquhai pigs compared to Duroc pigs. On day 28, the level of Mhp-specific antibody (reported as the sample mean/positive control mean (s/p) value) in the peripheral blood of Jiangquhai infected pigs reached at 0.85 ± 0.20, which was significantly higher than that of Duroc infected pigs (0.48 ± 0.19) (p < 0.01) (Table 1). Mhp-specific antibody was not detected in control pigs. Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 4/21 Table 1 Average daily weight gain and Mhp-specific antibody levels of experimental pigs. Groups N Average body weight (kg) ADG (g/d) Antibody levels (s/p) 0d 28d 0d 28d Jiangquhai infection 10 8.54 ± 0.77 12.26 ± 0.51 132.86 ± 14.56A 0.05 ± 0.03 0.85 ± 0.20A Jiangquhai control 10 8.46 ± 0.50 15.29 ± 0.76 244.11 ± 21.10B 0.05 ± 0.04 0.08 ± 0.07 Duroc infection 10 10.64 ± 0.83 18.41 ± 0.49 277.50 ± 25.81 0.02 ± 0.01 0.48 ± 0.19B Duroc control 10 10.53 ± 0.72 18.81 ± 0.58 295.89 ± 18.86 0.02 ± 0.01 0.04 ± 0.01 Notes. Different letters in the same column indicate significant differences of mean values (A and B) (p < 0.01). Table 1 Average daily weight gain and Mhp-specific antibody levels of experimental pigs. and Mhp-specific antibody levels of experimental pigs. Average body weight (kg) ADG (g/d) Antibody levels (s/p) 0d 28d 0d 28d 8.54 ± 0.77 12.26 ± 0.51 132.86 ± 14.56A 0.05 ± 0.03 0.85 ± 0.20A 8.46 ± 0.50 15.29 ± 0.76 244.11 ± 21.10B 0.05 ± 0.04 0.08 ± 0.07 10.64 ± 0.83 18.41 ± 0.49 277.50 ± 25.81 0.02 ± 0.01 0.48 ± 0.19B 10.53 ± 0.72 18.81 ± 0.58 295.89 ± 18.86 0.02 ± 0.01 0.04 ± 0.01 cate significant differences of mean values (A and B) (p < 0.01). Figure 1 Lung tissue of experimental pigs. (A) Jiangquhai infected pigs, (B) Jiangquhai control pigs, (C) Duroc infected pigs, (D) Duroc control pigs. Lung pathological tissues are indicated with arrows. Full-size DOI: 10.7717/peerj.7900/fig-1 Notes. Preliminary analysis and summary of RNA-Seq data quality We performed RNA-seq to analyze the transcriptional profile of lung tissue from the four experimental groups. We found that 87.54–96.47% of clean reads were mapped to the reference genome (Sus scrofa 11.1), and 80.19–93.33% were uniquely mapped (Table S2). The sample to sample distance heat map showed a good degree of similarity between all three replicates (Fig. S1), indicating that there were no significant differences in gene expression among the biological replicates. These results showed that the RNA-seq data were reliable and met the conditions for differential expression analysis. Eﬀect of Mhp infection on weight gain, antibody production, and lung lesions in Jiangquhai and Duroc pigs Different letters in the same column indicate significant differences of mean values (A and B) (p < 0.01). Figure 1 Lung tissue of experimental pigs. (A) Jiangquhai infected pigs, (B) Jiangquhai control pigs, (C) Duroc infected pigs, (D) Duroc control pigs. Lung pathological tissues are indicated with arrows. Full-size DOI: 10.7717/peerj.7900/fig-1 Figure 1 Lung tissue of experimental pigs. (A) Jiangquhai infected pigs, (B) Jiangquhai control pigs, (C) Duroc infected pigs, (D) Duroc control pigs. Lung pathological tissues are indicated with arrows. Full-size DOI: 10.7717/peerj.7900/fig-1 Figure 1 Lung tissue of experimental pigs. (A) Jiangquhai infected pigs, (B) Jiangquhai control pigs, (C) Duroc infected pigs, (D) Duroc control pigs. Lung pathological tissues are indicated with arrows. Full-size DOI: 10.7717/peerj.7900/fig-1 Our analysis of lung tissue on day 28 revealed that Jiangquhai infected pigs had more serious Mhp lung lesions (Fig. 1), a greater difference in Mhp lung lesion scores from Jiangquhai infected pigs compared to Duroc infected pigs (p < 0.01) (Fig. 2). Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 5/21 Figure 2 Lung lesion scores. Presence of pulmonary lesions was determined in Jiangquhai infected pigs and Duroc infected pigs. Different letters indicate significant differences (A and B) (p < 0.01). Error bars indicate the standard error. Full-size DOI: 10.7717/peerj.7900/fig-2 Figure 2 Lung lesion scores. Presence of pulmonary lesions was determined in Jiangquhai infected pigs and Duroc infected pigs. Different letters indicate significant differences (A and B) (p < 0.01). Error bars indicate the standard error. Figure 2 Lung lesion scores. Presence of pulmonary lesions was determined in Jiangquhai infected pigs and Duroc infected pigs. Different letters indicate significant differences (A and B) (p < 0.01). Error bars indicate the standard error. Full-size DOI: 10.7717/peerj.7900/fig-2 Figure 2 Lung lesion scores. Presence of pulmonary lesions was determined in Jiangquhai infected pigs and Duroc infected pigs. Different letters indicate significant differences (A and B) (p < 0.01). Error bars indicate the standard error. Full-size DOI: 10.7717/peerj.7900/fig-2 DEGs analysis and RT-qPCR validation Full-size DOI: 10.7717/peerj.7900/fig-3 Figure 4 The RT-qPCR identification of randomly selected DEGs and correlation with RNA-seq data. The X-axis is the name of genes and the Y -axis is the log2 Ratio (Treatmented group/Control group) rela- tive expression value. Full-size DOI: 10.7717/peerj.7900/fig-4 Terms and pathways associated with immunobiology were enriched in both breeds To determine the biological function of DEGs after Mhp infection in lung tissue, the common 1,669 DEGs were submitted to DAVID for GO analysis, which revealed enrichment of 281 GO terms (p < 0.05). The top 10 significant enrichments are shown in Fig. 5, among which cell adhesion, inflammatory response and immune response were the most significantly regulated by Mhp infection. Pathway analysis based on the KEGG database revealed the top 20 significant signaling pathways of the two breeds (Fig. 6). Many immune-related pathways were enriched, including the cytokine-cytokine receptor Figure 3 Venn diagram displaying the numbers of DEGs in the two groups. DEGs in infected Jiangquhai pigs and infected Duroc pigs were compared against their control groups. The numbers in the overlapping areas represent DEGs shared between the two groups. Full-size DOI: 10.7717/peerj.7900/fig-3 Jiangquhai pigs and infected Duroc pigs were compared against their control groups. The numbers in the overlapping areas represent DEGs shared between the two groups. Full-size DOI: 10.7717/peerj.7900/fig-3 Figure 4 The RT-qPCR identification of randomly selected DEGs and correlation with RNA-seq data. The X-axis is the name of genes and the Y -axis is the log2 Ratio (Treatmented group/Control group) rela- tive expression value. Full-size DOI: 10.7717/peerj.7900/fig-4 Figure 4 The RT-qPCR identification of randomly selected DEGs and correlation with RNA-seq data. The X-axis is the name of genes and the Y -axis is the log2 Ratio (Treatmented group/Control group) rela- tive expression value. Full-size DOI: 10.7717/peerj.7900/fig-4 Terms and pathways associated with immunobiology were enriched in both breeds To determine the biological function of DEGs after Mhp infection in lung tissue, the common 1,669 DEGs were submitted to DAVID for GO analysis, which revealed enrichment of 281 GO terms (p < 0.05). The top 10 significant enrichments are shown in Fig. 5, among which cell adhesion, inflammatory response and immune response were the most significantly regulated by Mhp infection. Pathway analysis based on the KEGG database revealed the top 20 significant signaling pathways of the two breeds (Fig. 6). Many immune-related pathways were enriched, including the cytokine-cytokine receptor Ni et al. DEGs analysis and RT-qPCR validation We analyzed the DEG profiles of lung tissues from Mhp-infected Jiangquhai and Duroc pigs by comparing infected and control animals of the same breed. Genes with relative expression levels that showed log2 FC > 1 (p < 0.05) were considered up-regulated, and those with log2 FC <−1 (p < 0.05) were considered down-regulated. Of the 2,250 DEGs detected in Jiangquhai pigs, 966 genes were up-regulated and 1,284 down-regulated. Of the 3,526 DEGs detected in Duroc pigs, 1,326 and 2,200 were up- and down-regulated, respectively (Fig. 3). Jiangquhai and Duroc pigs shared 1,669 DEGs in response to Mhp infection, with 632 up-regulated genes and 1,037 down-regulated genes. Further analysis showed that Duroc pigs had 694 uniquely up-regulated DEGs, which was two times higher than the number in Jiangquhai pigs (334 DEGs) (Fig. 3). To verify the RNA-seq results, eight genes were randomly selected for RT-qPCR analysis. Expression FCs determined by RT-qPCR analysis was compared against the profiles predicted by RNA-seq. The RT-qPCR results verified the changes in expression levels of the eight genes (Fig. 4), indicating that the RNA-seq data were reliable. Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 6/21 Figure 3 Venn diagram displaying the numbers of DEGs in the two groups. DEGs in infected Jiangquhai pigs and infected Duroc pigs were compared against their control groups. The numbers in the overlapping areas represent DEGs shared between the two groups. Full-size DOI: 10.7717/peerj.7900/fig-3 Figure 4 The RT-qPCR identification of randomly selected DEGs and correlation with RNA-seq data. The X-axis is the name of genes and the Y -axis is the log2 Ratio (Treatmented group/Control group) rela- tive expression value. Full-size DOI: 10.7717/peerj.7900/fig-4 Terms and pathways associated with immunobiology were enriched in both breeds To determine the biological function of DEGs after Mhp infection in lung tissue, the common 1,669 DEGs were submitted to DAVID for GO analysis, which revealed Figure 3 Venn diagram displaying the numbers of DEGs in the two groups. DEGs in infected Jiangquhai pigs and infected Duroc pigs were compared against their control groups. The numbers in the overlapping areas represent DEGs shared between the two groups. Full-size DOI: 10.7717/peerj.7900/fig-3 Figure 3 Venn diagram displaying the numbers of DEGs in the two groups. DEGs in infected Jiangquhai pigs and infected Duroc pigs were compared against their control groups. The numbers in the overlapping areas represent DEGs shared between the two groups. Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 DEGs analysis and RT-qPCR validation (2019), PeerJ, DOI 10.7717/peerj.7900 7/21 Figure 5 The top 10 gene ontology (GO) enrichments of common DEGs shared by Jiangquhai and Duroc pigs. The Y -axis is the name of each category, the X-axis is their –log10 (p-value). Full-size DOI: 10.7717/peerj.7900/fig-5 interaction (IL18 IL1R1) PI3K Akt signaling pathway (TLR2 ITGA1 ITGB7 PIK3R5) Figure 5 The top 10 gene ontology (GO) enrichments of common DEGs shared by Jiangquhai and Duroc pigs. The Y -axis is the name of each category, the X-axis is their –log10 (p-value). Full-size DOI: 10.7717/peerj.7900/fig-5 Figure 5 The top 10 gene ontology (GO) enrichments of common DEGs shared by Jiangquhai and Duroc pigs. The Y -axis is the name of each category, the X-axis is their –log10 (p-value). Full-size DOI: 10.7717/peerj.7900/fig-5 interaction (IL18, IL1R1), PI3K-Akt signaling pathway (TLR2, ITGA1, ITGB7, PIK3R5), and chemokine signaling pathway (CXCL8, CXCL13). Speciﬁc terms and pathways in Jiangquhai and Duroc pigs Speciﬁc terms and pathways in Jiangquhai and Duroc pigs Jiangquhai and Duroc pigs also had different responses to Mhp infection. The specific DEGs of Jiangquhai (581 DEGs) and Duroc pigs (1,857 DEGs) were separately submitted to DAVID for GO analysis. The results revealed 100 GO enrichments in Jiangquhai pigs and 314 GO enrichments in Duroc pigs. Table S3 shows the top 10 significant enrichments in Jiangquhai pigs, including cell adhesion, leukocyte migration and extracellular matrix disassembly. Table S4 lists the top 10 significant enrichments in Duroc pigs, including cilium assembly, cilium-dependent cell motility and interleukin-7-mediated signaling pathway. KEGG pathway analysis revealed 38 specific signaling pathways in Jiangquhai pigs (Table S5) and 54 specific signaling pathways in Duroc pigs (Table S6). Further analysis found that Duroc pigs had more immune-related pathways (18 immune-related pathways), including chemokine signaling pathway, NOD-like receptor signaling pathway, antigen processing and presentation, toll-like receptor signaling pathway, 253 DEGs were enriched Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 8/21 Figure 6 The top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of common DEGs shared by Jiangquhai and Duroc pigs. The Y -axis is the name of each category, the X-axis is their –log10 (p-value). The number of genes enriched in each category were shown at the top of each bar. Full-size DOI: 10.7717/peerj.7900/fig-6 Figure 6 The top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of common DEGs shared by Jiangquhai and Duroc pigs. The Y -axis is the name of each category, the X-axis is their –log10 (p-value). The number of genes enriched in each category were shown at the top of each bar. Full-size DOI: 10.7717/peerj.7900/fig-6 in those 18 pathways (Table 2). Jiangquhai pigs only identified 8 immune-related pathways, including leukocyte transendothelial migration, hematopoietic cell lineage, cell adhesion molecules, 99 DEGs were enriched in those 8 pathways (Table 2). Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 Analysis of important DEGs related to immune responses Analysis of important DEGs related to immune responses In this study, we mainly focused on the important DEGs related to immune responses. Based on gene clustering and specific KEGG pathways (Table 2), some DEGs that we identified were well-known components of the innate immune response, such as chemokines, interleukins, interferon response factors and complement components. Forty-five important specific DEGs related to innate immune responses had been identified in Duroc pigs (Fig. 7B), and only 20 important specific DEGs had been identified in Jiangquhai pigs (Fig. 7A). Furthermore, some immune-related genes were down-regulated in Jiangquhai pigs, such as adhesion molecules (PECAM1, CD34, CD7, CDH4), WNT molecules Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 9/21 Table 2 Important immune-related KEGG pathways of specific DEG Breeds Pathway terms Categories Cytosolic DNA-sensing pathway Immune system Intestinal immune network for IgA production Immune system Complement and coagulation cascades Immune system Chemokine signaling pathway Immune system NOD-like receptor signaling pathway Immune system RIG-I-like receptor signaling pathway Immune system Antigen processing and presentation Immune system Toll-like receptor signaling pathway Immune system T cell receptor signaling pathway Immune system TNF signaling pathway Signal transduction Jak-STAT signaling pathway Signal transduction cAMP signaling pathway Signal transduction NF-kappa B signaling pathway Signal transduction MAPK signaling pathway Signal transduction FoxO signaling pathway Signal transduction Cytokine-cytokine receptor interaction Signaling molecules and interaction Cell adhesion molecules (CAMs) Signaling molecules and interaction Duroc pigs Neuroactive ligand–receptor interaction Signaling molecules and interaction Complement and coagulation cascades Immune system Leukocyte transendothelial migration Immune system Natural killer cell mediated cytotoxicity Immune system Hematopoietic cell lineage Immune system Hippo signaling pathway Signal transduction cAMP signaling pathway Signal transduction Notch signaling pathway Signal transduction Jiangquhai pigs Cell adhesion molecules (CAMs) Signaling molecules and interaction Table 2 Important immune-related KEGG pathways of specific DEGs in Jiangquhai pigs and Duroc pigs. Analysis of important DEGs related to immune responses Pathway terms Categories Genes Cytosolic DNA-sensing pathway Immune system IFNB1, NFKBIA, CXCL10, IRF7, ZBP1, CCL5, CCL4, DDX Intestinal immune network for IgA production Immune system CCR9, CXCL12, ICOS, SLA-DRB1, CD28, LOC106504372, Complement and coagulation cascades Immune system C1QA, C1S, F7, BDKRB1, F2, THBD, VSIG4, C1QB, C1QC Chemokine signaling pathway Immune system CCR5, CCR1, XCR1, CXCR6, CCR9, CXCL2, NFKBIA, CX PTK2B, ADCY4, GRK5, JAK3, CXCR3, NFKBIB, LOC1102 ADCY1, PLCB2 NOD-like receptor signaling pathway Immune system NFKBIA, CCL5, PSTPIP1, TNFAIP3, IL6, MAPK10, TRIP6 RIG-I-like receptor signaling pathway Immune system IFNB1, NFKBIA, CXCL10, IRF7, ISG15, DHX58, DDX58, MAPK11 Antigen processing and presentation Immune system TAP1, SLA-DRB1, CALR, PDIA3, LOC106504372, CD8B, H LOC100523789 Toll-like receptor signaling pathway Immune system IFNB1, NFKBIA, CXCL10, IRF7, CD14, LY96, CCL5, IFNA MAP3K8, MAP2K6, MAPK11 T cell receptor signaling pathway Immune system NFKBIA, ICOS, PDCD1, AKT3, CD28, CD8B, IFNG, IL10 MAPK11, PAK6, NFKBIE TNF signaling pathway Signal transduction CXCL2, NFKBIA, CXCL10, CREB3L4, CCL5, AKT3, TNFA MAPK10, MAP3K8, MAP2K6, BIRC3, TRAF1, MAPK11 Jak-STAT signaling pathway Signal transduction IFNB1, IL7R, SOCS1, IFNAR2, IL27RA, AKT3, IL2RA, CSF IL15RA, IL20RB, LOC100736818, PIM1, CDKN1A, IL2RB, cAMP signaling pathway Signal transduction NFKBIA, TNNI3, CREB3L4, GABBR1, SSTR1, PLD1, AKT GRIA4, GRIA3, PDE4D, SUCNR1, LOC110255845, GRIN3 GLI3, PDE3B, GRIN2A, GRIN2B, CACNA1C, GIPR, CNG NF-kappa B signaling pathway Signal transduction NFKBIA, CXCL12, CD14, LY96, BCL2A1, TNFAIP3, CCL4 NFKB2, CARD11 MAPK signaling pathway Signal transduction CD14, NTF3, DDIT3, PLA2G4B, RPS6KA1, AKT3, HSP70 MAPK10, DUSP4, PRKCG, GADD45B, MAP3K8, MAP2K FGF12, NFKB2, CACNA1H, DUSP2, MAPK8IP2, MAPK1 CACNA1E FoxO signaling pathway Signal transduction CCNB2, IL7R, PCK2, CCNB1, AKT3, EGF, IL10, IL6, MAP CDKN1A, IRS2, PLK1, MAPK11 Cytokine-cytokine receptor interaction Signaling molecules and interaction CCR5, CCR1, XCR1, CXCR6, CCR9, CXCL2, IFNB1, CXC FRSF9, CD27, CCL4, IL2RA, FAS, CSF3, IFNG, EGF, IL10, IL15RA, IL20RB, LOC100736818, IL18RAP, IL25, IL2RB, C Cell adhesion molecules (CAMs) Signaling molecules and interaction ICOS, SELL, SLA-DRB1, OCLN, PDCD1, VCAN, CLDN1, MPZ, L1CAM, TIGIT, NLGN3, LOC100525346, ITGA8, N Neuroactive ligand–receptor interaction Signaling molecules and interaction P2RY2, BDKRB1, F2, GABBR1, GZMA, CRHR2, SSTR1, P HTR1B, PTH1R, TAAR1, LTB4R2, THRB, PTGDR, GRIA4 GABRB2, CNR1, LRRC74B, PARD3, GRIN3A, P2RX1, GR GRIN2B, GIPR Complement and coagulation cascades Immune system C5, MBL1, C6, PLAU, F5, SERPINA5, ITGAM, KNG1, SER Leukocyte transendothelial migration Immune system CLDN23, CLDN18, CLDN5, PECAM1, ITGAM, TXK, ESA Natural killer cell mediated cytotoxicity Immune system NCR1, IFNAR1, KLRK1, CD244, LOC100739080, SHC3, L Hematopoietic cell lineage Immune system CD4, GP1BB, CD34, CD19, ITGAM, CD7 Hippo signaling pathway Signal transduction PARD6B, BMP2, WNT10B, WNT16, GDF7, WNT11, TCF cAMP signaling pathway Signal transduction FXYD2, FOS, PDE4A, DRD2, GNAI1, LOC100738425, LO PDE4C Notch signaling pathway Signal transduction HES1, DTX2, RBPJL pigs Cell adhesion molecules (CAMs) Signaling molecules and interaction NECTIN1, CD4, CLDN23, CLDN18, CLDN5, PECAM1, C CNTNAP2 IFNB1, NFKBIA, CXCL10, IRF7, ISG15, DHX58, DDX58, TKFC, MAPK10, NFKBIB, TANK, MAPK11 TAP1, SLA-DRB1, CALR, PDIA3, LOC106504372, CD8B, HSP70.2, IFNG, PSME2, KLRD1, LOC100523789 IFNB1, NFKBIA, CXCL10, IRF7, CD14, LY96, CCL5, IFNAR2, AKT3, CCL4, IL6, MAPK10, MAP3K8, MAP2K6, MAPK11 NFKBIA, ICOS, PDCD1, AKT3, CD28, CD8B, IFNG, IL10, CTLA4, NFKBIB, MAP3K8, CARD11, MAPK11, PAK6, NFKBIE CXCL2, NFKBIA, CXCL10, CREB3L4, CCL5, AKT3, TNFAIP3, FAS, PTGS2, IL6, LTA, RIPK3, MAPK10, MAP3K8, MAP2K6, BIRC3, TRAF1, MAPK11 IFNB1, IL7R, SOCS1, IFNAR2, IL27RA, AKT3, IL2RA, CSF3, IFNG, IL10, IL2RG, IL6, JAK3, OSMR, IL15RA, IL20RB, LOC100736818, PIM1, CDKN1A, IL2RB, TSLP, IL22, PTPN2 NFKBIA, TNNI3, CREB3L4, GABBR1, SSTR1, PLD1, AKT3, HTR1B, AMH, ADCY4, MAPK10, GRIA4, GRIA3, PDE4D, SUCNR1, LOC110255845, GRIN3A, PPP1R1B, HCAR2, GRIA1, ADCY1, GLI3, PDE3B, GRIN2A, GRIN2B, CACNA1C, GIPR, CNGB1 NFKBIA, CXCL12, CD14, LY96, BCL2A1, TNFAIP3, CCL4, DDX58, PTGS2, LTA, BIRC3, TRAF1, NFKB2, CARD11 Signal transduction CD14, NTF3, DDIT3, PLA2G4B, RPS6KA1, AKT3, HSP70.2, FAS, EGF, FGF14, NTRK1, FGFR4, MAPK10, DUSP4, PRKCG, GADD45B, MAP3K8, MAP2K6, PTPN7, CACNA2D2, CACNA2D3, FGF12, NFKB2, CACNA1H, DUSP2, MAPK8IP2, MAPK11, CACNA1C, CACNA1I, MAP4K1, CACNA1E Analysis of important DEGs related to immune responses Breeds Pathway terms Categories Genes Cytosolic DNA-sensing pathway Immune system IFNB1, NFKBIA, CXCL10, IRF7, ZBP1, CCL5, CCL4, DDX58, IL6, RIPK3, POLR3D, NFKBIB Intestinal immune network for IgA production Immune system CCR9, CXCL12, ICOS, SLA-DRB1, CD28, LOC106504372, IL10, IL6, AICDA, IL15RA Complement and coagulation cascades Immune system C1QA, C1S, F7, BDKRB1, F2, THBD, VSIG4, C1QB, C1QC, VWF, SERPINE1, C7, CPB2, C1R Chemokine signaling pathway Immune system CCR5, CCR1, XCR1, CXCR6, CCR9, CXCL2, NFKBIA, CXCL10, CXCL12, CCL5, AKT3, CCL4, PTK2B, ADCY4, GRK5, JAK3, CXCR3, NFKBIB, LOC110255211, GNG3, PARD3, CXCR2, HCK, ADCY1, PLCB2 NOD-like receptor signaling pathway Immune system NFKBIA, CCL5, PSTPIP1, TNFAIP3, IL6, MAPK10, TRIP6, NFKBIB, BIRC3, MAPK11 RIG-I-like receptor signaling pathway Immune system IFNB1, NFKBIA, CXCL10, IRF7, ISG15, DHX58, DDX58, TKFC, MAPK10, NFKBIB, TANK, MAPK11 Antigen processing and presentation Immune system TAP1, SLA-DRB1, CALR, PDIA3, LOC106504372, CD8B, HSP70.2, IFNG, PSME2, KLRD1, LOC100523789 Toll-like receptor signaling pathway Immune system IFNB1, NFKBIA, CXCL10, IRF7, CD14, LY96, CCL5, IFNAR2, AKT3, CCL4, IL6, MAPK10, MAP3K8, MAP2K6, MAPK11 T cell receptor signaling pathway Immune system NFKBIA, ICOS, PDCD1, AKT3, CD28, CD8B, IFNG, IL10, CTLA4, NFKBIB, MAP3K8, CARD11, MAPK11, PAK6, NFKBIE TNF signaling pathway Signal transduction CXCL2, NFKBIA, CXCL10, CREB3L4, CCL5, AKT3, TNFAIP3, FAS, PTGS2, IL6, LTA, RIPK3, MAPK10, MAP3K8, MAP2K6, BIRC3, TRAF1, MAPK11 Jak-STAT signaling pathway Signal transduction IFNB1, IL7R, SOCS1, IFNAR2, IL27RA, AKT3, IL2RA, CSF3, IFNG, IL10, IL2RG, IL6, JAK3, OSMR IL15RA, IL20RB, LOC100736818, PIM1, CDKN1A, IL2RB, TSLP, IL22, PTPN2 cAMP signaling pathway Signal transduction NFKBIA, TNNI3, CREB3L4, GABBR1, SSTR1, PLD1, AKT3, HTR1B, AMH, ADCY4, MAPK10, GRIA4, GRIA3, PDE4D, SUCNR1, LOC110255845, GRIN3A, PPP1R1B, HCAR2, GRIA1, ADCY1, GLI3, PDE3B, GRIN2A, GRIN2B, CACNA1C, GIPR, CNGB1 NF-kappa B signaling pathway Signal transduction NFKBIA, CXCL12, CD14, LY96, BCL2A1, TNFAIP3, CCL4, DDX58, PTGS2, LTA, BIRC3, TRAF1, NFKB2, CARD11 MAPK signaling pathway Signal transduction CD14, NTF3, DDIT3, PLA2G4B, RPS6KA1, AKT3, HSP70.2, FAS, EGF, FGF14, NTRK1, FGFR4, MAPK10, DUSP4, PRKCG, GADD45B, MAP3K8, MAP2K6, PTPN7, CACNA2D2, CACNA2D3, FGF12, NFKB2, CACNA1H, DUSP2, MAPK8IP2, MAPK11, CACNA1C, CACNA1I, MAP4K1, CACNA1E FoxO signaling pathway Signal transduction CCNB2, IL7R, PCK2, CCNB1, AKT3, EGF, IL10, IL6, MAPK10, PRKAG2, GADD45B, CCNB3, CDKN1A, IRS2, PLK1, MAPK11 Cytokine-cytokine receptor interaction Signaling molecules and interaction CCR5, CCR1, XCR1, CXCR6, CCR9, CXCL2, IFNB1, CXCL10, CXCL12, CCL5, IL7R, IFNAR2, TN FRSF9, CD27, CCL4, IL2RA, FAS, CSF3, IFNG, EGF, IL10, IL2RG, AMH, IL6, LTA, OSMR, CXCR3 IL15RA, IL20RB, LOC100736818, IL18RAP, IL25, IL2RB, CXCR2, TSLP, IL22 Cell adhesion molecules (CAMs) Signaling molecules and interaction ICOS, SELL, SLA-DRB1, OCLN, PDCD1, VCAN, CLDN1, CD28, LOC106504372, CD8B, CTLA4, MPZ, L1CAM, TIGIT, NLGN3, LOC100525346, ITGA8, NCAM2, NEGR1 Duroc pigs Neuroactive ligand–receptor interaction Signaling molecules and interaction P2RY2, BDKRB1, F2, GABBR1, GZMA, CRHR2, SSTR1, P2RY6, TRH, TSPO, HRH4, OPRL1, HTR1B, PTH1R, TAAR1, LTB4R2, THRB, PTGDR, GRIA4, GRIA3, P2RX3, PTGIR, GRIK1, GABRB2, CNR1, LRRC74B, PARD3, GRIN3A, P2RX1, GRM2, GABRR3, GRIK2, GRIA1, GRIN2A, GRIN2B, GIPR Complement and coagulation cascades Immune system C5, MBL1, C6, PLAU, F5, SERPINA5, ITGAM, KNG1, SERPINF2 Leukocyte transendothelial migration Immune system CLDN23, CLDN18, CLDN5, PECAM1, ITGAM, TXK, ESAM, GNAI1, PLCG2 Natural killer cell mediated cytotoxicity Immune system NCR1, IFNAR1, KLRK1, CD244, LOC100739080, SHC3, LOC100523789, PLCG2 Hematopoietic cell lineage Immune system CD4, GP1BB, CD34, CD19, ITGAM, CD7 Hippo signaling pathway Signal transduction PARD6B, BMP2, WNT10B, WNT16, GDF7, WNT11, TCF7L1, GDF6, WTIP, FZD1 cAMP signaling pathway Signal transduction FXYD2 FOS PDE4A DRD2 GNAI1 LOC100738425 LOC110255845 LOC110255846 CACNA1F Jiangquhai pigs Important immune-related KEGG pathways of specific DEGs in Jiangquhai pigs and Duroc pigs. Duroc pigs CCNB2, IL7R, PCK2, CCNB1, AKT3, EGF, IL10, IL6, MAPK10, PRKAG2, GADD45B, CCNB3, CDKN1A, IRS2, PLK1, MAPK11 CCR5, CCR1, XCR1, CXCR6, CCR9, CXCL2, IFNB1, CXCL10, CXCL12, CCL5, IL7R, IFNAR2, TN- FRSF9, CD27, CCL4, IL2RA, FAS, CSF3, IFNG, EGF, IL10, IL2RG, AMH, IL6, LTA, OSMR, CXCR3, IL15RA, IL20RB, LOC100736818, IL18RAP, IL25, IL2RB, CXCR2, TSLP, IL22 ICOS, SELL, SLA-DRB1, OCLN, PDCD1, VCAN, CLDN1, CD28, LOC106504372, CD8B, CTLA4, MPZ, L1CAM, TIGIT, NLGN3, LOC100525346, ITGA8, NCAM2, NEGR1 P2RY2, BDKRB1, F2, GABBR1, GZMA, CRHR2, SSTR1, P2RY6, TRH, TSPO, HRH4, OPRL1, HTR1B, PTH1R, TAAR1, LTB4R2, THRB, PTGDR, GRIA4, GRIA3, P2RX3, PTGIR, GRIK1, GABRB2, CNR1, LRRC74B, PARD3, GRIN3A, P2RX1, GRM2, GABRR3, GRIK2, GRIA1, GRIN2A, GRIN2B, GIPR C5, MBL1, C6, PLAU, F5, SERPINA5, ITGAM, KNG1, SERPINF2 DN23, CLDN18, CLDN5, PECAM1, ITGAM, TXK, ESAM, GNAI1, PLCG2 NCR1, IFNAR1, KLRK1, CD244, LOC100739080, SHC3, LOC100523789, PLCG PARD6B, BMP2, WNT10B, WNT16, GDF7, WNT11, TCF7L1, GDF6, WTIP, FZD1 FXYD2, FOS, PDE4A, DRD2, GNAI1, LOC100738425, LOC110255845, LOC110255846, CACNA1F, PDE4C Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 Figure 7 Important immune-related genes showing specific expression in Jiangquhai pigs (A) and Duroc pigs (B). Genes have been arbitrarily positioned along the x-axis. Full-size DOI: 10.7717/peerj.7900/fig-7 (WNT11, WNT16). As for Duroc pigs, more immune-related genes were up-regulated, including chemokines (CCL4, CCL5, CCR1, CCR5, CXCL2, CXCL10, CXCL12, CXCR6), interferon response factors (IFNAR2, IFNG), and interleukins (IL6, IL10, IL15RA, IL2RA, IL18RAP, IL2RB, IL2RG, IL27RA, IL7R). Among these immune-related genes, CXCL10, CCL4, IL6 and IFNG were the most differently expressed genes. Figure 7 Important immune-related genes showing specific expression in Jiangquhai pigs (A) and Duroc pigs (B). Genes have been arbitrarily positioned along the x-axis. Full-size DOI: 10.7717/peerj.7900/fig-7 (WNT11, WNT16). As for Duroc pigs, more immune-related genes were up-regulated, including chemokines (CCL4, CCL5, CCR1, CCR5, CXCL2, CXCL10, CXCL12, CXCR6), interferon response factors (IFNAR2, IFNG), and interleukins (IL6, IL10, IL15RA, IL2RA, IL18RAP, IL2RB, IL2RG, IL27RA, IL7R). Among these immune-related genes, CXCL10, CCL4, IL6 and IFNG were the most differently expressed genes. Figure 7 Important immune-related genes showing specific expression in Jiangquhai pigs (A) and Duroc pigs (B). Genes have been arbitrarily positioned along the x-axis. Full-size DOI: 10.7717/peerj.7900/fig-7 Figure 7 Important immune-related genes showing specific expression in Jiangquhai pigs (A) and Duroc pigs (B). Genes have been arbitrarily positioned along the x-axis. Full-size DOI: 10.7717/peerj.7900/fig-7 (WNT11, WNT16). As for Duroc pigs, more immune-related genes were up-regulated, (WNT11, WNT16). Duroc pigs As for Duroc pigs, more immune-related genes were up-regulated, including chemokines (CCL4, CCL5, CCR1, CCR5, CXCL2, CXCL10, CXCL12, CXCR6), interferon response factors (IFNAR2, IFNG), and interleukins (IL6, IL10, IL15RA, IL2RA, IL18RAP, IL2RB, IL2RG, IL27RA, IL7R). Among these immune-related genes, CXCL10, CCL4, IL6 and IFNG were the most differently expressed genes. DISCUSSION Previous reports state that the primary effect of Mhp infection on pigs is reduced growth performance (Maes et al., 2008; Pointon, Byrt & Heap, 1985). However, the degree to which Mhp effect differs between Chinese local breeds and imported breeds (such as Duroc and Landrace), and the effect on Chinese local breeds is much more serious (Fang et al., 2015). Our results were consistent with previous reports, Jiangquhai pigs infected with Mhp exhibited poorer growth performance than Duroc pigs. Some studies have reported that Mhp infection causing a humoral immune response in pigs (Blanchard et al., 1992). Moreover, the level of antigen-specific antibody is an important indicator of MPS lesion Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 11/21 formation in animals, and increased antigen-specific antibody production may exacerbate MPS lung lesion severity (Borjigin et al., 2016; Davis et al., 1985; Katayama et al., 2011). Our results also showed Mhp-infected Jiangquhai pigs exhibited higher blood levels of Mhp antibody and more serious Mhp lung lesions. These results confirm that Jiangquhai pigs are more susceptible to Mhp infection, and Duroc pigs possess greater resistance. In addition, it was reported that the age and weight of pigs do not affect the susceptibility to Mhp infection (Piffer & Ross, 1984). Therefore, it is theoretically possible that genetic components contribute to Mhp resistance/susceptibility differs among breeds. formation in animals, and increased antigen-specific antibody production may exacerbate MPS lung lesion severity (Borjigin et al., 2016; Davis et al., 1985; Katayama et al., 2011). To gain insight into how the transcriptome profiles of different pig breeds vary in response to Mhp infection, we performed RNA-seq to analyze the transcriptional profiles of lung tissue from two breeds. We identified 2,250 and 3,526 DEGs in lung tissue from Jiangquhai pigs and Duroc pigs, respectively. Duroc pigs had 694 unique up-regulated DEGs, whereas Jiangquhai pigs only had 334. Taken together, these results indicate that the molecular interactions and signaling pathways following Mhp infection may be more complex in Duroc pigs. Our analysis revealed 1,669 DEGs shared between Jiangquhai and Duroc pigs in response to Mhp infection. These common DEGs also showed significant enrichment of many immune-relevant terms and pathways. Among these, many up-regulated immune-relevant genes were observed in both breeds that could play a role in resistance to Mhp infection. Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 DISCUSSION One TLR family member, TLR2, has a fundamental role in pathogen recognition, signal transmission and activation of innate immunity, and stimulation of inflammatory cytokine production (Yoshihiro et al., 2003). IL18 and IL1R1 are pro-inflammatory mediators involved in many cytokine-induced immune and inflammatory responses (Dale & Nicklin, 1999). PI3Ks (PIK3R5) are important enzymes involved in various signal transduction pathways, and play important roles in regulating cell growth, survival, death and chemotaxis (Margaria et al., 2019). Chemokines such as CXCL8 and CXCL13 can recruit immune cells to the site of infection (Strieter et al., 1999; Sun et al., 2006). Moreover, ITGA1 and ITGB7, which help recruit immune cells such as T, B, and natural killer (NK) cells, were up- regulated following Mhp infection (Campbell & Humphries, 2011; Cheli et al., 2007; Lim, Leung & Krissansen, 1998). These immune-related DEGs help clarify the immune response in pigs following Mhp infection. Comparison of the specific DEGs between Jiangquhai and Duroc pigs revealed some important DEGs related to immune responses were specifically altered in Jiangquhai or Duroc pigs, and these important DEGs enriched in immune-related pathways play an important role in the underlying host mechanism to defend against Mhp infection. Adhesion molecules are important signal transmitters of the immune system, they function as signal transduction, transported signals from outside to inside of cell (Kumar et al., 2016; Korytina et al., 2019), In this study, many adhesion molecules were specifically down-regulated in Jiangquhai pigs, including PECAM1, CD34, CD7 and CDH4. These adhesion molecules were significantly enriched in Cell adhesion molecules (CAMs) and Hematopoietic cell lineage pathway, have the function in pathogen recognition, signal transmission and regulating cell movement (Chasiotis et al., 2012; Samanta & Almo, 2015; Xiao et al., 2010). However, these adhesion molecules were down-regulated may reduce Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 host’s capacity for antigen presentation and processing. In addition, two WNT molecules (WNT11, WNT16) enriched in Hippo signaling pathway were identified in Jiangquhai pigs. WNT genes exert immune modulatory functions during pathogens infection (Brandenburg & Reiling, 2016), and could regulate the expression of immune response genes during challenge by pathogens, such as interferon genes, Toll-like receptors and MHC genes (Garcia-Rodriguez et al., 2017). WNT11 and WNT16 were down-regulated, which may induce the inhibition of its downstream signaling, including cell growth, survival and cell- cycle progression. DISCUSSION Therefore, thus results may interfere with Jiangquhai pigs to establish an effective immune response against Mhp infection. Chemokines constitute a large family of chemotactic molecules that are fundamentally involved in the inflammatory response by attracting immune cells to sites of inflammation, and promote the immune response and wound healing (Le et al., 2004; Zlotnik & Yoshie, 2012). Many chemokines are expressed in immune tissues and cells in pigs infected with Mhp (Li et al., 2014; Zhang et al., 2011). In this study, many specific up-regulated chemokines (CCL4, CCL5, CCR1, CCR5, CXCL2, CXCL10, CXCL12 and CXCR6) were identified in Duroc pigs, these chemokines were mainly enriched in cytokine- cytokine receptor interaction and chemokine signaling pathway. The two pathways are involved in specific functional tasks that recruit immune cells to induce inflammatory and adaptive immune responses (Carvalho et al., 2012; Hu et al., 2016). Therefore, these up-regulated chemokines could recruit more immune cells for pathogen defense, and Duroc pigs exhibited a larger chemotactic immune cell capacity. Among these chemokines, CXCL10 and CCL4 were most highly expressed. CXCL10 is an important regulator of pulmonary diseases (Gao et al., 2018; Tighe et al., 2011), and has the function of chemotactic monocytes/macrophages, T cells, and NK cells and promotion of T-cell adhesion to endothelial cells (Angiolillo et al., 1995; Dufour et al., 2002). CCL4 is an important chemoattractant for natural killer cells, monocytes and a variety of other immune cells, regulates immune response to pathogen infection (Bystry et al., 2001; Zhao et al., 2007). Therefore, CXCL10 and CCL4 maybe important chemokines involved in chemokine signaling pathway, and regulate the process of immune response to Mhp infection. Mhp stimulates host immune response by inducing macrophages to release pro- inflammatory cytokines, such as IL-2, IL-6, IL-8, IL10 and IL-1β (Fourour et al., 2019; Lorenzo et al., 2006). These pro-inflammatory cytokines are important players in innate and adaptive immunity, and affect immune balance by suppressing cell-mediated immunity (Zhang et al., 2018). In this study, we also identified some specific up-regulated interleukins (IL6, IL10, IL15R A, IL2RA, IL18RAP, IL2RB, IL2RG, IL27RA and IL7R) in Duroc pigs. These interleukin cytokines can modulate a broad spectrum of immune response processes, such as NOD-like receptor signaling pathway, toll-like receptor signaling pathway, TNF signaling pathway and Jak-STAT signaling pathway. Thus, activation of interleukin cytokines in Duroc pigs may induce additional immune cytokine production and immune cell recruitment for pathogen defense. Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 DISCUSSION In addition, interferon response factors have the function of activate macrophages, protect host cells to resist pathogen infection (Rodriguez et al., 2007). Two interferon response factors (IFNAR2, IFNG) were highly expressed in Duroc pigs, which may further activated macrophages and enhanced the ability to resistance Mhp Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 13/21 infection. Moreover, IL6 and IFNG were most highly expressed among these cytokines. IL-6 is a primary cytokine, which could activate macrophages to secrete inflammatory cytokines and chemokines (Yan et al., 2012), and responsible for Mhp clearance in lungs (Wu et al., 2008). IFNG had been detected in the lungs of pathogen-infected pigs and be postulated to be a necessary component for host control of pathogen (Zhang et al., 2011). Therefore, IL6 and IFNG were also important cytokines involved in the regulation of immune response to Mhp infection. Apoptosis is a mechanism of programmed cell death and is essential for the regulation of immune responses (Rantong & Gunawardena, 2015). Our results further revealed that the apoptosis-related gene (FAS) was specifically activated in Duroc pigs. The FAS gene has been reported to play a central role in the physiological regulation of programmed cell death and has been implicated in the pathogenesis of various diseases of the immune system (Strasser, Jost & Nagata, 2009). Previous studies have shown that the FAS gene involved in the regulation of inflammatory response in pulmonary (Liu et al., 2017). Therefore, the FAS gene activation can induce apoptosis and regulate the immune response in response to the Mhp infection. In addition, in the Duroc-specific DEGs involved in GO enrichments (cilium assembly, cilium-dependent cell motility), we found intraflagellar transport (IFT) genes, which can modulate primary cilia formation and function (Pedersen & Rosenbaum, 2008), such as IFT172 and IFT81. Primary cilia are found on the cell surface of almost every cell type, which play an important role in signaling and development (Singla & Reiter, 2006) and influence immune cell migration (Finetti, Onnis & Baldari, 2015). Therefore, intraflagellar transport genes may play an important role in the immune response to Mhp infection. Further elucidating the function remains to be a near future goal. Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 Competing Interests Competing Interests The authors declare there are no competing interests. The authors declare there are no competing interests. DNA Deposition The following information was supplied regarding the deposition of DNA sequences: The sequences are available at the NCBI Short Read Archive (SRA) under BioProject: PRJNA542576. Raw reads are available in the Sequence Read Archive database (SRR9114668–SRR9114679). Author Contributions • Ligang Ni conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables. • Chengyi Song conceived and designed the experiments. • Chengyi Song conceived and designed the experiments. • Xinsheng Wu analyzed the data, authored or reviewed drafts of the paper, approved the final draft. • Xuting Zhao analyzed the data. • Xuting Zhao analyzed the data. • Xiaoyan Wang performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft. • Bichun Li contributed reagents/materials/analysis tools. • Bichun Li contributed reagents/materials/analysis tools. • Yuan Gan performed the experiments. • Yuan Gan performed the experiments. Animal Ethics The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): Approval for the study was provided by the ethics committee of Yangzhou University (SYXK(Su) IACUC 2016–0131). CONCLUSIONS This is the first study to describe the transcriptional profiles of lung tissue from different pig breeds following Mhp infection. RNA-seq analysis identified 966 up-regulated and 1,284 down-regulated genes in Jiangquhai pigs compared to 1,326 up-regulated and 2,200 down-regulated genes in Duroc pigs. Both breeds shared some KEGG pathways, including cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, and chemokine signaling pathway. All of these may play important roles in Mhp infection resistance. In Duroc pigs, 1857 specific DEGs were identified, KEGG pathway analysis revealed 18 immune-related pathways. I Jiangquhai pigs, 581 specific DEGs were identified and eight immune-related pathways were identified. Compared to Jiangquhai pigs, chemokines, interferon response factors, interleukins, complement components, apoptosis-related molecule and other immune-related molecules were specifically activated in Duroc pigs, and they may help host enhance immune response and reduce Mhp susceptibility. The results of our analysis reveal an important role of genetics in the immune response to Mhp infection, and this should be investigated further to improve pig health during breeding. Ni et al. (2019), PeerJ, DOI 10.7717/peerj.7900 14/21 Funding This work was supported by the National Development and Reform Commission Special Breeding Projects (No. 2150299) and the Phoenix Talent Projects of Jiangsu Agri-animal Husbandry Vocational College (No. 201602). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: National Development and Reform Commission Special Breeding Projects: 2150299. National Development and Reform Commission Special Breeding Projects: 2150299. National Development and Reform Commission Special Breeding Projects: 2150299. Jiangsu Agri-animal Husbandry Vocational College: 201602. Jiangsu Agri-animal Husbandry Vocational College: 201602. Jiangsu Agri-animal Husbandry Vocational College: 201602. Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.7900#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.7900#supplemental-information. 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https://openalex.org/W2794331689	https://europepmc.org/articles/pmc5852795?pdf=render	English	null	The Role of the Slc39a Family of Zinc Transporters in Zinc Homeostasis in Skin	Nutrients	2,018	cc-by	9,811	The Role of the Slc39a Family of Zinc Transporters in Zinc Homeostasis in Skin Bum-Ho Bin 1,†, Shintaro Hojyo 2,†, Juyeon Seo 3, Takafumi Hara 4, Teruhisa Takagishi 4, Kenji Mishima 1 and Toshiyuki Fukada 1,4,5,* 1 Division of Pathology, Department of Oral Diagnostic Sciences, School of Dentistry, Showa University, Tokyo 142-8555, Japan; bbh82429@gmail.com (B.-H.B); mishima-k@dent.showa-u.ac.jp (K.M.) 2 Osteoimmunology, Deutsches Rheuma-Forschungszentrum, 10117 Berlin, Germany; Shintaro.Hojyo@drfz.de 1 Division of Pathology, Department of Oral Diagnostic Sciences, School of Dentistry, Showa University, Tokyo 142-8555, Japan; bbh82429@gmail.com (B.-H.B); mishima-k@dent.showa-u.ac.jp (K.M.) 2 Osteoimmunology, Deutsches Rheuma-Forschungszentrum, 10117 Berlin, Germany; Shintaro.Hojyo@drfz.de 3 AmorePaciﬁc Corporation R&D Center, Yongin, Gyeonggi-do 446-729, Korea; yeon8687@amorepaciﬁc.com 4 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8055, Japan; t-hara@ph.bunri-u.ac.jp (T.H.); t.takagishi@ph.bunri-u.ac.jp (T.T.) 5 RIKEN Center for Integrative Medical Sciences, Yokohama 230-0042, Japan * Correspondence: fukada@ph.bunri-u.ac.jp; Tel.: +81-88-602-8593 † These authors contributed equally to this work. Received: 20 December 2017; Accepted: 11 February 2018; Published: 16 February 2018 3 AmorePaciﬁc Corporation R&D Center, Yongin, Gyeonggi-do 446-729, Korea; yeon8687@amorepaciﬁc.com 4 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8055, Japan; t-hara@ph.bunri-u.ac.jp (T.H.); t.takagishi@ph.bunri-u.ac.jp (T.T.) 5 RIKEN Center for Integrative Medical Sciences, Yokohama 230-0042, Japan * Correspondence: fukada@ph.bunri-u.ac.jp; Tel.: +81-88-602-8593 † These authors contributed equally to this work. Received: 20 December 2017; Accepted: 11 February 2018; Published: 16 February 2018 4 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 7 t-hara@ph.bunri-u.ac.jp (T.H.); t.takagishi@ph.bunri-u.ac.jp (T.T.) 5 RIKEN Center for Integrative Medical Sciences, Yokohama 230-0042, Japan * Correspondence: fukada@ph.bunri-u.ac.jp; Tel.: +81-88-602-8593 † These authors contributed equally to this work. Received: 20 December 2017; Accepted: 11 February 2018; Published: 16 February 2018 Abstract: The ﬁrst manifestations that appear under zinc deﬁciency are skin defects such as dermatitis, alopecia, acne, eczema, dry, and scaling skin. Several genetic disorders including acrodermatitis enteropathica (also known as Danbolt-Closs syndrome) and Brandt’s syndrome are highly related to zinc deﬁciency. However, the zinc-related molecular mechanisms underlying normal skin development and homeostasis, as well as the mechanism by which disturbed zinc homeostasis causes such skin disorders, are unknown. Recent genomic approaches have revealed the physiological importance of zinc transporters in skin formation and clariﬁed their functional impairment in cutaneous pathogenesis. In this review, we provide an overview of the relationships between zinc deﬁciency and skin disorders, focusing on the roles of zinc transporters in the skin. We also discuss therapeutic outlooks and advantages of controlling zinc levels via zinc transporters to prevent cutaneous disorganization. Keywords: zinc; skin; homeostasis; transporter Nutrients 2018, 10, 219; doi:10.3390/nu10020219 nutrients nutrients 1. Introduction Zinc is an essential micronutrient obtained from food and drink [1]. Zinc is abundantly contained in mollusks such as oysters and in crustaceans such as crabs and crayﬁsh [2]. The biological role of zinc was ﬁrst revealed in 1869 by Raulin, who showed that zinc is essential for the growth of Aspergillus niger [1]. Subsequently, its importance for rodent growth was demonstrated using experimental rats in 1934 [1,3]. However, zinc’s physiological role in humans was unknown until 1961 when zinc deﬁciency in humans was discovered [4]. Zinc is associated with growth, gonad development, immune function and pregnancy outcome improvement, and hair loss prevention [1,5,6]. In 1961, growth retardation and hypogonadism observed in Iranian and Egyptian adults were reported to be associated with zinc deﬁciency, which was closely related to dietary habits [4,7,8]. Middle Eastern diets typically include bread and beans, which contain large quantities of phytate [9]. Phytate has a negative effect on zinc absorption, thereby causing zinc deﬁciency. Zinc deﬁciency is also observed in vegetarians, single people, alcoholics, dieters, pregnant women, and the malnourished in developing and developed countries [4]. In this review, we outline the characteristics of zinc transporters and identify skin phenotypes of their knockout mouse models and human skin genetic diseases caused Nutrients 2018, 10, 219; doi:10.3390/nu10020219 www.mdpi.com/journal/nutrients www.mdpi.com/journal/nutrients 2 of 14 Nutrients 2018, 10, 219 by mutations in zinc transporters. Furthermore, we discuss how zinc deﬁciency impacts normal skin development and homeostasis, based on the most recent research. by mutations in zinc transporters. Furthermore, we discuss how zinc deﬁciency impacts normal skin development and homeostasis, based on the most recent research. 2.1. Skin Structure The skin largely consists of three layers [10,11]. The epidermis functions as a barrier to protect the interior from direct contact with the external environment. The dermis supports the epidermis by ﬁlling the skin volume with ﬁbers. The hypodermis is present under the dermis and is composed of subcutaneous fat layers [10–12]. The epidermis is a cell layer composed of keratinocytes, the basal layer of which contains progenitor cells (called basal cells) at the interface with the dermal layer. These cells gradually proliferate perpendicularly to the basal layer. At the same time, they differentiate into spinous cells and induce keratinization while undergoing enucleation [10]. Spinous cells are ﬁrst differentiated from the basal layer and produce keratin, which contributes to tight cell-to-cell adhesion [10,11]. These cells then differentiate into granular cells that are rich in keratohyalin and eject lipids and proteins, as well as connect keratin ﬁbers with higher density [10–12]. Next, the granular cells immediately die after denucleation and form corneocytes that are eventually pushed up to the surface of the skin, resulting in a ﬁrm stratum corneum, the outermost layer of the dermal barrier. Most of the dermis consists of collagen, elastin, and the polysaccharide hyaluronan, which is produced by ﬁbroblasts [10–12]. There are various nerves, blood vessels, hair follicles, sweat glands, macrophages, and T-cells, which play important roles in the secondary function of skin sensation and immunity [4,13–15]. Upon aging or ultraviolet (UV) stimulation, the levels of these substances are decreased, leading to the production of matrix metalloproteinases (MMPs), which are degrading enzymes that reduce skin volume. The hypodermis is a subcutaneous organization of adipocyte-derived lipids [4,13–15]. Fat tissue is important for maintaining body temperature in humans. Unlike reptiles, which change temperature depending on the environment, humans, with a thin epidermis, develop fat tissue in the hypodermis to maintain body temperature and protect the body organs. Therefore, surplus energy can be stored in the hypodermis, and nerves and blood vessels larger than the dermis are safely preserved from external impact, completing the complex human body as an organic and safe system. 2.2. Zinc Transporters NC; nucleus, ER; endoplasmic reticulum, Zn; zinc. ZIP4 i a i te i ely tudied t a o te e e ed i e te o yte of the all i te ti e a d Figure 1. Zinc transporters and metallothionein (MT) are involved in intracellular zinc homeostasis. NC; nucleus, ER; endoplasmic reticulum, Zn; zinc. plays a first gate role in absorbing zinc into the body [26,27]. When zinc is deficient, the N‐terminus of ZIP4 is cleaved and the remaining truncated protein migrates to the cell surface to help absorb zinc [28–30]. ZIP4 is a causative gene for acrodermatitis enteropathica (AE), in which systemic zinc deficiency occurs because of impaired intestinal zinc transport [31]. Dozens of pathogenic mutations have been identified. Recent studies showed that single‐nucleotide polymorphisms (SNPs) in ZIP4 differ significantly among various regions of sub‐Saharan Africa, where people exhibit high sensitivity to zinc deficiency [32]. Some people have a leucine‐to‐valine (Leu372Val) replacement in the ZIP4 protein that allows for the expression of small amounts of ZIP4 on cell surfaces and thus transport a low amount of zinc, whereas others show the pathogenic mutations Leu372Pro and Leu372Arg, which prevent ZIP4 from migrating to the cell surface. Homozygous knockout of ZIP4 in an AE mouse model shows embryonic lethality [27], indicating that ZIP4 is essential for embryonic development in mice. A conditional Zip4‐knockout mouse in which a Zip4 allele is specifically deleted by Cre‐Lox recombination controlled under the villin promoter has also been generated [33]. These mice show collapse of the stem cell niche and integrity of the small intestine, but do not present any skin abnormality. ZIP10 is mainly localized to the cell surface and carries zinc in B‐cells [34,35]. A conditional Zip10‐knockout mouse in which a Zip10 allele is specifically deleted by Cre‐Lox recombination controlled under the keratin 14 promoter shows impaired skin barrier [36]. ZIP7 and ZIP13 are present in the endoplasmic reticulum (ER)/Golgi and play important roles in zinc homeostasis within these compartments [37,38]. A conditional Zip7‐knockout mouse under the collagen 1 promoter results in a thin epidermis; thus, both ZIP7 and ZIP10 are essential for skin homeostasis ZIP4 is an intensively studied transporter expressed in enterocytes of the small intestine and plays a ﬁrst gate role in absorbing zinc into the body [26,27]. 2.2. Zinc Transporters When zinc is deﬁcient, the N-terminus of ZIP4 is cleaved and the remaining truncated protein migrates to the cell surface to help absorb zinc [28–30]. ZIP4 is a causative gene for acrodermatitis enteropathica (AE), in which systemic zinc deﬁciency occurs because of impaired intestinal zinc transport [31]. Dozens of pathogenic mutations have been identiﬁed. Recent studies showed that single-nucleotide polymorphisms (SNPs) in ZIP4 differ signiﬁcantly among various regions of sub-Saharan Africa, where people exhibit high sensitivity to zinc deﬁciency [32]. Some people have a leucine-to-valine (Leu372Val) replacement in the ZIP4 protein that allows for the expression of small amounts of ZIP4 on cell surfaces and thus transport a low amount of zinc, whereas others show the pathogenic mutations Leu372Pro and Leu372Arg, which prevent ZIP4 from migrating to the cell surface. Homozygous knockout of ZIP4 in an AE mouse model shows embryonic lethality [27], indicating that ZIP4 is essential for embryonic development in mice. A conditional Zip4-knockout mouse in which a Zip4 allele is speciﬁcally deleted by Cre-Lox recombination controlled under the villin promoter has also been generated [33]. These mice show collapse of the stem cell niche and integrity of the small intestine, but do not present any skin abnormality. promoter results in a thin epidermis; thus, both ZIP7 and ZIP10 are essential for skin homeostasis [39]. In contrast to ZIPs, ZnTs are unique secondary transporters with Y‐type and transport zinc using a concentration gradient of a partner transport substrate such as hydrogen ions [40,41]. As in the ZIP family, the ZnT group is present on the cell surface or within the cells depending on the cell type, and thus plays an indispensable role in zinc export [17]. As ZIPs and ZnTs, several transporters and channels were also found to mediate zinc influx. Although their main substrates do not appear to be zinc, transient receptor potential (TRP) channels, ZIP10 is mainly localized to the cell surface and carries zinc in B-cells [34,35]. A conditional Zip10-knockout mouse in which a Zip10 allele is speciﬁcally deleted by Cre-Lox recombination controlled under the keratin 14 promoter shows impaired skin barrier [36]. ZIP7 and ZIP13 are present in the endoplasmic reticulum (ER)/Golgi and play important roles in zinc homeostasis within these compartments [37,38]. A conditional Zip7-knockout mouse under the collagen 1 promoter results in a thin epidermis; thus, both ZIP7 and ZIP10 are essential for skin homeostasis [39]. 2.2. Zinc Transporters Intracellular zinc homeostasis is tightly regulated by zinc transporters and metal binding proteins, known as metallothioneins (MTs) (Figure 1) [16]. Because zinc is a metal ion that cannot pass through the cell wall, where lipid is abundant, cells must use carriers to maintain intracellular zinc homeostasis [16,17]. There are two types of zinc transporters: ZIP, which transports zinc into cells from extracellular regions or the luminal side of intracellular compartments and is dependent on zinc concentration, and ZnT, which transports zinc to the exterior of cells or the lumen side from cell cytoplasm. There are 14 ZIP and 10 ZnT family members in humans, and their expression patterns and intracellular locations vary depending on the cell type, developmental stage, and Zn status [18]. Currently, the structure of the ZIP family has not been clariﬁed, but it is thought to possess a domain that penetrates the cell membranes approximately eight times and constitutes a homodimer or a heterodimer with other ZIP members [19,20]. Both ends of ZIP family member peptides face the extracellular or luminal side, with a variety of N-terminal domains, while the intracellular domain has two lengths [19]. In particular, the two domains contain a large number of histidine residues that can bind zinc. Proteoliposome studies using bacterial homologs have suggested that ZIP family members have transport mechanisms such as channels that are independent of other ionic concentrations or adenosine triphosphate (ATP) [21]. In some cases, the ﬁlter allowing the passage of zinc is thought to ﬂexibly transport other metals with physicochemical properties similar to 3 of 14 Nutrients 2018, 10, 219 those of zinc such as cadmium. For instance, ZIP14 is responsible for transporting iron and manganese in the liver [22–24]. ZIP8 and ZIP14 are symporters that carry metal and biscarbonate ions [25]. properties similar to those of zinc such as cadmium. For instance, ZIP14 is responsible for transporting iron and manganese in the liver [22–24]. ZIP8 and ZIP14 are symporters that carry metal and biscarbonate ions [25]. Figure 1. Zinc transporters and metallothionein (MT) are involved in intracellular zinc homeostasis. NC; nucleus, ER; endoplasmic reticulum, Zn; zinc. ZIP4 is an intensively studied transporter expressed in enterocytes of the small intestine and Figure 1. Zinc transporters and metallothionein (MT) are involved in intracellular zinc homeostasis. NC; nucleus, ER; endoplasmic reticulum, Zn; zinc. Figure 1. Zinc transporters and metallothionein (MT) are involved in intracellular zinc homeostasis. 2.4. Zinc Levels in Skin The expression of MTs is induced by metal-responsive transcription factor 1 (MTF1) in a zinc concentration-dependent manner. Therefore, many studies have indirectly monitored the quantity of zinc in cells and tissues by measuring MT expression levels [19,54,55]. MT in the skin is mainly accumulated in progenitor cells and initial spinous cells at the bottom of the epidermis near the basal layers [56]. MTs are also found in out-root sheath cells of hair follicles and epidermal stem cells [57], which are undifferentiated cells with common features and show strong proliferative capacity when necessary [11,12]. Given that zinc is required as a structural component or activating cofactor for over 300 enzymes and other proteins related to cell proliferation, survival, and differentiation, undifferentiated cells with proliferative capacity contain high amounts of zinc [17,46]. It is known that zinc has excellent efﬁcacy for treating and regenerating skin wounds. Indeed, zinc deﬁciency causes delayed growth with skin anomalies [1,58]. Therefore, zinc is crucial for epidermal stem cells that regenerate inter-follicular epidermis, sebaceous gland, and hair follicle cells by rapidly migrating and dividing basal cells in the early stages of differentiation and scar tissue formation. Treatment with materials that thicken the epidermis have been shown to increase the amount of MT1 in such regions [52,57]. Fibroblasts are sparse in the dermis and do not undergo rapid cell division, suggesting that the dermis contains less zinc than the epidermis. In fact, zinc is present at 60 µg/g in the epidermis and 40 µg/g in the dermis [13,59]. Furthermore, it has been reported that the upper dermis contains higher zinc levels than the lower dermis [59]. This difference may be attributed, in part, to mast cells that contain a large quantity of zinc in the granules. Recently, the skin zinc concentration was measured by synchrotron radiation high energy X-ray ﬂuorescence [60]. This study suggested that the greatest amount of dermal zinc exists in the stratum spinosum, a ﬁnding that somewhat differs from those of previous studies that showed a large quantity of zinc in spinous and granular cells compared to basal cells. Given that it is difﬁcult to accurately measure the concentration of speciﬁc metals without interference from other metals, a new method for precise measurement of zinc concentration should be developed. 2.2. Zinc Transporters In contrast to ZIPs, ZnTs are unique secondary transporters with Y-type and transport zinc using a concentration gradient of a partner transport substrate such as hydrogen ions [40,41]. As in the ZIP family, the ZnT group is present on the cell surface or within the cells depending on the cell type, and thus plays an indispensable role in zinc export [17]. As ZIPs and ZnTs, several transporters and channels were also found to mediate zinc inﬂux. Although their main substrates do not appear to be zinc, transient receptor potential (TRP) channels, divalent metal transporter (DMT1), N-methyl-D-aspartate receptors (NMDA), amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPA-Rs), and voltage-dependent calcium channels (VDCCs), are reported to transport zinc [42,43]. However, their involvement in skin development and homeostasis is unknown. 4 of 14 Nutrients 2018, 10, 219 2.3. MTs MTs were ﬁrst puriﬁed from the cortex of horse kidneys in 1957 [44]. MTs are small proteins (molecular weight less than 7 kDa) containing more than 33% cysteine residues and facilitate the storage of zinc, cadmium, and copper, etc. with high thermal stability [45,46]. MTs are important for acquiring resistance to epithelial apoptosis mediated by reactive oxygen species, possibly through their antioxidant activities [15,47]. Currently, more than 10 isoforms have been identiﬁed in humans, located in both the cytoplasm and nucleus [48–50]. Mice lacking Mt1 or Mt2 exhibit no speciﬁc skin abnormalities [51]. However, the epidermal swelling caused by cholera toxin or UV-B irradiation is not observed in Mt1/2-double knockout mice [52]. In a mouse wound healing model, zinc enrichment in hair follicles was found in parallel with increased MT1 and MT2 expression during the wound healing process [53], suggesting that MT1 and MT2 play important roles in epidermal proliferation in certain situations. Nutrients 2018, 10, 219 Nutrients 2018, 10, 219 When ZnT2 function is impaired, mammary epithelial cells do not release zinc into breast tissue [63]. Therefore, patients with transient neonatal zinc deﬁciency cannot expect to increase zinc concentrations in the breast milk unless zinc is prescribed. Mice carrying mutations involving ZnT4 exhibit lethal milk (lm) syndrome, causing dermatitis and alopecia, but this is irrelevant to humans. Thus, clinical features according to the zinc transport species involved differ among species [64]. Although zinc deﬁciency is diagnosed by direct measurement of zinc concentrations in the blood, accuracy can be low and perturbed even under healthy conditions. Therefore, the development of new zinc biomarkers has been widely pursued for more precise measurement of zinc levels (e.g., erythrocyte linoleic acid:dihomo-y-linolenic acid (LA:DGLA) [65]. Zinc deﬁciency initially causes skin problems such as dermatitis, alopecia (thin and sparse hair), acne, eczema, dry, scaling skin, delayed wound healing, and oral ulceration, as well as problems such as stomatitis. [1,17,58,66]. Dermatitis due to zinc deﬁciency is termed as AE. Brandt’s syndrome also includes autosomal recessive metabolic disorders associated with AE, which is accompanied by inherited genetic abnormalities with low zinc distribution. In AE patients, personality disorders are observed, with blistering of the skin (dry skin), hair loss from the scalp, eyebrows, and eyelashes, glossitis, and pustules. Therefore, secondary infections can be easily caused by pathogens such as Staphylococcus aureus and fungi such as Candida albicans in the weakened epidermis due to zinc deﬁciency. Recent reports revealed that keratinocytes produce excess amounts of ATP, which induces inﬂammation upon contact with irritants in individuals with zinc-deﬁcient skin [67]. In zinc-deﬁcient mice, Langerhans cells (LCs), which can neutralize harmful ATP by surface CD39, are greatly reduced in the skin, resulting in augmented skin inﬂammation by ATP. In AE patients, the number of LCs is also decreased, and is recovered by zinc treatment. The effects of zinc deﬁciency have also been studied on a cellular basis. When keratinocytes in monolayer culture are scratched, zinc is released from these cells [68]. Subsequently, zinc binds to zinc-sensing receptors and induces cell proliferation in an autocrine/paracrine manner, suggesting that zinc is required for skin regeneration. In fact, the removal of zinc by N,N,N,N tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) during keratinocyte culture induces caspase-3 activity and DNA fragmentation followed by apoptosis [69]. Zinc also has an anti-inﬂammatory effect and reduces tumor necrosis factor-α production by keratinocytes [59]. Nutrients 2018, 10, 219 A recent study demonstrated that R1 and EVER2 proteins found in epidermodysplasia verruciformis (EV) patients with high susceptibility to papillomaviruses causing skin cancer interact with ZnT1 to control the zinc balance in keratinocytes, suggesting the importance of maintaining intracellular zinc homeostasis in skin cancer induced by viral infection [70]. 2.5. Zinc Deﬁciency in Skin There are two major causes of zinc deﬁciency in humans. First, zinc deﬁciency is caused by a diet that is low in zinc content. Approximately 17% of the world’s population is confronting health risks related to zinc deﬁciency, particularly in developing countries. However, in even developed countries, vegetarians, pregnant women, and singles also suffer from zinc deﬁciency. Second, zinc deﬁciency can be caused by genetic defects. In 1988, it was reported that babies with severe skin diseases and hair loss were diagnosed with transient neonatal zinc deﬁciency [61], but recovered by eating normal infant foods [62]. Their mothers have a variety of mutations in alleles of the zinc transporter ZnT2 [63]. 5 of 14 3.1. Epidermis ZIP4 and ZIP10 support p63 activity for adult epidermal homeostasis. ZIP4 and ZIP10 supply zinc to epidermal master regulator p63 for their activity. Zinc deﬁciency leads to improper function of p63, resulting in epidermal hypoplasia. Zinc is depicted as a brown circle. Figure 2. ZIP4 and ZIP10 support p63 activity for adult epidermal homeostasis. ZIP4 and ZIP10 supply zinc to epidermal master regulator p63 for their activity. Zinc deficiency leads to improper function of p63, resulting in epidermal hypoplasia. Zinc is depicted as a brown circle. Of note, the murine and human epidermis have several different characteristics. Overall, the Figure 2. ZIP4 and ZIP10 support p63 activity for adult epidermal homeostasis. ZIP4 and ZIP10 supply zinc to epidermal master regulator p63 for their activity. Zinc deﬁciency leads to improper function of p63, resulting in epidermal hypoplasia. Zinc is depicted as a brown circle. and thick‐layered structure, and has a rete ridge structure because of the diversity of the number of spinous layers. The patterns of epidermal stem cells in the basal layer also differ between mouse and human. Therefore, the use of mouse models in human epidermal studies should be carefully assessed. ZIP10 also plays important roles in epidermal development [36]. In situ hybridization analysis using mouse whole body sections showed that ZIP10 expression appears in hair follicles near embryonic day 14 (E14) during epidermal development, after which it gradually increases. In a postnatal phase after the completion of epidermis formation, ZIP10 expression is decreased. In the epidermis, ZIP10 accumulates in the outer root sheath region of the hair follicle, which is rich in Of note, the murine and human epidermis have several different characteristics. Overall, the human epidermis is thicker than the mouse epidermis [11,12]. The stratum corneum shows a solid and thick-layered structure, and has a rete ridge structure because of the diversity of the number of spinous layers. The patterns of epidermal stem cells in the basal layer also differ between mouse and human. Therefore, the use of mouse models in human epidermal studies should be carefully assessed. epidermis, ZIP10 accumulates in the outer root sheath region of the hair follicle, which is rich in various Lgr6‐positive epidermal stem cells that migrate to the sebaceous glands, hair follicles, and wound space, when epidermis is regenerated upon injury. Consistent with this, next‐generation sequencing analysis confirmed that Zip10‐expressing cells co‐express Lgr6. 3.1. Epidermis Zinc transporters known for their role in the epidermis are ZIP4 and ZIP10. ZIP4 is actively expressed in human but not in mouse skin [33,71]. Recent skin equivalent experiments with human keratinocytes demonstrated the importance of ZIP4 in epidermis formation [56]. When ZIP4 is knocked-down by siRNA, epidermal hypoplasia occurs, accompanied by decreased activity of p63, an master regulator with a zinc binding site that promotes the proliferation and differentiation of epidermal progenitor cells in epithelium formation (Figure 2) [56,72,73]. When zinc is deﬁcient, nuclear translocation of p63 is disturbed, followed by abnormal epidermal formation (Figure 2) [56]. Detailed analysis revealed that the 205th Cys among the four Cys residues in the zinc binding site plays an important role in post-modiﬁcation of p63, suggesting that zinc deﬁciency affects the DNA binding afﬁnity of p63 involved in signal transduction. Since Zn affects the structure and functions of many proteins [17], ZIP4 may not only target the p63, but also inﬂuence the structure and activity of other zinc binding enzymes for epidermal growth. With regard to this issue, further studies are needed to clarify the relationship between ZIP4 and AE. Interestingly, it was recently found that natural products 6 of 14 Nutrients 2018, 10, 219 such as soybean extracts increase the expression of ZIP4 [74]. Therefore, this can be a starting point for controlling the rate of zinc absorption. needed to clarify the relationship between ZIP4 and AE. Interestingly, it was recently found that natural products such as soybean extracts increase the expression of ZIP4 [74]. Therefore, this can be a starting point for controlling the rate of zinc absorption. such as soybean extracts increase the expression of ZIP4 [74]. Therefore, this can be a starting point for controlling the rate of zinc absorption. needed to clarify the relationship between ZIP4 and AE. Interestingly, it was recently found that natural products such as soybean extracts increase the expression of ZIP4 [74]. Therefore, this can be a starting point for controlling the rate of zinc absorption. Figure 2. ZIP4 and ZIP10 support p63 activity for adult epidermal homeostasis. ZIP4 and ZIP10 supply zinc to epidermal master regulator p63 for their activity. Zinc deficiency leads to improper function of p63, resulting in epidermal hypoplasia. Zinc is depicted as a brown circle. Of note, the murine and human epidermis have several different characteristics. Overall, the Figure 2. 3.1. Epidermis A conditional knockout mouse line in which Zip10 was specifically deleted in Keratin14‐expressing cells was born with severe epidermal hypoplasia (Figure 3a,b). In these mice, the p63‐positive basal layer formed normally, and slight stratification of the dorsal part of the epidermis occurred by E14. However, such mice fail to form a thick and firm epidermis following rapid proliferation and differentiation of epidermal progenitor cells after E14 when the expression of both ZIP10 and p63 is increased in wild‐type mice (Figure 3b,c). Further detailed analysis revealed that ZIP10 augments the activity of p63 by regulating its nuclear translocation (Figure 3c). ZIP10 also plays important roles in epidermal development [36]. In situ hybridization analysis using mouse whole body sections showed that ZIP10 expression appears in hair follicles near embryonic day 14 (E14) during epidermal development, after which it gradually increases. In a postnatal phase after the completion of epidermis formation, ZIP10 expression is decreased. In the epidermis, ZIP10 accumulates in the outer root sheath region of the hair follicle, which is rich in various Lgr6-positive epidermal stem cells that migrate to the sebaceous glands, hair follicles, and wound space, when epidermis is regenerated upon injury. Consistent with this, next-generation sequencing analysis conﬁrmed that Zip10-expressing cells co-express Lgr6. A conditional knockout mouse line in which Zip10 was speciﬁcally deleted in Keratin14-expressing cells was born with severe epidermal hypoplasia (Figure 3a,b). In these mice, the p63-positive basal layer formed normally, and slight stratiﬁcation of the dorsal part of the epidermis occurred by E14. However, such mice fail to form a thick and ﬁrm epidermis following rapid proliferation and differentiation of epidermal progenitor cells after E14 when the expression of both ZIP10 and p63 is increased in wild-type mice (Figure 3b,c). Further detailed analysis revealed that ZIP10 augments the activity of p63 by regulating its nuclear translocation (Figure 3c). 7 of 14 Nutrients 2018, 10, 219 Expression level! in epidermis ZIP10 p63 <Development> E14 P1 <Homeostasis> E14~Neo ~E8 E8~E14 Postnatal p63+ K14 K14 K14 K1, K10 Filagrin P63+ K14 Filagrin K1, K10 ZIP10+ ZIP10+++ Zinc p63+++ ZIP10+ Zinc p63+ ZIP10/E-cadherin/! Hoechst E14.5 E17.5 b Control Dorsal Ventral C G S B C G S B Zip10K14! * B C G S B x400! x400! x400! x400! 2cm! Zip10K14! Control! 2cm! a c DC P Epi Mes E14.5 Mes Epi E17.5 P2 Figure 3. ZIP10 reinforces p63 function for epidermis development. 3.1. Epidermis (a) ZIP10 deﬁciency leads to epidermal hypoplasia in mice. (b) Hematoxylin and eosin staining revealed dorsal epidermal hypoplasia and ventral embryonic epidermis (asterisk) in P1 Zip10K14 mice. C, corniﬁed layer; G, granular layer; S, spinous layer; B, basal layer. (c) Model for ZIP10’s involvement in p63 function during epidermis development. ZIP10, whose expression is elevated from E14, contributes to the activities of increased p63 by supplying zinc. This allows p63 to properly bind to DNA for initiating gene expression for epidermis development, including cell proliferation and stratiﬁcation. DC, dermal condensate; Epi, epithelium; Mes, mesenchyme; P, placode (Scale bar, 100 µm). Red (E17.5) and arrows (P2) indicate the ZIP10 protein expression. Modiﬁed from Bin et al. [36]. 2cm! Zip10K14! Control! 2cm! a b Control Dorsal Ventral C G S B C G S B Zip10K14! * B C G S B x400! x400! x400! x400! b a Expression level! in epidermis ZIP10 p63 <Development> E14 P1 <Homeostasis> E14~Neo ~E8 E8~E14 Postnatal p63+ K14 K14 K14 K1, K10 Filagrin P63+ K14 Filagrin K1, K10 ZIP10+ ZIP10+++ Zinc p63+++ ZIP10+ Zinc p63+ ZIP10/E-cadherin/! Hoechst E14.5 E17.5 c DC P Epi Mes E14.5 Mes Epi E17.5 P2 c Figure 3. ZIP10 reinforces p63 function for epidermis development. (a) ZIP10 deﬁciency leads to epidermal hypoplasia in mice. (b) Hematoxylin and eosin staining revealed dorsal epidermal hypoplasia and ventral embryonic epidermis (asterisk) in P1 Zip10K14 mice. C, corniﬁed layer; G, granular layer; S, spinous layer; B, basal layer. (c) Model for ZIP10’s involvement in p63 function during epidermis development. ZIP10, whose expression is elevated from E14, contributes to the activities of increased p63 by supplying zinc. This allows p63 to properly bind to DNA for initiating gene expression for epidermis development, including cell proliferation and stratiﬁcation. DC, dermal condensate; Epi, epithelium; Mes, mesenchyme; P, placode (Scale bar, 100 µm). Red (E17.5) and arrows (P2) indicate the ZIP10 protein expression. Modiﬁed from Bin et al. [36]. 3.2. Dermis Zinc transporters known to be important in the dermis are ZIP 7 and ZIP 13 [37,38]. Both ZIP7 and ZIP13 are present in skin cells and are mainly located in the ER and Golgi, respectively [19,37,38]. These two zinc transporters are closest to each other when a phylogenetic tree of ZIP family members is constructed. ZIP13 is also close to the zinc transporter of insects/bacteria [19]. ZIP13 is the smallest of the mammalian zinc transporters classiﬁed in the LIV-1 family, and thus has a shorter N-terminus and intracellular domain 2 than other transporters. The most abundant amino acid in ZIP7 is histidine, which is a characteristic of the LIV-1 family whose role is unknown; this is also the case for the methionine-rich motif that is largely conserved in copper transporters for copper binding and storage [19,75]. During functional analysis of zinc carriers, the ﬁrst association of the zinc transporter with skin was found in Zip13-knockout mice [38]. The Zip13-knockout mice showed impaired collagen production with abnormal morphology of collagen-producing cells and shrunken cartilage with defective chondrocyte differentiation. The number of collagen ﬁbers in Zip13-knockout mice was remarkably reduced as visualized under an electron microscope, and skin cracking and 8 of 14 Nutrients 2018, 10, 219 swelling phenomena were also remarkable because of severely decreased skin strength. This abnormal collagen production was reﬂected in the mouse face features with an enophthalmos-like appearance, eye blindness, and down-slanting palpebral ﬁssures. The ﬁbroblasts isolated from Zip13-knockout mice exhibited dysfunction in the BMP/TGF-β signaling pathway, which is essential for collagen formation. In Zip13-knockout mice, the nuclear translocation of SMAD transcription factors in the BMP/TGF-β signaling pathway was impaired, while remaining intact in terms of their phosphorylation status (Figure 4a). Examination of the zinc distribution in Zip13-deﬁcient ﬁbroblasts with an electron probe X-ray micro analyzer revealed that the nuclear zinc content was reduced, while that in the Golgi was increased [38,76]. a Golgi P ZIP13 Zinc Col! BMPR/TGFβR SMAD SMAD SMAD SMAD Fibroblast NC P a b Golgi P ZIP13 Zinc Col! BMPR/TGFβR SMAD SMAD SMAD SMAD Fibroblast NC P Figure 4. Ehlers–Danlos syndrome spondylodysplastic type 3 is caused by impaired collagen production due to ZIP13 dysfunction. (a) ZIP13 supplies zinc (brown circle) to SMAD proteins for their nuclear translocation. Phosphorylatoin is depicted as circled P. (b) The elder affected sib is shown at age 22 years. Nutrients 2018, 10, 219 Apoptosis Lumen Cytoplasm Zinc PDI c c Zip7-KO ER Control ER Figure 5. ZIP7 is essential for dermis formation. (a) Five-week-old female wild-type (WT) and Zip7Col1 mice. (b) The thickness of the Azan-stained Zip7Col1 dermis is reduced. M, muscle; S, Subcutis. (c) Models for the involvement of ZIP7 in ER function. When ZIP7 is dysregulated (right), the luminal zinc level is elevated, which would induce zinc-dependent aggregation of PDIs. Therefore, protein folding and disulﬁde bond formation would proceed aberrantly, leading to unfolded protein responses. ER, endoplasmic reticulum; KO, knockout; PDI, protein disulﬁde isomerase; UPR, unfolded protein response. Modiﬁed from Bin et al. [37]. Figure 5. ZIP7 is essential for dermis formation. (a) Five-week-old female wild-type (WT) and Zip7Col1 mice. (b) The thickness of the Azan-stained Zip7Col1 dermis is reduced. M, muscle; S, Subcutis. (c) Models for the involvement of ZIP7 in ER function. When ZIP7 is dysregulated (right), the luminal zinc level is elevated, which would induce zinc-dependent aggregation of PDIs. Therefore, protein folding and disulﬁde bond formation would proceed aberrantly, leading to unfolded protein responses. ER, endoplasmic reticulum; KO, knockout; PDI, protein disulﬁde isomerase; UPR, unfolded protein response. Modiﬁed from Bin et al. [37]. Nutrients 2018, 10, 219 Nutrients 2018, 10, 219 Nutrients 2018, 10, 219 ZIP7 is ubiquitously expressed in the ER of nearly all mammalian tissues [17,18] and maintains zinc homeostasis in this intracellular organelle, and the ER acts as a zinc reservoir, although a study showed that ZIP7 also exists in the Golgi [81]. Zip13-knockout mice display speciﬁc abnormalities in connective tissues irrespective of ZIP7 expression [37], suggesting that ZIP13 and ZIP7 play distinct physiological roles in connective tissue development. Similar to in Zip13-knockout mice, Zip7-conditional knockout mice, in which Cre-Lox recombination is under the control of the collagen promoter, dysfunction of connective tissues including the dermis and cartilage is observed (Figure 5a,b) [37,38]. The width of the growth plate in Zip7-knockout mice is reduced with a normal columnar structure, while that of Zip13-knockout mice is disorganized. Unlike Zip13-knockdown cells, Zip7-knockdown cells show elevated ER stress with increased zinc concentrations in the ER and protein aggregation of protein disulﬁde isomerase (PDI) [37]. In a Zip7-deﬁcient environment, PDI does not function properly because of persistent sticking of zinc on the protein. In fact, Zip7-deﬁcient cells fail to achieve the classical protein folding function in the ER, leading to ER stress, unfolded protein response, cell growth inhibition, and apoptosis (Figure 5c). Taken together, this series of studies indicate that proper intracellular zinc distributions in the ER and Golgi by ZIP7 and ZIP13, respectively, are critical for collagen synthesis. ContZip7Col1 HE Azan! Control Zip7Col1 200μm! 200μm! HE Azan! S S M M PDI PDI PDI PDI PDI Control ER ZIP7 Zip7-KO ER ZIP7 Protein folding, ! Disulﬁde bonding ER dysfunction UPR,! Apoptosis Lumen Cytoplasm Zinc PDI a c b Figure 5. ZIP7 is essential for dermis formation. (a) Five-week-old female wild-type (WT) and Zip7Col1 mice. (b) The thickness of the Azan-stained Zip7Col1 dermis is reduced. M, muscle; S, Subcutis. (c) Models for the involvement of ZIP7 in ER function. When ZIP7 is dysregulated (right), the luminal zinc level is elevated, which would induce zinc-dependent aggregation of PDIs. Therefore, protein folding and disulﬁde bond formation would proceed aberrantly, leading to unfolded protein responses. ER, endoplasmic reticulum; KO, knockout; PDI, protein disulﬁde isomerase; UPR, unfolded protein response. Modiﬁed from Bin et al. [37]. Zip7Col1 HE Azan! Control Zip7Col1 200μm! 200μm! HE Azan! S S M M b b a PDI PDI PDI PDI PDI Control ER ZIP7 Zip7-KO ER ZIP7 Protein folding, ! Disulﬁde bonding ER dysfunction UPR,! 3.2. Dermis Patient exhibits short stature with mildly shortened trunk, antimongoloid eye slant with lack of periorbital tissue, thin and ﬁnely wrinkled skin on the palms of the hands. BMPR/TGFβR, BMP receptor/TGF-β receptor; Col, collagen. Modiﬁed from Fukada et al. [38]. a b b Fibroblast Figure 4. Ehlers–Danlos syndrome spondylodysplastic type 3 is caused by impaired collagen production due to ZIP13 dysfunction. (a) ZIP13 supplies zinc (brown circle) to SMAD proteins for their nuclear translocation. Phosphorylatoin is depicted as circled P. (b) The elder affected sib is shown at age 22 years. Patient exhibits short stature with mildly shortened trunk, antimongoloid eye slant with lack of periorbital tissue, thin and ﬁnely wrinkled skin on the palms of the hands. BMPR/TGFβR, BMP receptor/TGF-β receptor; Col, collagen. Modiﬁed from Fukada et al. [38]. The phenotypes of Zip13-knockout mice are similar to those of patients with spondylocheiro dysplastic form of Ehlers–Danlos syndrome (Ehlers–Danlos syndrome spondylodysplastic type 3, EDSSPD3), with an extremely thin layer of dermis and many wrinkles. EDSSPD3 patients have similar phenotypes such as sagging of the eyes, cracking of the skin, and prominent blood vessels (Figure 4b) [38,76,77]. To date, two mutated Gly64Asp and ∆Phe-Leu-Ala residues in ZIP13 protein have been identiﬁed in patients with EDSSPD3. Each of these mutations results in rapid clearance of ZIP13 protein due to ER-associated degradation via valprotein (VCP)/ubiquitination/proteasome machinery [78,79]. Thus, the compounds that increase the activity of ZIP13 or slow the degradation of ZIP13 are considered as therapeutic drugs that can increase collagen synthesis. Since ZIP13 is not only expressed in the Golgi, but also in putative zinc stores [80], another potential mechanism of EDSSPD3 development may be due to non-smooth zinc transport from zinc stores to the cytoplasm in the absence of ZIP13, resulting in low zinc levels in the cytoplasm. If this is the case, this condition also may induce ER stress, as the cytoplasm is a main zinc source for zinc homeostasis in the ER. 9 of 14 3.3. Hypodermis Zip7- and Zip13-knockout mice display reduced thickness of subcutaneous fat and dermal layers [37,38]. In EDSSPD3 patients, the subcutaneous fat layer is also signiﬁcantly reduced in the aging skin [38]. Adipocytes play a major role in the formation of subcutaneous fat layers, and ﬁbroblasts produce collagen for dermal formation. Thus, the abnormality of fat layers in Zip7- and Zip13-knockout mice may be attributed to impaired differentiation of mesenchymal stem cells and the generation of connective tissue cells. In fact, Zip7-deﬁciency in human mesenchymal stem cells 10 of 14 Nutrients 2018, 10, 219 leads to ER stress, resulting in defects in ﬁbrogenic and chondrogenic lineage differentiation [37]. However, although Zip13-deﬁcient mice show defective formation of connective tissue [38], the effect of mesenchymal stem cells on adipocyte differentiation has not been investigated. Recent research demonstrated that the inguinal white adipose tissue (iWAT) in Zip13-knockout mice shows a browning phenotype that reﬂects increased energy expenditure, thereby reducing iWAT mass. Further analysis revealed that Zip13-deﬁciency causes stabilization of C/EBP-β, an essential transcription factor for adipocyte browning, and thus promotes this process. These data suggest that the ZIP13-zinc axis plays a speciﬁc role in clearing C/EBP-β proteins to inhibit adipocyte browning [82]. Abbreviations Abbreviations AE acrodermatitis enteropathica ATP adenosine triphosphate EDSSPD3 Ehlers–Danlos syndrome spondylodysplastic type 3 iWAT inguinal white adipose tissue MTs metallothioneins ZIPs Zrt- and Irt-like proteins ZnTs zinc transporters Abbreviations AE acrodermatitis enteropathica ATP adenosine triphosphate EDSSPD3 Ehlers–Danlos syndrome spondylodysplastic type 3 iWAT inguinal white adipose tissue MTs metallothioneins ZIPs Zrt- and Irt-like proteins ZnTs zinc transporters 4. Conclusions Skin plays an important role as a primary defense line to protect the human body from the external environment. Healthy skin is an important factor that affects social activities and quality of life. It has long been known that zinc is important for maintaining healthy skin. Studies using knockout mouse models have demonstrated that speciﬁc zinc signaling axes mediated by individual zinc transporters play crucial roles in skin homeostasis and development. Aging is associated with reduced zinc levels in the human body. Therefore, the development of technology to control the versatile functions of zinc transporters in relation to anti-aging will contribute to regeneration of aged skin. Acknowledgments: This study was supported by KAKENHI (17H04011) of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), the Vehicle Racing Commemorative Foundation, the Mitsubishi Foundation, and Takeda Science Foundation to Toshiyuki Fukada. Amorepaciﬁc Corporation R&D Center provided ﬁnancial support to Juyeon Seo. We also thank Andrea Superti-Furga (University of Lausanne) for use of the human case images [30]. Author Contributions: Bum-Ho Bin, Shintaro Hojyo, Juyeon Seo, Takafumi Hara, Teruhisa Takagishi, Kenji Mishima, and Toshiyuki Fukada wrote the paper. Toshiyuki Fukada approved the paper. Conﬂicts of Interest: The authors declare no conﬂicts of interest. References Getting under the skin of epidermal morphogenesis. Nat. Rev. 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https://openalex.org/W4322743707	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/645451D316BD647592ADEAD6536DF851/S1755773923000024a.pdf/div-class-title-how-governance-shaped-military-responses-to-the-covid-19-pandemic-div.pdf	English	null	How governance shaped military responses to the COVID-19 pandemic	European political science review	2,023	cc-by	6,916	European Political Science Review (2023), 15, 628–640 doi:10.1017/S1755773923000024 RESEARCH NOTE Abstract Most countries deployed their military in some capacity to combat the COVID-19 pandemic. We present original data on early pandemic-related deployments, identifying seven types of deployment: logistic oper- ations, enforcement, international involvement, border protection, information provision, intelligence operations, and domestic protection. We find that military deployments are shaped by capacity and elec- toral considerations, even after accounting for cross-country differences in perceptions of the military. Countries with elected leaders were significantly more likely to deploy the military for border protection. Incumbents facing reelection were especially sensitive to electoral concerns, becoming significantly less likely to deploy the military for domestic enforcement when facing an imminent election. Keywords: civil-military relations; military deployment; pandemic How governance shaped military responses to the COVID-19 pandemic Nick Dietrich1,* , Kristine Eck2 and Chiara Ruffa3 1Data Analytics Program, Ohio Wesleyan University, Ohio, USA, 2Department of Peace and Conflict Research, Uppsala University, Uppsala, Sweden and 3Centre for International Studies (CERI), Sciences Po, Paris, France E-mail: dietrich.nicholas@gmail.com (Received 26 August 2022; revised 21 December 2022; accepted 16 January 2023; first published online 01 March 2023) (Received 26 August 2022; revised 21 December 2022; accepted 16 January 2023; first published online 01 M © The Author(s), 2023. Published by Cambridge University Press on behalf of European Consortium for Political Research. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. © The Author(s), 2023. Published by Cambridge University Press on behalf of European Consortium for Political Research. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons org/licenses/by/4 0/), which 1For Israel, see: https://www.timesofisrael.com/elite-idf-tech-unit-working-to-develop-medical-equipment-protective-gear/; for Italy, see: https://edition.cnn.com/2020/03/20/europe/italy-military-coronavirus-intl/index.html; For Morocco, see: https:// www.jeuneafrique.com/913138/politique/coronavirus-larmee-marocaine-sur-le-pied-de-guerre/. 1For Israel, see: https://www.timesofisrael.com/elite-idf-tech-unit-working-to-develop-medical-equipment-protective-gear/; for Italy, see: https://edition.cnn.com/2020/03/20/europe/italy-military-coronavirus-intl/index.html; For Morocco, see: https:// www.jeuneafrique.com/913138/politique/coronavirus-larmee-marocaine-sur-le-pied-de-guerre/. https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press Introduction The COVID-19 pandemic was an unprecedented test of the military’s role during emergencies. Nearly every country in the world was affected by the pandemic between March and July of 2020, and a majority of countries responded in this initial phase by deploying the military. These deploy- ments, however, varied significantly in scope and character. In Israel, military units manufactured ventilators and personal protective equipment; in Italy, soldiers patrolled the streets of major cities to enforce restrictions on movement; and in Morocco, the military announced lockdowns from loudspeakers on tanks.1 p In this paper, we have two goals. First, we document the variation in early pandemic-related deployments with an original dataset covering every country with a standing military during the initial pandemic response. Our dataset captures seven distinct categories of pandemic-related deployments, listed here from most to least prevalent: logistic operations, enforcement operations, international involvement, border protection, information provision, intelligence operations, and domestic protection. Second, we demonstrate that capacity and governance explain a significant amount of the variation in pandemic-related deployments, shaping the way that countries choose to respond to the pandemic militarily. 1For Israel, see: https://www.timesofisrael.com/elite-idf-tech-unit-working-to-develop-medical-equipment-protective-gear/; for Italy, see: https://edition.cnn.com/2020/03/20/europe/italy-military-coronavirus-intl/index.html; For Morocco, see: https:// www.jeuneafrique.com/913138/politique/coronavirus-larmee-marocaine-sur-le-pied-de-guerre/. © The Author(s), 2023. Published by Cambridge University Press on behalf of European Consortium for Political Research. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press 629 How governance shaped military responses to the COVID-19 pandemic Overall, three quarters of countries with a standing military responded with a deployment at the outset of the pandemic. Logistics operations and enforcement of lockdowns, mandates, or restrictions on movement were the most common kinds of deployments. Countries with par- ticularly high numbers of cases were more likely to deploy the military, but the mission of these deployments was shaped by governance. States with elected leaders were more likely to deploy the military in a ‘behind the scenes’ capacity, relying especially on logistic support and border protection. Incumbents facing reelection were particularly sensitive to electoral concerns and avoided domestic enforcement operations especially when the next election was imminent.2 Military deployments in crisis situations Securitization theory argues that when an issue is considered a security threat it is framed as exis- tential and, therefore, critical to survival (Buzan et al., 1998). In these situations, the threat is ele- vated above normal political processes. Audiences empower securitizing actors (like politicians) to take exceptional measures to mitigate these threats, including extraordinary allocations of resources (human, financial, etc.) (Kuehn and Levy, 2021). The COVID-19 pandemic presents an unusual case for securitization. The pandemic emerged as a threat to nearly every country in the world during a short period of time. It quickly became a domestic security issue: securitized language, such as being ‘at war’ with the virus, and the politics of exceptionalism were hallmarks to many countries’ pandemic response (Kirk and McDonald, 2021). Indeed, we are not the first to demonstrate that a majority of countries deployed the mili- tary in some fashion when responding to the pandemic (Erickson, Kljajíc and Shelef, 2022). y p g p j j Regulations of types and levels of military deployment are a function of a plethora of institu- tional, historical idiosyncratic factors that affect the military’s role in a particular country (Passos and Acácio, 2021; Bove et al., 2020: 264). Due to their totalitarian past, countries like Germany, Italy or Japan impose severe restrictions in their use of the military, particularly in domestic func- tions. Yet even those countries utilized the military in the pandemic. Despite cross-national differ- ences in the military’s role in society, we expect that all countries with a military have the potential to mobilize following the shock of the pandemic, but that those mobilizations might take different forms.3 for the state s reputation (Flynn et al., 2019: 753; Fazal, 2020). 3For instance, we do not expect Germany—for which the use of the military is heavily restricted—to utilize the military at the same level as Israel, which uses its military extensively in domestic functions. p ( y ) 3For instance, we do not expect Germany—for which the use of the military is heavily restricted—to uti the same level as Israel, which uses its military extensively in domestic functions. https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press 2These countries might choose an alternative policy tool, like health diplomacy activities, with potentially beneficial effects for the state’s reputation (Flynn et al., 2019: 753; Fazal, 2020). 3For instance, we do not expect Germany—for which the use of the military is heavily restricted—to utilize the military at the same le el as Israel hich uses its militar e tensi el in domestic functions 2These countries might choose an alternative policy tool, like health diplomacy activities, with potentially beneficial effects for the state’s reputation (Flynn et al., 2019: 753; Fazal, 2020). Capacity What shaped military responses to the COVID-19 pandemic? Securitization theory would suggest that, as a first step, state capacity to manage the virus played a role. Securitization is a response to an emergency situation; we therefore expect that the perceived emergency is influenced by the severity of the pandemic and will predict deployments in response to the virus. Hypothesis 1: All types of deployments are more likely in countries where the pandemic is more severe. pp 5Some deployments that we categorize as ‘behind the scenes’ deployments are, in fact, highly visible. We draw the distinc- tion, however, based on whether a deployment has potential effects on civilians’ rights. Highly-visible logistics operations do not violate norms of civilian control in the same way as military enforcement. Hypothesis 1: All types of deployments are more likely in countries where the pandemic is more severe. The threat of the pandemic is additionally related to the state’s pre-existing capacity to manage the virus. A country with a poor health care infrastructure faces a greater existential threat from the pandemic. https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press 630 Nick Dietrich, Kristine Eck and Chiara Ruffa The relationship between health capacity and military responses to the pandemic is not straightforward; even a country with excellent pre-existing health capacity may view the pandemic as an existential threat and mobilize the military to respond to it. A country with low health capac- ity, meanwhile, may face a greater threat from the pandemic, but may not have the infrastructure in place for the military to assist with pandemic response. A crucial factor at play in this context is the popularity of the military. In countries where the military is very popular, its deployment may enhance public support for the elected leaders. Previous research has shown, for instance, that former French President Hollande extensively deployed the military domestically in an antiterrorist function to boost his low public support (Bove et al., 2020). In these contexts, we expect that public leaders may weigh the benefit of deploying the military domestically against the cost of doing so. Those considerations are specific to each country in terms of political culture and popularity of the military.4 We do not predict that pre-existing levels of health capacity will make a state more or less likely to deploy the military in response to the pandemic. Rather, we expect that health capacity will affect the type of mission deployment. Countries with low domestic health capacity will be forced to use the military to shore up deficiencies in domestic infrastructure. Countries with high health capacity, meanwhile, can use the military in other roles. Hypothesis 2: Countries with a high level of domestic healthcare capacity are more likely to deploy the military in a non-domestic capacity, including international deploy- ments or deployments to protect the border. 4Please refer also to online Appendix 3 for further elaboration on this. https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press Data We collected original data on pandemic-related military deployments taking place from the begin- ning of the pandemic through August 2020 in order to focus on states’ initial securitization responses. On a country-by-country basis, we conducted targeted searches for news articles on Google using the following search string: military AND (COVID OR Corona) AND [country name].6 We documented military actions reported in mainstream news media, government sour- ces, or non-governmental organization sources (for example, the World Health Organization). In cases where information was sparse, we supplemented the initial search with additional searches of the Factiva news database.7 Media reports and government press statements constituted the majority of our raw material, which consisted of some 1500 separate reports.8 j y p p Take the case of Sudan, which exemplifies the data landscape. We relied on seven articles to code Sudan as engaging in logistics and enforcement activities. An article from All Africa described the military enforcement of an April 2020 lockdown, relying on statements from various leaders (including a TV appearance by the Minister of Health), interviews of residents, and social media.9 Additional news reports from the Agence France Press newswire, the European Council of Foreign Relations, and others, provide additional information on military enforcement and logis- tics (which involved assisting health authorities in preparing a quarantine center). We choose to highlight Sudan because it is considered to be a ‘closed autocracy’, that is, a country which not only restricts rights but also fails to hold multiparty elections.10 In that sense, one might presume that it could be a ‘hard case’ for obtaining information on government military responses to COVID. We did not find, however, that information landscapes varied noticeably across different regime types. This is likely to be the case for several reasons. First, autocracies generally do not have the capacity to enforce complete censorship11; even a highly capable and well-resourced state like China has struggled to control its citizens’ spread of information and opinions (Chang et al., 2022). Second, autocracies usually pick and choose which topics they consider to be threatening (King et al., 2013). Autocratic governments would be unlikely to restrict information on their own policy responses for which they sought citizen cooperation. 6We coded all countries included in the Correlates of War system membership datasets. 7https://www.dowjones.com/professional/factiva/. 8These are available upon request. 9All Africa, 2020, ‘Sudan: Military Employed to Enforce Anti-Covid-19 Measures’. 1 May. Available at: https://allafrica. com/stories/202005040361.html. 10See https://www.v-dem.net/documents/14/dr_2020_dqumD5e.pdf. 11North Korea is the exception; it is the only country in our dataset with missing data. 12The means of implementing these policies, however, might warrant censure, for example when excessive violence was used to enforce freedom of movement restrictions. Measuring the level of violence used by security forces in enforcing COVID measures or the level of popular resistance to COVID policies might face the informational challenges (and autocratic bias) well-known to researchers on contentious politics (Dietrich and Eck, 2020), but the same problems do not obtain for our topic. Hypothesis 4: Countries where incumbent leaders are facing an imminent election are particu- larly likely to avoid deploying the military in a ‘boots on the ground’ capacity. Hypothesis 4: Countries where incumbent leaders are facing an imminent election are particu- larly likely to avoid deploying the military in a ‘boots on the ground’ capacity. Electoral politics Securitization, as a political process, will unfold differently depending on electoral politics. Kenwick and Maxey (2022) find that democratically elected leaders use the military in ways that bolster their public approval and improve their chances of political survival. Elected leaders have incentives to avoid unpopular actions and take actions that curry favor with the electorate. We therefore distinguish between ‘boots on the ground’ deployments—which are highly visible and civilian-facing—and ‘behind the scenes’ deployments—which assist with pandemic response in a way that does not create interaction between military personnel and the average citizen. We argue that ‘boots on the ground’ deployments are riskier for democratically elected leaders because they give the impression that the pandemic is out of control. Because using the military directly to manage citizens breaks norms of civilian control, it is likely to lead to negative assessments of leadership.5 Hypothesis 3: Countries with elected leaders are more likely to deploy the military in a ‘behind the scenes’ capacity—to provide logistics, border protection, or intelligence—and less likely to deploy the military in a ‘boots on the ground’ capacity—to enforce lockdowns or protect domestic infrastructure. Even among elected leaders, there is considerable variation in sensitivity to electoral concerns. A leader facing a reelection campaign has more to lose from an unpopular pandemic response than does a leader who does not need to run for reelection in the near future. Pulejo and Querubín (2021) find evidence of such effects for other kinds of pandemic responses (unrelated to military https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press 631 How governance shaped military responses to the COVID-19 pandemic deployments). We postulate that leaders who are eligible to run for another term and face an election in the near future will be particularly sensitive to electoral concerns. deployments). We postulate that leaders who are eligible to run for another term and face an election in the near future will be particularly sensitive to electoral concerns. Data In fact, our source material indicates that government statements were often the primary sources for media reports, as governments— democratic and autocratic alike—sought to inform the populace of the new policy in place, as well as the repercussions which would be incurred for non-compliance. Finally, because even OECD democracies were responding with lockdowns and other restrictive policies, autocracies had no reason to fear that the policy itself would result in international censure.12 https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press 632 Nick Dietrich, Kristine Eck and Chiara Ruffa In order to meet the inclusion criteria for our dataset, a deployment must be explicitly moti- vated by the COVID-19 pandemic. We do not include deployments motivated by factors other than the pandemic or deployments that were planned or initiated before the onset of the pan- demic. In practice, distinguishing pandemic-related deployments from other cases was generally straightforward because of the public nature of large-scale deployments and pandemic responses. We are aware of one other effort to collect data on military responses to the COVID-19 pan- demic: Erickson et al. (2022) similarly present original data on military deployments. Our approach differs in the way that we disaggregate deployments. Erickson et al. (2022) disaggregate deployments into logistic, healthcare, coercion, and policymaking. We disaggregate deployments into seven distinct categories. These include international deployments—which Erickson et al. (2022) explicitly exclude—and border deployments, which may be of particular interest to schol- ars studying the political implications of pandemics (Kenwick and Simmons, 2020).13 13The papers explore differing explanatory variables, increasing their complementarity. 14In some rare cases the military has taken the lead in logistic operations; for instance in Mexico, Guatemala, and Colombia (Passos and Acácio, 2021). 15See, for example, Norway’s deployment of the military to assist in border control after an outbreak in neighboring Sweden: https://www.reuters.com/article/us-health-coronavirus-sweden-cases-idINKBN27Q1U5. 13The papers explore differing explanatory variables, increasing their complementarity. 14 https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press 14In some rare cases the military has taken the lead in logistic operations; for instance in Mexico, Guatemala, (Passos and Acácio, 2021). 15See, for example, Norway’s deployment of the military to assist in border control after an outbrea Sweden: https://www.reuters.com/article/us-health-coronavirus-sweden-cases-idINKBN27Q1U5. Types of deployments https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press How governance shaped military responses to the COVID-19 pandemic How governance shaped military responses to the COVID-19 pandemic 633 Table 1. Summary of COVID-related military action Statistic N Mean St. Dev. Min Max Any Deployment 194 0.74 0.44 0 1 Logistics 194 0.58 0.50 0 1 Enforcement 194 0.41 0.49 0 1 Intl. Involvement 194 0.19 0.39 0 1 Border Protection 194 0.19 0.39 0 1 Information Provision 194 0.09 0.29 0 1 Intelligence 194 0.07 0.25 0 1 Domestic Protection 194 0.07 0.25 0 1 (as in Morocco) or military personnel communicating the gravity of the disease among their home communities (as in India). Intelligence Operations: We define intelligence operations as military intelligence activities related to COVID-19, including technological development (such as tracking apps or facial rec- ognition software) and the provision of analysis. Examples include preventing or countering the spread of misinformation (as in the UK), developing contact tracing apps for smartphones (as in Switzerland), or ‘cyber patrols’ to monitor social media for misinformation or other COVID-related communication (as in Argentina). Domestic Protection: We define domestic protection as domestic military deployment to pro- tect critical infrastructure, including security targets, personal protective equipment production sites, etc., motivated with reference to COVID-19. Our categorization captures seven distinct kinds of deployment. Within these seven types of deployment, one might construct aggregate categories—those that are civilian-facing and grant more power to the military, for example (Gibson-Fall, 2021). We leave the categories disaggre- gated for our analysis, but explore alternative codings in online Appendix 2. Types of deployments We define the categories of pandemic-related deployments below in decreasing order of preva- lence. Table 1 shows the proportion of countries reporting each of these kinds of deployments, and Figure 1 shows their geographic distribution. Logistics: We define logistic operations as military involvement in public health/medical logis- tics relating to COVID-19, including providing human resources support to these activities. Involvement is sufficient; the military may or may not take the lead.14 Civilian military personnel, including medical staff and logistics staff, count as ‘military involvement’ for the purposes of this variable. Logistics can include aiding healthcare personnel with testing, constructing new hospitals or field hospitals, manufacturing or distributing medical supplies for domestic use, or military personnel treating patients. Enforcement: We define enforcement operations as military involvement in the enforcement of domestic restrictions on the freedom of movement motivated with reference to COVID-19, such as quarantines and lockdowns. This can include, for example, clearing shops and public spaces or checking on people’s permission to be out of doors. We consider the military involved in enforce- ment even if they share enforcement authority with civilian police. y y p International Involvement: We define international involvement as military involvement out- side of the national borders, motivated with reference to COVID-19. This variable includes cases where the military (1) is involved with the repatriation of citizens from foreign countries or (2) is involved in providing assistance to one or more foreign countries in any form, be it material (for example, sending respirators) or human resources (for example, sending military medical doctors) or providing transportation (for example, air lifts). Border Protection: We define border protection as military deployment to conduct or assist in border protection motivated with reference to COVID-19. Involvement is sufficient; the military need not take the lead. Pandemic-related border protection can involve pandemic-specific duties, like checking test results or travel documents, or it might involve standard border procedures motivated with respect to COVID-19.15 Information Provision: We define information provision as the military providing information to the populace on COVID-19-related issues, such as government COVID-19 policies or citizen obligations. This can include, for example, announcing a lockdown using loud speakers on tanks xample, Norway’s deployment of the military to assist in border control after an outbreak in neighboring //www.reuters.com/article/us-health-coronavirus-sweden-cases-idINKBN27Q1U5. https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press 16The data represent a cross-section of 196 countries. Including a large number of independent variables in the model with so few observations would create unstable results and increase statistical uncertainty in our estimates. Research design We examine the associations between pandemic-related military deployments and capacity and gov- ernance using a series of logistic regressions. In each model, the dependent variable is a pandemic- related military deployment. We model each type of deployment separately. We chose this modeling strategy because the various kinds of deployment are not mutually exclusive; a state can, for example, mobilize the military to assist in logistics, enforce lockdowns, both, or neither. This process is best modeled as a set of parallel decisions to mobilize in various capacities. We additionally include a model where the dependent variable is any kind of military deployment. This variable is coded 1 if the country in question deployed the military for any of the seven kinds of deployment we measured. We first estimate a series of models predicting pandemic-related military deployments among all countries with a standing military. We then estimate a second series of models among presi- dential democracies with scheduled elections in order to examine the influence of electoral politics on pandemic-related deployments. p p y Our modeling strategy is designed to identify associations between governance/capacity and pandemic-related deployments. Our models are not intended to capture every possible factor that might affect the decision to deploy the military. Instead, we include measures of our theoretical concepts of interest (governance and capacity) and potential confounders—variables that might affect both governance/capacity and the decision to deploy the military. Our models are thus designed to estimate unbiased associations between governance/capacity and deployments with- out overparameterizing the model.16 https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press Nick Dietrich, Kristine Eck and Chiara Ruffa 634 ure 1. Global pandemic military deployments through July 2020. Figure 1. Global pandemic military deployments through July 2020. Independent variables e independent variables in our analysis measure aspects of capacity and governance. Capacity: We conceptualize capacity as two-sided: a state’s capacity to manage the pandemic increases with the resources available to it to control the virus, and decreases with the severity of the pandemic. We measure pandemic severity using data from the World Health Organization https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press How governance shaped military responses to the COVID-19 pandemic 635 (2020b) on the cumulative number of COVID cases in the country through July 31, 2020.17 COVID cases are logged due to their skewed distribution. We measure a state’s healthcare capacity as the number of hospital beds per capita at the outset of the pandemic (World Health Organization, 2020a). We additionally include gross domestic product (GDP) per capita in current USA dollars as a general indicator of a country’s financial resources (The World Bank, 2015). Governance: We are particularly interested in how electoral politics—particularly the need for leaders to consider the consequences of their actions in future elections—influence the state deci- sion to deploy the military in response to the COVID-19 pandemic. We therefore distinguish between democracies and non-democracies. The variable democracy is a binary variable measur- ing regime type (Marshall et al., 2016). It is coded 1 for democratic nations and 0 for all others.18 Within the subset of democratic countries with scheduled elections, we include additional var- iables to measure variation in electoral pressure. This subset consists of 65 presidential democra- cies with constitutionally scheduled elections, as defined by Pulejo and Querubín (2021). Election proximity measures the time until the next scheduled election from the first recorded COVID case in the country. Values of this variable are measured in days and converted to years for ease of interpretation. Values of this variable are multiplied by −1 so that higher values correspond to more proximate elections. A country with an upcoming election in six months, for example, would have an Election Proximity value of −0.5. Incumbent can run is a binary variable measuring whether the incumbent leader is eligible for reelection. We interact this variable with election proximity to test our expectation that leaders facing electoral pressure are less likely to deploy the military in a ‘boots on the ground’ capacity. Independent variables Incumbent leaders who are ineligible for reelection, or who will not go up for reelection in the immediate future, are less likely to respond to electoral considerations than those who are eligible and whose elections are imminent. Additional variables: Perceptions of the military are likely to differ between countries due to cultural and historical differences in the military’s role in society. These differences in military popularity might shape leaders’ decisions to deploy the military—a leader has more to lose and less to gain, for example, by deploying an unpopular military. We therefore include a measure of perceptions of military corruption from the Global Corruption Barometer (Transparency International, 2013). Military corruption is measured as an ordinal scale derived from survey data. Respondents are asked, ‘To what extent do you perceive the following categories in this country to be affected by corruption?’ and then prompted to answer the question with respect to the military. Responses are provided on an ordinal scale where 1 represents ‘not at all corrupt’ and 5 represents ‘extremely corrupt’. Values of this variable are averages of the ordinal responses given by all respondents within a country. All models additionally include a measure of population density, measured as the total popu- lation divided by the land area of the country in square kilometers (The World Bank, 2015). Population density has the potential to affect both the spread of the virus within a country and that country’s military response. It could therefore cause omitted variable bias if excluded from the models. This variable is logged due to its skewed distribution. 17We do not include cases after July 31, 2020 to avoid counting cases that took place after our time period. This is the same approach used by Erickson et al. (2022). 18We use a binary measure of democracy because we expect regime type to matter only to the extent to which leaders face the prospect of electoral accountability. We explore continuous measures of regime type in Appendix 2. https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press Results The results among all countries are displayed in Table 2. The severity of the pandemic is associated with an increase in most kinds of pandemic-related military deployments. Democratic countries 17We do not include cases after July 31, 2020 to avoid counting cases that took place after our time period. This is the same approach used by Erickson et al. (2022). 18We use a binary measure of democracy because we expect regime type to matter only to the extent to which leaders face t include cases after July 31, 2020 to avoid counting cases that took place after our time period. This is the same d by Erickson et al. (2022). binary measure of democracy because we expect regime type to matter only to the extent to which leaders face f electoral accountability. We explore continuous measures of regime type in Appendix 2. https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press 19It is worth noting that leaders facing imminent elections might reduce coercive policies in general—not just those that involve the military. Investigating non-military pandemic-related coercive policies is outside the scope of this article, but is a potentially valuable avenue for future research. In 0. 0. 0.0 0 In 0. 0. 0.0 0 In 0. 0 0.0 0 riable rder Protection Information Provision 0.12 (0.06) 0.11 (0.09) 1.30** (0.51) 0.57 (0.56) −0.02 (0.01) −0.05* (0.03) −0.40 (0.37) −0.08 (0.45) 0.11 (0.17) −0.12 (0.26) −0.31** (0.15) −0.05 (0.20) −1.52 (2.03) −1.06 (2.57) 194 194 180.48 120.43 How governance shaped military responses to the COVID-19 pandemic 637 Figure 2. Prevalence of pandemic-related military deployments by regime type. Figure 2. Prevalence of pandemic-related military deployments by regime type. were more likely to deploy the military to protect the border. Additional health capacity in the form of hospital beds per capita was associated with an increase in international deployments and a decrease in deployments for information provision. Figure 2 shows the relative frequency of each kind of pandemic-related deployment as a per- centage of democracies and non-democracies. Democracies deployed the military more often than non-democracies for each type of deploy- ment that we measured. This finding is in line with theories in civil-military relations that suggest that leaders in non-democracies are aware of the risks attached to deploying the military domestically because they increase the dependence of leaders on the military (Svolik, 2012). Note, however, that this difference is only statistically significant for border protection when con- trolling for other covariates. Despite lacking statistical significance when controlling for other covariates, there is a pronounced difference in logistic deployments by regime type; three quarters of democracies deployed the military to conduct logistics operations, compared to fewer than half of non-democracies. Next, we model the prevalence of pandemic-related deployments among the subset of democ- racies with scheduled elections. Note that we test only for enforcement operations, logistic sup- port, and any kind of deployment in these models due to the sparse number of occurrences of the less-frequent military deployments in this restricted sample. The results of these models are shown in Table 3. Among this subset of democratic countries with scheduled elections, pandemic severity was associated with an increase in deployments of all kinds and logistic deployments. Domestic health capacity was also a significant predictor of logistics. Strikingly, the interaction between election proximity and reelection-eligible incumbents was strongly and significantly associated with a reduction in enforcement operations.19 We visualize the probability of enforcement deployments as a function of time until the next election for eligible incumbents in Figure 3. https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press In 0. 0. 0.0 0 19It is worth noting that leaders facing imminent elections might reduce coercive policies in general—not just those that involve the military. Investigating non-military pandemic-related coercive policies is outside the scope of this article, but is a potentially valuable avenue for future research. https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press Nick Dietrich, Kristine Eck and Chiara Ruffa 638 Table 3. COVID military action, countries with scheduled elections (logistic regression) Dependent variable: Any Deployment Logistics Enforcement COVID Cases (log) 0.47* 0.54 0.06 (0.26) (0.22) (0.14) Election Proximity −0.03 0.31 0.18 (0.37) (0.36) (0.28) Incumbent Can Run −1.38 −1.21 −1.18 (1.89) (1.53) (1.17) Incumbent x Proximity −1.97 −0.71 −0.77 (1.29) (0.48) (0.38) Hospital Beds (pc) 0.06 0.07* 0.005 (0.04) (0.04) (0.02) Military Corruption 0.65 0.53 0.26 (0.78) (0.59) (0.51) GDPpc (log) 0.13 0.04 0.09 (0.47) (0.39) (0.29) Population Density 0.61 0.21 0.53 (0.43) (0.30) (0.27) Constant −10.21 −7.61** −3.93 (4.43) (3.84) (3.10) Observations 65 65 65 AIC 54.09 72.63 95.66 Note: p < 0.1; p < 0.05; **p < 0.01. Table 3. COVID military action, countries with scheduled elections (logistic regression) Figure 3. Probability of military enforcement as a function of time until next election. Note: Predicted probability of a military deployment to enforce lockdowns or restrictions on movement as a function of time until next election. Predicted probabilities are calculated using the enforcement model in Table 3 for a country where the incumbent is eligible to run for reelection. Control variables are held at their mean value. Figure 3. Probability of military enforcement as a function of time until next election. Note: Predicted probability of a military deployment to enforce lockdowns or restrictions on movement as a function of time until next election. Predicted probabilities are calculated using the enforcement model in Table 3 for a country where the incumbent is eligible to run for reelection. Control variables are held at their mean value. Figure 3. Probability of military enforcement as a function of time until next election. Note: Predicted probability of a military deployment to enforce lockdowns or restrictions on movement as a function of time until next election. Predicted probabilities are calculated using the enforcement model in Table 3 for a country where the incumbent is eligible to run for reelection. Control variables are held at their mean value. https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press Conclusions When COVID-19 hit, most countries in the world made use of their militaries from the outset: 73% of countries deployed the military in some form, demonstrating that countries still consider the military a central instrument for responding to emergencies. 58% of countries deployed the military for logistics and 41% for enforcing lockdowns. The widespread reliance on military deployments conceals extensive variation in the missions and tactics of these deployments. Some kinds of military responses—like intelligence operations or protection of domestic infrastructure—were relatively rare. Our findings indicate that countries use military deployments to increase domestic capacity in an emergency. The shape of these deployments, however, is largely a function of electoral politics. Democracies were particularly likely to deploy the military in a ‘behind the scenes’ capacity, par- ticularly through deployments to the border.20 Further research should continue to scrutinize the factors driving specific deployments as well as the differences we find between democracies and non-democracies. The popularity of the mili- tary, or its historical role within the country, is likely to influence deployments (Heinecken, 2021; Levy, 2021).21 The global COVID-19 pandemic is a unique opportunity to observe how various governments respond militarily in crisis situations. We hope that our dataset is a worthwhile contribution to scholars attempting to understand military operations in peacetime and domestic military deploy- ments. We additionally find support for recent work theorizing that civilian political officials use the military in ways that bolster their own political image and chances of electoral survival (Kenwick and Maxey, 2022). Our findings offer evidence from an unprecedented emergency sit- uation about the ways that civilian control shapes military deployment. Supplementary material. To view supplementary material for this article, please visit https://doi.org/10.1017/ S1755773923000024. Acknowledgements. We thank Chiara Tulp for her excellent research assistance during the data collection, Nguyen Ha for her help in the initial data cleaning phase and the editors and anonymous reviewers for their helpful and constructive feed- back. All remaining errors are our own. We gratefully acknowledge financial support from the Swedish Research Council (grant number 2017-02139) that made the data collection possible and the Royal Swedish Academy in Letters, History and Antiquities. In 0. 0. 0.0 0 There is a substantively large reduction in the probability of an enforcement operation as the election draws nearer for leaders who are eligible to run for reelection. Notably, this relationship does not hold for incumbents who are ineligible to run for reelection. This pattern is also not present among logistics deployments. It seems that there was a sharp reduction in enforcement https://doi.org/10.1017/S1755773923000024 Published online by Cambridge University Press 639 How governance shaped military responses to the COVID-19 pandemic operations among electoral democracies only when the incumbent leader was eligible to run for reelection and the election was temporally proximate. operations among electoral democracies only when the incumbent leader was eligible to run for reelection and the election was temporally proximate. 20In Appendix 2, we include an additional model specification to test for associations between democracy and ‘behind the scenes’ deployments in aggregate. Cite this article: Dietrich N, Eck K, and Ruffa C (2023). How governance shaped military responses to the COVID-19 pandemic. 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https://openalex.org/W2326387720	https://zenodo.org/records/1405816/files/2312ijesa08.pdf	English	null	Speaker Recognition Model Based on Generalized Gamma Distribution Using Compound Transformed Dynamic Feature Vector	International journal of embedded systems and applications	2,012	cc-by	2,772	International Journal of Embedded Systems and Applications (IJESA) Vol.2, No.3, September 2012 International Journal of Embedded Systems and Applications (IJESA) Vol.2, No.3, September 2012 KEYWORDS Speaker identification, MFCC, LPC, Generalized Gamma, Shifted Delta coefficients ABSTRACT In this paper, we present an efficient speaker identification system based on generalized gamma distribu- tion. This system comprises of three basic operations, namely speech features classification and metrics for evaluation. The features extracted using MFCC are passed to shifted delta cepstral coefficients (SDC) and then applied to linear predictive coefficients (LPC) to have effective recognition. To demonstrate our me- thod, a database is generated with 200 speakers for training and around 50 speech samples for testing. Above 90% accuracy reported. K Suri Babu1, Srinivas Yarramalle2, Suresh Varma Penumatsa3 K Suri Babu1, Srinivas Yarramalle2, Suresh Varma Penumatsa 1Scientist, NSTL (DRDO),Govt. of India, Visakhapatnam, India 2 Dept. of IT, GITAM University, Visakhapatnam. India. 3 Aadikavi Nannaya University, Rajahmundry, India.. {1suribabukorada2000@gmail.com, 2sriteja.y@gmail.com } 2. FEATURE EXTRACTION In the proposed work we have considered the speech signals with frame amount of 20 to 30 Ms. and the window analysis is shifted by 10ms. Each frame is transformed using cepstral coeffi- cients such as linear prediction coding and Mel frequency cepstral coefficients (MFCC) MFCC s are considered as they are based on the known variation of human ear critical band width with the frequency, each frame is transformed into 12 MFCC and a normalized energy parameter each frame consist of 39 columns including first and second derivatives, i.e. Delta and double Delta. In feature extraction the speech waves stored in wav format each converted to a parametric form. The speech signals remains stationary between the time intervals 5 Ms. to 100 Ms. and the changes observed over long periods i.e. 0.2sec or more. Therefore to identify the speech varia- tion in short time sequence, cepstral analysis is mostly preferred hence MFCC are considered Linear prediction coding (LPC) coefficient helps to extract signal more effectively in the presence of noise and when the speech signal is of very short duration .So in this thesis we have exploited MFCC combined with LPC to have effective feature vector identification. In speech analysis, significant information spread over few 100s of milliseconds there may be overlaps and the speech signals are not completely separated in-time. These overlaps may result in to ambiguities at the time of classification to overcome this it is assumed to extract the features between the frequencies 2 to 16 Hz, a maximum of 4 Hz. In order to distinguish these signals in the overlapping situations Delta features are mostly pre- ferred. In delta coefficients we obtained the derivative to estimate the differences in the speech trajectories. Delta-Delta coefficients are also considered for every longer temporal context. But these features will be effective for short term speech samples, for long term features shifted delta coefficients (SDC) are well proffered. The features obtained from MFCC are converted to shifted delta coefficients. It is observed that the features obtained from MFCC followed by SDC outper- form MFCC followed by delta. SDC reflects the dynamic cepstral features along with pseudo- Prosodic feature behavior. 1. INTRODUCTION With the recent advancements in Technology, lot of information can be stored in the databases, in any of the format such as audio, video or text. Therefore, searching the exact information is diffi- cult task [1]. Automatic indexing to the multimedia content can solve this problem. To retrieve speech signal from this Meta data is a crucial task. The speech signal to be retrieved is considered and is divided into small streams (segments) and the features are to be extracted. In order to extract features, MFCC are mostly proffered [3], [4] since they are less vulnerable to noise and give less variability. In order to have effective recog- nition it is needed to extract the first and second order time derivatives of cepstral features, that is delta and delta-delta features[5], but these features will be effective for short term speech sam- ples, for long term features shifted delta coefficients (SDC) are well proffered [6], [7], [8]. Hence in this paper, we develop a model for speaker identification, where the features obtained from MFCC are converted to shifted delta coefficients and also by converting MFCC to delta coefficients. It is observed that the features obtained from MFCC followed by SDC outperform MFCC followed by delta. 75 DOI : 10.5121/ijesa.2012.2308 International Journal of Embedded Systems and Applications (IJESA) Vol.2, No.3, September 2012 International Journal of Embedded Systems and Applications (IJESA) Vol.2, No.3, September 2012 The paper is organized as follows, the section-2 of the paper discuses about feature extraction, in section-3 generalized gamma distribution is proposed. Section -4 deals with experimental results. Finally, in section-5 conclusions are presented. 3. SPEAKER RECOGNITION ALGORITHM The steps to be followed for recognizing the speaker effectively are given under Step1: Step1: Obtain the training set by recording the speech voices in a .wav form Step2: Pre-emphasis the speech signals to remove silence and noise. Step3: Identify the compound feature vectors feature vector of these speech signals by using MFCC, LPC, SDC, Delta, and Delta-Delta. Step1: Step7: Evaluation metrics such as Acceptance Rate (AR), False Acceptance Rate (FAR), and Missed Detection Rate (MDR) are calculated to find the accuracy of speaker recognition. 4. GENERALIZED GAMMA MIXTURE MODEL Today most of the research in speech processing is carried out by using Gaussian mixture model, but the main disadvantage with Gaussian mixture model is that it relies exclusively on the ap- proximation and low in convergence, and also if Gaussian mixture model is used, the speech and the noise coefficients differ in magnitude [7]. To have a more accurate feature extraction, maxi- mum posterior estimation models are to be considered [8]. Hence in this paper, a generalized gamma distribution is utilized for classifying the speech signal. Generalized gamma distribution represents the sum of n-exponential distributed random variables both the shape and scale para- meters have non-negative integer values [9]. Generalized gamma distribution is defined in terms of scale and shape parameters [10]. The generalized gamma mixture is given by , , , , = (1) (1) Where, k and c are the shape parameters, a is the location parameter, b is the scale parameter and gamma is the complete gamma function [11]. The shape and scale parameter of the generalized gamma distribution helps to classify the speech signal and identify the speaker accurately. Where, k and c are the shape parameters, a is the location parameter, b is the scale parameter and gamma is the complete gamma function [11]. The shape and scale parameter of the generalized gamma distribution helps to classify the speech signal and identify the speaker accurately. Step1: Obtain the training set by recording the speech voices in a .wav form Pre-emphasis the speech signals to remove silence and noise. : Identify the compound feature vectors feature vector of these speech signals by using MFCC, LPC, SDC, Delta, and Delta-Delta. 76 International Journal of Embedded Systems and Applications (IJESA) Vol.2, No.3, September 2012 Step4: Generate the probability density function (PDF) of the generalized gamma distribution for all the trained data set. Step5: Same procedure is followed for test sequence. Step6: Find the range of speech of test signal in the trained set. Step7: Evaluation metrics such as Acceptance Rate (AR), False Acceptance Rate (FAR), and Missed Detection Rate (MDR) are calculated to find the accuracy of speaker recognition. International Journal of Embedded Systems and Applications (IJESA) Vol.2, No.3, September 2012 Step4: Generate the probability density function (PDF) of the generalized gamma distribution for all the trained data set. Step5: Same procedure is followed for test sequence. Step6: Find the range of speech of test signal in the trained set. Step7: Evaluation metrics such as Acceptance Rate (AR), False Acceptance Rate (FAR), and Missed Detection Rate (MDR) are calculated to find the accuracy of speaker recognition tep4: Generate the probability density function (PDF) of the generalized gamma distribution for all the trained data set. 5. EXPERIMENTAL RESULTS During the training phase, the signal must be preprocessed and the features are extracted using MFCC. In order to have an effective recognition system we have sampled the data into short speech samples of different time frames and the MFCC features that are extracted are converted delta coefficients and shift delta coefficients. It is observed that MFCC combined delta coeffi- cients could not effectively recognize the speech samples as compared to that of MFCC combined with SDC. The output is then fed to LPC (linear predictive coefficients). The features extracted are then given as input to the classifier that is generalized gamma distribution, using these feature set, the generalized gamma distribution is effectively recognized. The speech samples that are ob- tained from MFCC-SDC-LPC, it can also be seen that as and when the sample size is increased, these features that are extracted helps to classify the speakers most effectively. The results are presented in both tabular and graphical formats. 5. EXPERIMENTAL RESULTS 77 nternational Journal of Embedded Systems and Applications (IJESA) Vol.2, No.3, September 2012 International Journal of Embedded Systems and Applications (IJESA) Vol.2, No.3, September 2012 Fig.1: Effect of Recognition accuracy with trained dataset Fig.2: Effect of Recognition accuracy with Speech duration 0 20 40 60 80 100 120 0 100 200 300 400 accuracy of recognition no of trained speakers Effect of recognition accuracy with trained no of speakers MFCC- DELTA-LPC MFCC-SDC- LPC 0 20 40 60 80 100 120 0 20 40 percentage of correctness frame amount(sec) Effect of recognition accuracy with speech duration MFCC- DELTA-LPC 0 20 40 60 80 100 120 0 5 10 15 20 accuracy of recognition Effect of recognition accuracy with test speech duration MFCC- DELTA- LPC y pp ( ) p Fig.1: Effect of Recognition accuracy with trained dataset 0 20 40 60 80 100 120 0 100 200 300 400 accuracy of recognition no of trained speakers Effect of recognition accuracy with trained no of speakers MFCC- DELTA-LPC MFCC-SDC- LPC Fig.1: Effect of Recognition accuracy with trained dataset 0 20 40 60 80 100 120 0 100 200 300 400 accuracy of recognition no of trained speakers Effect of recognition accuracy with trained no of speakers MFCC- DELTA-LPC MFCC-SDC- LPC Fig.1: Effect of Recognition accuracy with trained dataset Fig.2: Effect of Recognition accuracy with Speech duration 0 20 40 60 80 100 120 0 20 40 percentage of correctness frame amount(sec) Effect of recognition accuracy with speech duration MFCC- DELTA-LPC Fig.2: Effect of Recognition accuracy with Speech duration 0 20 40 60 80 100 120 0 20 40 percentage of correctness frame amount(sec) Effect of recognition accuracy with speech duration MFCC- DELTA-LPC Fig.2: Effect of Recognition accuracy with Speech duration Fig.3: Effect of recognition accuracy with test Speech duration 0 20 40 60 80 100 120 0 5 10 15 20 accuracy of recognition test utterance length(sec) Effect of recognition accuracy with test speech duration MFCC- DELTA- LPC Fig.3: Effect of recognition accuracy with test Speech duration 0 20 40 60 80 100 120 0 5 10 15 20 accuracy of recognition test utterance length(sec) Effect of recognition accuracy with test speech duration MFCC- DELTA- LPC Fig.3: Effect of recognition accuracy with test Speech duration 78 78 Table 1: Statistical data showing accuracy % No of trained speakers MFCC- DELTA-LPC 0 to 50 50 to 100 100 to 300 MFCC-SDC- LPC 0 to 50 50 to 100 100 to 300 Table 1: Statistical data showing accuracy % No of trained speakers Frame amount (sec) Test utterance length (sec) Recognition Accuracy 0 to 50 0 to 5 0 to 5 Less than 60 50 to 100 5 to 10 5 to 10 Around 100 to 300 10 to 30 10 to 15 Above 62 0 to 50 0 to 5 0 to 5 Less Than 80 50 to 100 5 to 10 5 to 10 Around 85 100 to 300 10 to 30 10 to 15 Above 90 Recognition Accuracy (%) Less than 60 Around 60 Above 62 Less Than 80 Around 85 Above 90 From the above figures and table (Fig.1 to Fig.3 and Table 1), it could be easily MFCC-SDC-LPC outperforms MFCC seen in the developed model. 5. EXPERIMENTAL RESULTS From the above figures and table (Fig.1 to Fig.3 and Table 1), it could be easily LPC outperforms MFCC-Delta-LPC and over all recognition rate is above 90% is From the above figures and table (Fig.1 to Fig.3 and Table 1), it could be easily seen that the LPC and over all recognition rate is above 90% is 5. PERFORMANCE EVALUATION VALUATION In order to evaluate the performance of the developed model various metrics such as Acceptance Rate (AR), False Acceptance Rate (FAR), and Missed Detection Rate (MDR) are considered. The various formulas for evaluating the metrics are given below. In order to evaluate the performance of the developed model various metrics such as Acceptance (AR), False Acceptance Rate (FAR), and Missed Detection Rate (MDR) are considered. The various formulas for evaluating the metrics are given below. In order to evaluate the performance of the developed model various metrics such as Acceptance (AR), False Acceptance Rate (FAR), and Missed Detection Rate (MDR) are considered. The " #$% " &'%%%( (') " = +, -+ +. /0 +, -+ +. 1/21 ∗100 " = +, -+ +. 1/21 +, -+ +. 1/0 +, -+ +. 1/21 ∗100 (') " = +, -+ +. 67110 + 2+8-79 +*, -+ +. 1/21 ∗100 100 The developed model is tested for accuracy using the above metrics mentioned in equations The developed model is tested for accuracy using the above metrics mentioned in equations The developed model is tested for accuracy using the above metrics mentioned in equations he developed model is tested for accuracy using the above metrics mentioned in equations he developed model is tested for accuracy using the above metrics mentioned in equations he developed model is tested for accuracy using the above metrics mentioned in equations p y g q Fig .4: feature vector performance 0 20 40 60 80 100 AR p y g q Fig .4: feature vector performance evaluation FAR MDR MFCC-DELTA-LPC MFCC-SDC-LPC p y g q LPC Fig .4: feature vector performance Fig .4: feature vector performance evaluation Fig .4: feature vector performance Fig .4: feature vector performance evaluation 79 79 International Journal of Embedded Systems and Applications (IJESA) Vol.2, No.3, September 2012 The above Fig.4 shows the performance with metrics as Acceptance rate(AR) ,False Acceptance Rate(FAR) and Missed Detection Rate(MDR) by considering Various combinations of feature vectors as MFCC-DELTA-LPC, ,MFCC-SDC-LPC and it shows performance comparison of fea- ture vectors using generalized gamma distribution with three metrics . From the above Fig.4, It can be clearly seen that MFCC-SDC-LPC feature vector out performs than all the combinations of feature vectors. 5. CONCLUSIONS In this paper, we have developed a new model for speaker identification based on generalized gamma distribution. The speeches are extracted using MFCC are combined with delta coeffi- cients followed by LPC and also MFCC combined with SDC followed by LPC. The model is demonstrated a database of 200 samples and tested with 50 samples, the accuracy is around 90% and proved to be efficient model. REFERENCES [1] Marko kos, Damjan Vlaj,Zdravko Kacic,(2011) “Speaker’s gender classification and segmentation using spectral and cepstral feature averaging”, 18th International Conference on Systems, Signals and Image Processing - IWSSIP 2011. [1] Marko kos, Damjan Vlaj,Zdravko Kacic,(2011) “Speaker’s gender classification and segmentation using spectral and cepstral feature averaging”, 18th International Conference on Systems, Signals and Image Processing - IWSSIP 2011. [2] J.Razik,C.SEnac,D.Fohr,O.Mella and N Parlangeau-Valles,(2003) “comparision of two speech/Music segmentation systems for audio indexing on Web”,in Proc WMSCI’03,Florida,USA,July2003. [3] Corneliu Octavian.D,I.Gavat,(2005), “Feature Extraction Modeling &Training Strategies in conti- nuous speech Recognition For Roman Language”, EU Proceedings of IEEE Xplore,EUROCN- 2005,pp-1424-1428. [4] Sunil Agarwal et al,(2010), “Prosodic Feature Based Text-Dependent Speaker Recognition Using ma- chine Learning Algorithm”,International Journal of Engg.sc &Technology, Vol:2(10), 2010,pp5150- 5157. [5] Dayana Ribas Gonzalez,Jose R.Calvo de Lara(2009), “Speaker verification with shifted delta cepstral features:Its Pseudo-Prosodic Behaviour”, proc I Iberian SLTech 2009. 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https://openalex.org/W2413475812	http://www.ajnr.org/content/ajnr/37/11/1983.full.pdf	English	null	Bidirectional Changes in Anisotropy Are Associated with Outcomes in Mild Traumatic Brain Injury	American journal of neuroradiology	2,016	cc-by	9,068	ABSTRACT BACKGROUND AND PURPOSE: Mild traumatic brain injury results in a heterogeneous constellation of deﬁcits and symptoms that persist in a subset of patients. This prospective longitudinal study identiﬁes early diffusion tensor imaging biomarkers of mild traumatic brain injury that signiﬁcantly relate to outcomes at 1 year following injury. MATERIALS AND METHODS: DTI was performed on 39 subjects with mild traumatic brain injury within 16 days of injury and 40 controls; 26 subjects with mild traumatic brain injury returned for follow-up at 1 year. We identiﬁed subject-speciﬁc regions of abnormally high and low fractional anisotropy and calculated mean fractional anisotropy, axial diffusivity, radial diffusivity, and mean diffusivity across all white matter voxels brain-wide and each of several white matter regions. Assessment of cognitive performance and symptom burden was performed at 1 year. RESULTS: Signiﬁcant associations of brain-wide DTI measures and outcomes included the following: mean radial diffusivity and mean diffusivity with memory; and mean fractional anisotropy, radial diffusivity, and mean diffusivity with health-related quality of life. Signif- icant differences in outcomes were found between subjects with and without abnormally high fractional anisotropy for the following white matter regions and outcome measures: left frontal lobe and left temporal lobe with attention at 1 year, left and right cerebelli with somatic postconcussion symptoms at 1 year, and right thalamus with emotional postconcussion symptoms at 1 year. CONCLUSIONS: Individualized assessment of DTI abnormalities signiﬁcantly relates to long-term outcomes in mild traumatic brain injury. Abnormally high fractional anisotropy is signiﬁcantly associated with better outcomes and might represent an imaging correlate of postinjury compensatory processes. ABBREVIATIONS: AD axial diffusivity; EZ-MAP Enhanced Z Score Microstructural Assessment of Pathology; FA fractional anisotropy; HRQoL health- related quality of life; hFA high fractional anisotropy; lFA low fractional anisotropy; MD mean diffusivity; mTBI mild traumatic brain injury; PCS postconcussion symptoms; RD radial diffusivity; TBI traumatic brain injury M ild traumatic brain injury (mTBI) is associated with a hetero- geneousconstellationofdeficitsandsymptomsthatpersistfor the long term in 20% of patients who experience concussion.1 The syndrome can entail cognitive impairment, most prominently in memory, attention, and executive function, and postconcussion symptoms (PCS) and limitations in daily functioning. Bidirectional Changes in Anisotropy Are Associated with Outcomes in Mild Traumatic Brain Injury X S.B. Strauss, X N. Kim, X C.A. Branch, X M.E. Kahn, X M. Kim, X R.B. Lipton, X J.M. Provataris, X H.F. Scholl, X M.E. Zimmerman, and X M.L. Lipton X S.B. Strauss, X N. Kim, X C.A. Branch, X M.E. Kahn, X M. Kim, X R.B. Lipton, X J.M. Provataris, X H.F. Sch X M.E. Zimmerman, and X M.L. Lipt X S.B. Strauss, X N. Kim, X C.A. Branch, X M.E. Kahn, X M. Kim, X R.B. Lipton, X J.M. Provataris, X H.F. Scholl, X M.E. Zimmerman, and X M.L. Lipton and M.L. Lipton R.B. Lipton, J.M. Provataris, H.F. Scholl, M.E. Zimmerman S.B. Strauss, N. Kim, C.A. Branch, M.E. Kahn, M. Kim, of October 23, 2024. This information is current as of October 23, 2024. This information is current as http://www.ajnr.org/content/37/11/1983 https://doi.org/10.3174/ajnr.A4851 doi: 2016, 37 (11) 1983-1991 AJNR Am J Neuroradiol ORIGINAL RESEARCH ADULT BRAIN Please address correspondence to Michael L. Lipton, MD, PhD, FACR, Albert Ein- stein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Ave MRRC, Room 219C, Bronx, NY 10461; e-mail: michael.lipton@einstein.yu.edu Received August 4, 2015; accepted after revision April 25, 2016. From the Department of Radiology (S.B.S., M.L.L.), Monteﬁore Medical Center, Bronx, New York; Departments of Radiology (N.K., M.L.L.), Physiology and Biophys- ics (C.A.B.), Epidemiology and Population Health (M.K., R.B.L.), and Psychiatry and Behavioral Sciences (M.L.L.), The Gruss Magnetic Resonance Research Center (N.K., C.A.B., M.E.K., H.F.S., M.L.L.), The Saul R. Korey Department of Neurology (R.B.L., M.E.Z.), and The Dominick P. Purpura Department of Neuroscience (M.L.L.), Albert Einstein College of Medicine, Bronx, New York; and Department of Emergency Medicine (J.M.P.), Jacobi Medical Center, Bronx, New York. This work was supported by a National Institutes of Health grant NS082432-03 (M.L.L.). Please address correspondence to Michael L. Lipton, MD, PhD, FACR, Albert Ein- stein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Ave MRRC, Room 219C, Bronx, NY 10461; e-mail: michael.lipton@einstein.yu.edu Indicates open access to non-subscribers at www.ajnr.org Indicates article with supplemental on-line photos. http://dx.doi.org/10.3174/ajnr.A4851 AJNR Am J Neuroradiol 37:1983–91 Nov 2016 www.ajnr.org 1983 Received August 4, 2015; accepted after revision April 25, 2016. Exclusion Criteria Exclusion Criteria Focal neurologic deﬁcits History of head injury (based on history and medical record) Thirty-nine subjects with mTBI were prospectively enrolled from 2 urban emergency departments. Chronic posttraumatic abnormal ﬁndings on CT/MRI Hospitalization due to the current head injury History of a neurodevelopmental or neurologic disorder Inclusion and exclusion criteria for subjects are detailed in Table 1. Subjects were evaluated in the emergency depart- ment within 48 hours of injury, and di- agnosis of mTBI/concussion was made by an emergency department physician. If CT was performed for clinical care of the current head injury, then an American Board of Radiology Certificate of Added Quali- fication–certified neuroradiologist reviewed the CT images. Subjects were excluded if skull fracture or any acute or chronic posttraumatic abnormality such as gliosis, localized encepha- lomalacia, or remote hemorrhage was identified. rent head injury ntal or neurologic Inclusion and exclusion criteria for subjects are detailed in Table 1. Subjects were evaluated in the emergency depart- ment within 48 hours of injury, and di- agnosis of mTBI/concussion was made by an emergency department physician. Major psychiatric disorder Illicit drug use within 30 days Major psychiatric disorder Illicit drug use within 30 days overwhelming consensus of 120 published studies indicates that despite methodologic heterogeneity, abnormally low fractional anisotropy (FA) derived from DTI is characteristic of patients with mTBI.5 Cross-sectional associations of DTI abnormalities and functional outcomes support the clinical significance of these imaging findings.6-8 Despite strong evidence supporting the ability of DTI to detect clinically salient traumatic axonal injury pathology and its poten- tial to identify patients at risk for poor long-term outcomes, to date DTI has not yet yielded a validated prognostic biomarker for several reasons: First, only a small number of longitudinal studies have assessed the relationship between early imaging and later outcomes.9,10 Second, most studies define outcomes as perfor- mance on tests of cognitive function.7,8,11,12 Only a few have ex- amined the relationship between early imaging and subjective measures, which may more closely approximate real-world func- tioning, such as PCS10,11 and health-related quality of life (HRQoL).13 The morbidity of mTBI in real-world settings may reflect deficits not captured by standard formal cognitive testing, such as multimodal processing and divided attention.14 Third, image-analysis approaches have largely (though not exclusively) used group-level delineation of DTI measures. Exclusion Criteria However, delinea- tion of ROIs at the group level, whether on an a priori basis or by using a voxelwise analysis, is insensitive to the unique spatial dis- tribution of traumatic axonal injury, which is likely present in each patient. Finally, most studies report the association of low FA with poor mTBI outcomes7,8 but do not report abnormally high FA. The few studies assessing the functional significance of abnor- mally high FA do so at a relatively short follow-up.9,15 The objec- tive of this study was to address current gaps in knowledge by characterizing the relationship between acute diffusion abnor- malities in patients with uncomplicated mTBI and their 1-year functional outcomes, including cognition, PCS, and HRQoL. To address the importance of spatial heterogeneity of traumatic ax- onal injury across subjects, we used an individualized approach to identification of abnormality in each patient with mTBI, En- hanced Z Score Microstructural Assessment of Pathology (EZ-MAP),16,17 and we considered both regional and brain-wide measures and their relationship outcome. If identified prospec- tively, those with worse prognosis could be targeted for studies of interventions designed to improve outcomes following mTBI. Forty healthy volunteers were recruited from the community via printed advertisements. The inclusion criterion for controls was 18–70 years of age. Exclusion criteria were the same as those for subject enrollment. This study used a prospective, within-subjects design: The role of the group of 40 controls was to provide imaging for comparison, to allow identification of abnormal FA. In this longitudinal study, imaging was performed within 16 days of injury (median, 7.5 days; range, 1–16 days), and cognitive function, PCS, and HRQoL were assessed at 1 year following injury. Data Acquisition Imaging. Imaging was performed by using a 3T MR imaging scan- ner (Achieva TX; Philips Healthcare, Best, the Netherlands) with a 5-channel head coil (SENSE Head Coil; Philips Heathcare). T1- weighted whole-head structural imaging was performed by using sagittal 3D magnetization-prepared rapid acquisition of gradient echo imaging (TR/TE, 9/4.6 ms; FOV, 240 mm2; matrix, 240 240; section thickness, 1 mm). T2-weighted whole-head imaging was performed by using axial 2D turbo spin-echo imaging (TR/ TE, 4000/100 ms; FOV, 240 mm2; matrix, 384 512; section thickness, 4.5 mm) and axial 2D fluid-attenuated inversion recov- ery turbo spin-echo (TR/TE, 1100/120 ms; TI, 2800 ms; FOV, 240 mm2; matrix, 384 512; section thickness, 4.5 mm; number of signals acquired, 1). DTI was performed by using single-shot spin-echo echo-planar imaging (TR/TE, 3800/88 ms; FOV, 240 mm2; matrix, 112 89; section thickness, 4.5 mm; independent diffusion-sensitizing directions, 32; b800 s/mm2 images). Outcome Measures Cognition. Tests of cognitive function were administered to all subjects, by using IntegNeuro (Brain Resource Company, Sydney, Australia), a computerized battery of cognitive tasks.18 A sum- mary z score was computed for each of 3 cognitive domains (ex- ecutive function, episodic memory, and attention), selected for study because of their known associations with the mTBI syn- drome, by using an international database of 5000 age-, sex-, and education-matched healthy individuals, as detailed in Table 2. ABSTRACT M Notwithstanding earlier constructs that frame PCS as largely fac- titious or psychogenic and without a biologic basis, it is now widely understood that even uncomplicated mTBI-related dysfunction re- sults from structural pathology such as traumatic axonal injury.2,3 The inability of imaging techniques such as CT and MR imaging to detect traumatic axonal injury has led to delayed understanding of the clinical mTBI syndrome, despite human (eg, Bigler, 20042) and animal (eg, Mac Donald et al, 20074) studies delineating trauma- related histopathology following even mild head trauma. More recently, DTI has become an established means for the detection of human traumatic axonal injury pathology in vivo; the Indicates open access to non-subscribers at www.ajnr.org Indicates article with supplemental on-line photos. http://dx.doi.org/10.3174/ajnr.A4851 AJNR Am J Neuroradiol 37:1983–91 Nov 2016 www.ajnr.org 1983 Indicates open access to non-subscribers at www.ajnr.org Indicates article with supplemental on-line photos. http://dx.doi.org/10.3174/ajnr.A4851 AJNR Am J Neuroradiol 37:1983–91 Nov 2016 www.ajnr.org 1983 1983 Table 1: Subject inclusion and exclusion criteria Inclusion Criteria Exclusion Criteria Age 18–70 years Focal neurologic deﬁcits Availability for testing within 2 weeks of concussion History of head injury (based on history and medical record) Glasgow Coma Scale 13–15 Chronic posttraumatic abnormal ﬁndings on CT/MRI Loss of consciousness 20 minutes Hospitalization due to the current head injury Posttraumatic amnesia 24 hours History of a neurodevelopmental or neurologic disorder English or Spanish proﬁciency Major psychiatric disorder Illicit drug use within 30 days Table 1: Subject inclusion and exclusion criteria Inclusion Criteria Table 1: Subject inclusion and exclusion criteria ance Portability and Accountability Act. All subjects provided written, informed consent for participation in the study. Exclusion Criteria Subject Enrollment and Study Design Subject Enrollment and Study Design This study was approved by the Einstein Institutional Review Board and was conducted in accordance with the Health Insur- 1984 Strauss Nov 2016 www.ajnr.org 1984 Table 2: Cognitive domains and component cognitive tasks Cognitive Domain Constituent Cognitive Tasks Executive Function Digit Span Backward Switching of attention (digits/letters) Verbal interference Executive maze task Episodic Memory Verbal list learning task (immediate recall, delayed recall, and recognition) Attention Digit Span Forward Continuous Performance Task Switching of attention (digits) Visual memory within-subject and nonlinear subject-to-template registration steps as previously reported.17 This spatial normalization procedure has beenshowntoberobustacrosssubjects.26 Nonetheless,theresultsof each registration are critically assessed by viewing each stage of the registration output, with particular assessment of the alignment of brain surface; deep structures including the brain stem, corpus callo- sum, and fornix; and gray/white margins in both the deep gray mat- terstructuresandatthecorticalmargin.Theselandmarksmustalign within2voxeldimensionsfortheregistrationtobeaccepted,though alignment is typically nearly exact. White Matter Segmentation. The FMRIB Automated Segmenta- tion Tool (FAST; http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FAST)27 was used to generate a white matter mask for the 3D T1-weighted tem- plate brain images. This mask was eroded by 3 voxels to eliminate locations most at risk of misregistration and was used to restrict sub- sequent statistical analysis of FA to white matter voxels. Premorbid intelligence was estimated by using the Spot-the- Word test, which is a measure of word recognition ability and lexical decision-making, with good reliability (0.88) and conver- gent validity (0.60–0.86).19 Postconcussion Symptoms and Health-Related Quality of Life Outcome Measures Subregion Segmentation of White Matter. While the analysis was performed in the Montreal Neurological Institute template space, the Johns Hopkins University white matter atlas28 was adapted and used for segmentation of white matter subregions. This segmentation procedure eliminates potential observer bias introduced by intra- and interrater variance inherent in manually delineated ROIs. With the FMRIB Linear Image Registration Tool (FLIRT; http://www.fmrib.ox.ac.uk/),27 the T1-weighted tem- plate for the Johns Hopkins University white matter atlas was registered to the T1-weighted Montreal Neurological Institute template used for DTI analysis. The resulting transformation ma- trix was applied to the white matter segmentation volume of the Johns Hopkins University white matter atlas to bring it into registra- tion with the Montreal Neurological Institute template. Individual Johns Hopkins University regions were combined to generate larger white matter regions used for analysis, as detailed below. At 1 year postinjury, the Rivermead Post Concussion Symptoms Questionnaire20 was administered to assess PCS, and the Sickness Impact Profile21 was administered to assess HRQoL. Patients were classified as having postconcussion syndrome on the basis of out- comes at 1 year rather than 3 or 6 months; thus, those with postcon- cussionsyndromethatlasted1yearwerenotclassifiedinthisman- ner.Thisclassificationrestrictsthepostconcussionsyndromepatient group to those with more persistent and chronic symptoms. The Rivermead Post Concussion Symptoms Questionnaire consists of a series of 16 symptoms; subjects are asked to rate the severity for each item, relative to preinjury experience, on a scale from 0 to 4. Symptoms are categorized into cognitive, somatic, or emotional factors; and each factor is used as an independent out- come measure.22 The Sickness Impact Profile includes 68 questions pertaining to daily functioning and is graded on a dichotomous scale (0 or 1) yielding 5 subscores. The physical dimension is assessed as “so- matic autonomy” and “mobility control”; the psychological di- mension, as “psychological autonomy and communication”; and the social dimension, as “mobility range” and “social behavior.”23 Adjustment for Demographic Covariates. Before lesion detec- tion, multiple linear regression analysis was performed to adjust for the effects of age, sex, and education as detailed in Kim et al.17 Regression coefficients were determined from control subjects only, to avoid potential interaction effects of mTBI on the afore- mentioned putative risk factors, and were applied to the voxels within each subject’s FA image, where covariate effects on indi- vidual voxels were significant at P .05 and 100 significant voxels formed a contiguous cluster. Data Analysis All analyses were performed after transforma- tion of diffusion parameter images to match a high-resolution T1- weightedtemplate(MontrealNeurologicalInstitute).25Theregistra- tion process includes correction for EPI distortions and linear Table 3: Demographic and injury features of subjects and controls Subjects Controls Mean age (yr) (range) 38.5 (24–64) 38.85 (20–60) Sex 10 Men (38.5%) 21 Men (52.5%) 16 Women (61.5%) 19 Women (47.5%) Mean years of education (range) 14.3 (8–24) 16.4 (2–26) Mechanism of injury Motor vehicle crash: 3 NA Sports accident: 2 Fall: 7 Assault: 7 Falling object: 7 Posttraumatic amnesia 2/26 Loss of consciousness 10/26 Note:—NA indicates not applicable. Table 3: Demographic and injury features of subjects and controls Subjects Controls Mean age (yr) (range) 38.5 (24–64) 38.85 (20–60) Sex 10 Men (38.5%) 21 Men (52.5%) 16 Women (61.5%) 19 Women (47.5%) Mean years of education (range) 14.3 (8–24) 16.4 (2–26) Mechanism of injury Motor vehicle crash: 3 NA Sports accident: 2 Fall: 7 Assault: 7 Falling object: 7 Posttraumatic amnesia 2/26 Loss of consciousness 10/26 Note:—NA indicates not applicable. Table 3: Demographic and injury features of subjects and controls normal clusters were further delineated by applying a cluster size threshold determined on the basis of the Gaussian random field theory. This technique has been optimized and validated previ- ously for assessment of individual subjects, and the EZ-MAP showed greater robustness in varying control samples compared with the z score and the 1-versus-many t test, approaches that have been adopted in other single-subject analyses of FA imaging data in mTBI.16,17 The EZ-MAP thus generated was thresholded with 2 criteria: EZ 1.96 for each voxel and cluster size P value 1% (corrected for multiple comparisons by the Gaussian random field theory).16,17 A subject FA value from an abnormal FA lesion detected by EZ-MAP is in the range of magnitude: y x 1.96 Kx, where Kx sdx ˆBx. Because ˆBx is 1 as aforementioned, the value of SD (K) from the control mean in the EZ-MAP is 1.96. Validation of the EZ-MAP method in Kim et al17 serves as the premise for its application in the current study. Note:—NA indicates not applicable. and was used to evaluate the monotonic association of brain-wide early imaging variables and long-term (1 year) outcomes. Study Subjects: Sample Size, Demographics, Mechanisms of Injury, and Baseline Functional Status Thirty-nine subjects with mTBI and 40 control subjects met the inclusion criteria. Subjects with mTBI underwent DTI within 16 days of injury. Twenty-six subjects returned for 1-year follow-up and were included in the analysis. During the time elapsed be- tween enrollment and follow-up, 6 of the 26 subjects moved to locations too far from the testing center to allow on-site, comput- erized cognitive assessment; they were, however, available to com- plete the Rivermead Post Concussion Symptoms Questionnaire and Sickness Impact Profile by phone interview. Data Analysis In addition, a regional analysis of abnormal FA was performed to facilitate investigation of structure-function associations, de- spite the fact—which is an expected feature of traumatic brain injury (TBI) pathology—that not all subjects exhibited abnor- malities in the same brain location. Student 2-sample t tests were used to compare long-term outcomes between groups of subjects who did-versus-did not exhibit hFA or lFA within a given ana- tomic region (listed above). False Discovery Rate Control for Statistical Analyses False Discovery Rate Control for Statistical Analyses The total number of hypotheses tested for association between imaging measures and long-term outcomes are as follows: In the brain-wide assessment of abnormal diffusion metrics across all areas of hFA and lFA in each subject, 5 imaging parameters (FA lesion volume, mean FA, mean AD, mean RD, and mean MD) were correlated with 12 outcomes for each total hFA and total lFA, totalling 120 (5 2 12) tests. For regional analyses, we exam- ined the difference between the 2 groups in 12 outcome measures for each of 9 brain regions, in which subjects were classified by the presence or absence of hFA and lFA within each brain region; 216 (2 9 12) tests were required for these analyses. Thus, in total, 336 comparisons were performed across the entire study. We grouped the 336 comparisons into 10 subgroups by 5 outcome categories (memory, attention, executive, PCS, and HRQoL) and analysis approaches, brain-wide and regional. The significance of individual tests was determined by using the Benjamini Hochberg method at a false discovery rate 0.15 for each subgroup.30,31 Regional Imaging Measures. We selected 9 white matter regions known to be susceptible to mTBI and/or to serve functions asso- ciated with mTBI morbidity, as follows: the left frontal lobe, right frontal lobe,8 left temporal lobe, right temporal lobe,10 left thala- mus, right thalamus,12 left cerebellum and right cerebellum,29 and corpus callosum (On-line Fig 2).7 Two new class variables were generated, representing the fol- lowing: a) the presence (1) or absence (0) of hFA in each region, and b) the presence (1) or absence (0) of lFA for each region (On-line Fig 2). The 2 class variables were independently gener- ated for each region. As a result, it is possible that hFA and lFA lesions will coexist within a single brain region. The 2 subgroups of subjects classified by using each class variable (a or b) were tested for significant differences in long-term outcomes. RESULTS Study Subjects: Sample Size, Demographics, Mechanisms of Injury, and Baseline Functional Status Calculation of Imaging Variables The procedure for calculating brain-wide and regional DTI measures for subsequent analysis is summarized in On-line Figs 1 and 2. Brain-Wide Imaging Measures. Each subject-specific EZ-MAP of abnormal FA regions was segregated into 2 separate maps: 1) all voxels showing abnormally high FA, indicated below as hFA, and 2) all voxels showing abnormally low FA, referred to below as lFA. These maps were then used as masks and were applied to each subject’s DTI parameter images: FA, axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD). Mean FA, AD, RD, and MD were thus calculated separately across all hFA voxels and across all lFA voxels, yielding 2 measures for each diffusion pa- rameter in each subject. Additionally, the total volume (number of 1-mm3 voxels) of hFA and lFA was also computed for each subject (On-line Fig 1). Data Analysis Neuroradiology Assessment. An American Board of Radiology Certificate of Added Qualification–certified neuroradiologist re- viewed structural MR images for posttraumatic pathology, in- cluding hemorrhage, extra-axial collection, contusion, or trau- matic axonal injury. EZ-MAP Analysis for FA Lesion Detection. The Enhanced Z Score Microstructural Assessment of Pathology17 is a method for delineating abnormal regions in individual patients with mTBI. The EZ-MAP is based on a whole-brain voxelwise z score of a subject Z Score y x sdx , calculated with the mean (x) and SD [sd (x)] from healthy controls at each voxel, where we denote SD from healthy controls as sd(x). We here omit the voxel index for notation convenience. The EZ-MAP is more ro- bust than standard z score analysis because it incorporates esti- mated sampling variance of individual z scores by using a boot- strap procedure, which is finally calculated as EZ Z Score ˆ Bx . ˆBx is 1 due to sample-to-sample variation of z scores.17 Ab- AJNR Am J Neuroradiol 37:1983–91 Nov 2016 www.ajnr.org 1985 EZ-MAP Analysis for FA Lesion Detection. The Enhanced Z Score Microstructural Assessment of Pathology17 is a method for delineating abnormal regions in individual patients with mTBI. The EZ-MAP is based on a whole-brain voxelwise z score of a subject Z Score y x sdx , calculated with the Calculation of Diffusion Parameter Images. The 32 diffusion- weighted image sets (32 b800 s/mm2 images) were corrected for head motion and eddy current effects by using an affine registra- tion algorithm, with the b0 s/mm2 image as the target, and ten- sor fitting was performed at each voxel by using the FMRIB Dif- fusion Toolbox (http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FDT).24 mean (x) and SD [sd (x)] from healthy controls at each voxel, where we denote SD from healthy controls as sd(x). We here omit the voxel index for notation convenience. The EZ-MAP is more ro- bust than standard z score analysis because it incorporates esti- mated sampling variance of individual z scores by using a boot- d hi h i fi ll l l d EZ Z Score Image Registration. 1986 Strauss Nov 2016 www.ajnr.org Statistical Analyses: Relationship between Early Imaging and Long-Term Outcomes All statistical analyses were conducted in SPSS (Version 22.0, Re- leased 2013; IBM, Armonk, New York). Both brain-wide and re- gional imaging measures (as defined above) were used in distinct analyses. Table 3 details demographic and injury characteristics of the 26 subjects and 40 controls used in the analysis. We found no The Spearman rank correlation analysis is robust to outliers 1986 Strauss Nov 2016 www.ajnr.org 1986 Strauss Nov 2016 www.ajnr.org FIG 1. Outcomes in mTBI. A, Cognitive outcomes. The red dotted line indicates impairment in the given domain, deﬁned as a z score less than 1.5. B, Health-related quality of life (Sickness Impact Proﬁle), grouped according to dimension. C, Postconcussion symptoms (Rivermead Post Concussion Symptoms Questionnaire). D, Postconcussion syndrome diagnosis. PAC indicates psychological autonomy and communication. FIG 1. Outcomes in mTBI. A, Cognitive outcomes. The red dotted line indicates impairment in the given domain, deﬁned as a z score less than 1.5. B, Health-related quality of life (Sickness Impact Proﬁle), grouped according to dimension. C, Postconcussion symptoms (Rivermead Post Concussion Symptoms Questionnaire). D, Postconcussion syndrome diagnosis. PAC indicates psychological autonomy and communication. and the 13 subjects lost to follow-up (note that loss of conscious- ness data were available for only 12/13 lost to follow-up). significant difference in age (t 0.134, P .894), sex (2 1.247, P .264), or years of education (t 1.919, P .059) between the 26 subjects and 40 controls. Nevertheless, potential effects of age, sex, and education were addressed by voxelwise regression adjustment, with application of regression coefficients to all FA voxels in which demographic covariate effects were sig- nificant at P .05 across 100 contiguous voxels.16 All subjects were diagnosed with mTBI in the emergency department, and no abnormalities were identified on conventional CT or MR imag- ing. In this relatively young sample, with a mean age of 38.5 years, changes of microvascular ischemia and stroke were not observed. AJNR Am J Neuroradiol 37:1983–91 Nov 2016 www.ajnr.org 1987 Relationship between Microstructural Abnormalities and Functional Outcomes Whole White Matter Assessment. Brain-Wide Imaging Mea- sures and Cognitive Performance. Imaging measures were as- sociated with 1-year memory performance, but not with atten- tion or executive function. The associations with memory were significant for higher RD from areas of hFA at baseline ( 0.562, P .015) and higher MD from areas of hFA at baseline ( 0.488, P .040) (On-line Fig 3). Brain-Wide Imaging Measures and PCS. Neither hFA nor lFA were associated with PCS when averaged across the whole brain. FIG 2. Associations between regional imaging measures and long- term cognitive outcomes. A, Subjects with high FA in the left frontal lobe perform signiﬁcantly better on tasks of attention at 1 year postinjury than do those without it (t 2.985, P .008). B, Subjects with high FA in left temporal lobe perform signiﬁcantly better on tasks of attention at 1 year postinjury than do those without it (t 3.524, P .002). Brain-Wide Imaging Measures and HRQoL. Higher MD and higher RD from areas of lFA ( 0.513, P .009; 0.514, P .009) were significantly associated with worse somatic autonomy. Lower mean FA from regions of lFA were significantly associated with worse psychologic autonomy and communication ( 0.596, P .002) and worse emotional stability ( 0.581, P .002) at 1 year postinjury (On-line Fig 4). FA, AD, RD, and MD from regions of hFA were not significantly associated with HRQoL. Brain-Wide Imaging Measures and HRQoL. Higher MD and higher RD from areas of lFA ( 0.513, P .009; 0.514, P .009) were significantly associated with worse somatic autonomy. Lower mean FA from regions of lFA were significantly associated with worse psychologic autonomy and communication ( 0.596, P .002) and worse emotional stability ( 0.581, P .002) at 1 year postinjury (On-line Fig 4). FA, AD, RD, and MD from regions of hFA were not significantly associated with HRQoL. neither of these approaches acknowledges nor has the ability to characterize the widely recognized substantial spatial variation in injury location, which is a principal feature of mTBI.33 As a result, these standard approaches will include areas with and without tissue injury in the imaging measures they study. Loss of Consciousness and Microstructural Abnormalities Loss of Consciousness and Microstructural Abnormalities Ten of 26 of subjects experienced loss of consciousness at the time of injury. Mean hFA and mean lFA values within 16 days of injury were not significantly different between those with and without loss of consciousness at the time of injury (t 0.904, P .375; t 0.923, P .365, respectively). Relationship between Microstructural Abnormalities and Functional Outcomes We therefore used a technique that specifically identifies abnormalities in each individual patient (EZ-MAP), without dilution by values from normal tissue (inevitable with group-level delineation). Regional White Matter Assessment. Regional Imaging Mea- sures and Cognitive Performance. Subjects with hFA in the left frontal and left temporal white matter performed better than those without hFA in these regions on tasks of attention at 1 year postinjury (t 2.985, P .008 and t 3.322, P .004, respec- tively) (Fig 2). We have previously demonstrated the robustness17 and appli- cation16 of the EZ-MAP to the detection of microstructural ab- normalities in patients with mTBI. In this study, we further dem- onstrate that the EZ-MAP method is highly effective in identifying brain pathology related to long-term outcomes. Similar to using the EZ-MAP, various other studies have used individual subject- level procedures33,34 to detect pathology in DTI datasets from patients with mTBI. Our approach differs in that we used regres- sion adjustment for covariates and EZ-MAP bootstrap resam- pling, to better characterize the population variance. Imaging measures summarized over brain subregions, such as the frontal lobe, surpassed whole-brain summary measures (eg, mean FA across all abnormal white matter voxels) in identifying relation- ships between DTI measures and functional outcomes. Although this result may be related to differences in the statistical approach between the whole-brain measures and subregion measures (cor- relation analysis versus dichotomous analysis, respectively), it po- tentially reveals an important phenomenon: Structure-function relationships are more effectively detected when measures are ex- tracted from delimited brain regions relevant to a particular func- Regional Imaging Measures and PCS. Subjects with hFA in the right thalamus white matter experienced fewer emotional postconcussion symptoms than did those without hFA in this region (t 0.398, P .003). Subjects with hFA in the left or right cerebellar hemisphere experienced fewer somatic postcon- cussion symptoms than did those without abnormality (t 3.365, P .003; t 3.38, P .003, respectively) (Fig 3A, -C). Regional Imaging Measures and HRQoL. There were no sig- nificant differences in HRQoL between individuals with and without hFA or lFA within individual regions. Functional Outcomes at 1 Year: Cognitive Function, HRQoL, and PCS in lFA (0.625, 0.567) regions than in hFA regions (0.517, 0.357) (P .001, P .001). AD was not significantly different between hFA (0.805) and lFA (0.740) regions. Loss of Consciousness and Microstructural Abnormalities Ten of 26 of subjects experienced loss of consciousness at the time of injury. Mean hFA and mean lFA values within 16 days of injury were not significantly different between those with and without loss of consciousness at the time of injury (t 0.904, P .375; t 0.923, P .365, respectively). Functional Outcomes at 1 Year: Cognitive Function, HRQoL, and PCS Figure 1A depicts a range of z scores for each of the 3 cognitive domains, with impairment defined as z score values of 1.5 SDs below the mean for the given domain. Figure 1B shows the prev- alence of symptom endorsement related to HRQoL according to dimension (social, psychological, and physical). Figure 1C, -D demonstrate the prevalence of symptom endorsement related to PCS and the percentage of those meeting the criteria for the diag- nosis of postconcussion syndrome based on the Rivermead Post Concussion Symptoms Questionnaire cutoff criterion.32 Preinjury cognitive ability was estimated through administra- tion of the Spot-the-Word test, a measure of reading achieve- ment, a “hold” ability that is resistant to the effects of brain in- jury.19 We found no difference between the 26 subjects and a subset of 18 controls (those for whom data were available) on the Spot-the-Word test (t 1.152, P .256), indicating that the subjects’ preinjury intellectual functioning was not significantly different from that of controls. There were no differences in age (t 1.353, P .184), sex (2 0.834, P .361), years of educa- tion (t 1.227, P .228), Spot-the-Word scores (t 0.019, P .985), and loss of consciousness at the time of injury (2 1.805, P .179) between the 26 subjects included in the analysis Microstructural Abnormalities at the Time of mTBI Using the procedures described above, we detected regions of hFA in 24/26 subjects (mean total volume 9398 L; maximum 27,660 L) and lFA in 25/26 subjects (mean total volume 6923 L, maximum 39,686 L). All subjects showed at least 1 region of hFA or lFA, though not all showed both. The mean hFA aver- aged across all subjects (0.641) was significantly higher than the mean lFA (0.278, P .001). MD and RD were significantly higher FIG 2. Associations between regional imaging measures and long- term cognitive outcomes. A, Subjects with high FA in the left frontal lobe perform signiﬁcantly better on tasks of attention at 1 year postinjury than do those without it (t 2.985, P .008). B, Subjects with high FA in left temporal lobe perform signiﬁcantly better on tasks of attention at 1 year postinjury than do those without it (t 3.524, P .002). 1988 Strauss Nov 2016 www.ajnr.org DISCUSSION This prospective, longitudinal study leverages individualized as- sessment of DTI16,17 to demonstrate associations between early imaging and 1-year mTBI outcomes. Imaging studies of mTBI outcomes generally use either a pri- ori ROI or group-level voxelwise comparisons of subjects and controls to extract imaging measures for study. Most important, 1988 Strauss Nov 2016 www.ajnr.org tion and not diluted by measures from regions not specifically FIG 3. Associations between regional imaging measures and long- term functional outcomes. A, Subjects with hFA in the right thalamus have signiﬁcantly fewer emotional postconcussion symptoms at 1 year than those without it (t 3.398, P .003). Of those with hFA in the right thalamus, only 2 subjects reported emotional postconcus- sion symptoms. The asterisk indicates an outlier. B, Subjects with hFA in the left cerebellar white matter have signiﬁcantly fewer somatic postconcussion symptoms at 1 year than those without hFA in the left cerebellum (t 3.365, P .003). Of those with hFA in the left cerebellum, only 1 subject reported somatic postconcussion symp- toms. The asterisk indicates an outlier. C, Subjects with hFA in the right cerebellar white matter have signiﬁcantly fewer somatic post- concussion symptoms at 1 year than do those without high FA in the right cerebellum (t 3.380, P .003). Of those with hFA in the left cerebellum, only 2 reported somatic postconcussion symptoms. proaches. Whereas hFA delineated at the group level might rep- resent damage to common areas injured across subjects, regions of abnormally hFA identified at the individual level might reflect subject-specific compensatory mechanisms that enhance diffu- sion anisotropy through structural or functional changes, such as myelination or increased synaptogenesis at the level of the den- dritic spine, which is mediated by actin polymerization.35 Nota- bly, studies have shown that short-term reversible increases in anisotropy develop with cognitive training, presumably through similar neuroplastic mechanisms.36 Relating structural changes to their functional consequences is important in understanding the import of imaging findings and characterizing injury effects in patients with TBI. Thus, the re- gional analyses we performed are most salient and reveal several interesting structure-function relationships. Notably, most liter- ature on neuroanatomic structure-function relationships focuses on gray matter. DISCUSSION However, white matter axons form the infrastruc- ture of distributed neural networks, which underlie the domains of higher functioning so commonly impacted by the white matter injury that follows TBI.37,38 We found significant regional associ- ations of frontal and temporal hFA with performance on tasks of attention, tested by using the Digit Span Forward, Continuous Performance Task, and switching of attention (digits) tasks; the latter 2 depend on processing speed and therefore index neural network function. Moreover, the frontal39-41 and temporal lobes42 both directly play an important role in attention. The sig- nificant association of hFA in the thalamus with emotional PCS is not surprising, given the susceptibility of the thalamus to mTBI and its role as the major relay network of the brain, with extensive limbic and prefrontal connectivity.43,44 We also identified associ- ations of cerebellar abnormalities with somatic PCS, including dizziness, nausea/vomiting, double vision, and blurry vision, which reflect the role of the cerebellum in balance and spatial orientation. FIG 3. Associations between regional imaging measures and long- term functional outcomes. A, Subjects with hFA in the right thalamus have signiﬁcantly fewer emotional postconcussion symptoms at 1 year than those without it (t 3.398, P .003). Of those with hFA in the right thalamus, only 2 subjects reported emotional postconcus- sion symptoms. The asterisk indicates an outlier. B, Subjects with hFA in the left cerebellar white matter have signiﬁcantly fewer somatic postconcussion symptoms at 1 year than those without hFA in the left cerebellum (t 3.365, P .003). Of those with hFA in the left cerebellum, only 1 subject reported somatic postconcussion symp- toms. The asterisk indicates an outlier. C, Subjects with hFA in the right cerebellar white matter have signiﬁcantly fewer somatic post- concussion symptoms at 1 year than do those without high FA in the right cerebellum (t 3.380, P .003). Of those with hFA in the left cerebellum only 2 reported somatic postconcussion symptoms FIG 3. Associations between regional imaging measures and long- term functional outcomes. A, Subjects with hFA in the right thalamus have signiﬁcantly fewer emotional postconcussion symptoms at 1 year than those without it (t 3.398, P .003). Of those with hFA in the right thalamus, only 2 subjects reported emotional postconcus- sion symptoms. The asterisk indicates an outlier. DISCUSSION B, Subjects with hFA in the left cerebellar white matter have signiﬁcantly fewer somatic postconcussion symptoms at 1 year than those without hFA in the left cerebellum (t 3.365, P .003). Of those with hFA in the left cerebellum, only 1 subject reported somatic postconcussion symp- toms. The asterisk indicates an outlier. C, Subjects with hFA in the right cerebellar white matter have signiﬁcantly fewer somatic post- concussion symptoms at 1 year than do those without high FA in the right cerebellum (t 3.380, P .003). Of those with hFA in the left cerebellum, only 2 reported somatic postconcussion symptoms. In addition to FA, which characterizes overall coherence of diffusion direction, we explored the utility of AD, RD, and MD. Preclinical experimental studies posit low AD as a marker for intra-axonal injury and high RD as a marker for transaxonal/ axolemmal injury and demyelination.4 We found that high RD, but not low AD, significantly correlated with worse outcomes. We may not have identified abnormally low AD as a correlate of out- comes because the expected decrease in AD may be masked by increased AD related to gliosis and edema, which evolve in the late acute setting, the timeframe during which our subjects underwent DTI (mean, 7 days).45 In a postmortem analysis of patients with multiple sclerosis, increased RD correlated with the severity of demyelination and decreased axonal attenuation, presumably be- cause the 2 are interrelated.46 We found that mean RD, but not mean AD, was significantly associated with memory and somatic autonomy in correlation analyses, consistent with this pattern. Because high RD may be a manifestation of transaxonal injury, as suggested by preclinical studies,47 it may indicate more severe and irreparable axonal pathology and therefore might serve as a better early predictor of long-lasting dysfunction. tion and not diluted by measures from regions not specifically supporting that function. Using the EZ-MAP approach, we found regions of hFA, in addition to regions of lFA, in almost all subjects. A minority of prior studies, most of which used group-level techniques to iden- tify abnormalities, also reported high FA and have attributed this finding to cytotoxic edema, altered myelin sheath water compo- sition, or inflammation. Thus, hFA has been considered an addi- tional biomarker for injury pathology due to mTBI.6,9,15 In con- trast, our use of individual-level delineation of abnormalities reveals associations of hFA with better long-term outcomes. AJNR Am J Neuroradiol 37:1983–91 Nov 2016 www.ajnr.org 19 Disclosures: Mimi Kim—RELATED: Grant: National Institutes of Health; UNRELATED: Consultancy: Lupus Foundation of America; Grants/Grants Pending: National Insti- tutes of Health. Richard B. Lipton—RELATED: Grant: National Institutes of Health R01; UNRELATED: Consultancy: serves as consultant and advisory board member or has received honoraria from Alder, Allergan, American Headache Society, Auto- nomic Technologies, Avanir Pharmaceuticals, Boehringer Ingelheim, Boston Scien- tiﬁc, Bristol Myers Squibb, Colucid Pharmaceuticals, Dr Reddy’s Laboratories, Elec- troCore, Eli Lilly, eNeura Therapeutics, Informa, Merck & Co, Novartis, Pﬁzer, Teva, and Vedanta; Grants/Grants Pending: receives research support from the National DISCUSSION These associations may reflect differences in the type of abnor- mality identified by using individual-level versus group-level ap- Several limitations of this study should be considered. Aniso- tropic voxels with 4.5-mm section thickness limit spatial resolu- tion and may lead to spuriously low FA, particularly when multi- ple crossing fibers are included within a single voxel.48 Nonetheless, this limitation applies equally to mTBI and control subjects, who underwent identical imaging procedures; therefore, we do not expect a systematic bias to result. Despite the standard- ization of imaging, this limitation may partially account for the absence of significant associations between the presence of low FA and outcomes on the regional level. Moreover, the nature of the interdependence of the various DTI parameters should be noted because for instance, increased AD and RD might lead to high MD but not low FA, and an increase in AD alone would affect MD and FA. As such, primary identification of abnormality based on a summary measure such as FA may ultimately be of less value than a measure such as RD and may serve as the basis for future study. In our study, of the 39 subjects who underwent acute imaging, 26 completed PCS and HRQoL assessment (33% attrition) and 20 were available for cognitive testing (49% attrition). Attrition in mTBI cohorts is a particular challenge to research in head trauma because subjects are disinclined to follow-up either because they do not perceive themselves as injured or because of injury-related dysfunction and stress. The rate of attrition we encountered is well within the range reported in mTBI studies (35%–88%),49-51 and we did not identify demographic or injury-severity differences between those who did or did not complete follow-up. Nonethe- less, the possibility of bias due to selective attrition cannot be completely excluded. PCS and HRQoL are assessed by self-report, and studies have shown that symptoms related to postconcussion syndrome are common in other disease conditions, such as chronic pain,52 and in healthy controls.53 Thus, a further poten- tial limitation is that symptom measures were not tested in the control sample. DISCUSSION We addressed this issue by excluding subjects with preexisting conditions that might cause symptoms, includ- ing prior head injury, and by using tools that have been exten- sively validated and widely applied to TBI.20,21 Ultimately, the shortcomings of subjective assessment tools underscore the im- portance of other, more objective indicators of impairment that might not otherwise be appreciated. This limitation is thus a ma- jor motivation for the current study. Finally, a future step might be to evaluate similar effects in a larger cohort of patients with mTBI and in a group of healthy controls. Institutes of Health: PO1AG003949 (Program Director), PO1AG027734 (Project Leader), RO1AG025119 (Investigator), RO1AG022374–06A2 (Investigator), RO1AG034119 (Investigator), RO1AG12101 (Investigator), 23NS05140901A1 (Mentor), and K23NS47256 (Mentor), and the National Headache Foundation; Payment for Lectures (including service on Speakers Bureaus): lecture honoraria for Continuing Medical Education–accredited lectures from the American Headache Society, the American Academy of Neurology, and the Headache Cooperative of New England; Payment for Development of Educational Presentations: American Headache So- ciety, Comments: Chronic Migraine Education Program; Stock/Stock Options: eNeura Therapeutics. Michael L. Lipton—UNRELATED: Expert Testimony: Various (on behalf of defendants and plaintiffs); Grants/Grants Pending: Resurrecting Lives Foundation, Comments: grant for research on imaging in blast TBI; Patents (planned, pending or issued): image-processing methodology (patent pending); Royalties: Springer (MRI physics book); Other: the Dana Foundation, Comments: grant for study of imaging in sports TBI. *Money paid to the institution. REFERENCES 1. Ruff R. Two decades of advances in understanding of mild trau- matic brain injury. J Head Trauma Rehabil 2005;20:5–18 CrossRef Medline 2. Bigler ED. Neuropsychological results and neuropathological find- ings at autopsy in a case of mild traumatic brain injury. J Int Neu- ropsychol Soc 2004;10:794–806 Medline 3. Johnson VE, Stewart W, Smith DH. Axonal pathology in traumatic brain injury. Exp Neurol 2013;246:35–43 CrossRef Medline 3. Johnson VE, Stewart W, Smith DH. Axonal pathology in traumatic brain injury. 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https://openalex.org/W4230135829	https://zenodo.org/records/1938471/files/article.pdf	English	null	UNAUTHORISED PUBLICITY.	Lancet	1,902	public-domain	1,639	1080 were 6386. The detailed figures are : Bombay city, 56, as against 37 ; Bombay Presidency, 5424, as against 3929 ; Madras Presidency, 264 ; Bengal, 14, as against 11 ; United Provinces, 416, as against 288; Punjab, 101, as against 55 ; Mysore, 785, as against 811 ; Hyderabad State, 242, as against 45 ; and Berar, 242, as against 97. Only six deaths were recorded in Calcutta. These figures give indications of what may be expected to happen and taken in comparison with the figures for last year present a gloomy prospect for the ensuing cold season. were 6386. The detailed figures are : Bombay city, 56, as against 37 ; Bombay Presidency, 5424, as against 3929 ; Madras Presidency, 264 ; Bengal, 14, as against 11 ; United Provinces, 416, as against 288; Punjab, 101, as against 55 ; Mysore, 785, as against 811 ; Hyderabad State, 242, as against 45 ; and Berar, 242, as against 97. Only six deaths were recorded in Calcutta. These figures give indications of what may be expected to happen and taken in comparison with the figures for last year present a gloomy prospect for the ensuing cold season. soll." It seems to me that their objections are unanswerable. Both these authors point to the discrepancy existing between Professor Runeberg’s gloomy view of the fatal influence of syphilis, and the leniency of his practical proposals for the admission of syphilitics to life assurance. This incongruity is the natural consequence of the vague and arbitrary character of his computations. Perhaps the recent investi- gation by Martius and Knabe will, if carried out on a sufficiently large scale, lead to more useful results. Possibly Dr. Weber’s estimation of the influence of syphilis on the death-rate comes nearer the mark-at least, with regard to British life assurance offices. For the practical purposes of life assurance companies such statistics are worthless. As Dr. Weber has adopted Professor Runeberg’s " primitive mode of investigation " his figures only afford another instance of syphilis statistics as they should not be." " year present gloomy prospect ensuing " The attention of Government has been drawn to the advisability of improving the arrangements for the supply of nurses to the hospitals in Calcutta." So runs a recent notice. 1080 The extensions of the Presidency General Hospital and of the Medical College, Eden, and other hospitals have necessitated a change in the system hitherto in vogue according to which the nursing arrangements have been chiefly managed by a committee of ladies. It is now proposed to form a large influential and repre- sentative committee from which sub-committees will be formed to advise on matters of detail. There will still be a committee of ladies who will visit the hospitals and deal with all matters affecting the appointment and control of the nursing staff. The financial assistance of the Government will be continued. Rules for management and control have been drawn up and will come into force at an early date. The previous committee of ladies has rendered great services for a period of over 40 years. Compared with administrations at home it is to be noticed that the chief medical officer of each hospital is well represented on the committees and has considerable power over the nurses of his institution. The chief medical officer to a Government hospital in India is a much more important man than the senior member of a medical staff at home, and this greater power applies to the whole administration of his hospital. syphilis they I am, Sirs, yours faithfully, , , yours faithfully, Welbeck-street, W., Oct. 13th, 1902. GEORGE OGILVIE. , , y faithfully, Welbeck-street, W., Oct. 13th, 1902. GEORGE OGILVIE. UNAUTHORISED PUBLICITY. To the Editors of THE LANCET. SIRS,-I have received a marked copy of the Herald of the Golden Age which seems to be a vegetarian newspaper ; in it I find an opinion attributed to me set out in a very objectionable way, with my titles and appointments. Would you allow me to say that this has been done without my authority ? I believe that some time ago I received a card purporting to be from a medical practitioner, asking me my opinion upon certain questions with respect to diet. This I answered, and from this a portion has been picked out and printed as above described. I am, Sirs, yours faithfully, hfully, ROBERT SAUNDBY. am, Sirs, yours faithfully, Birmingham, Oct. 13th, 1902. ROBERT SAUNDBY. am, Sirs, yours faithfully, Birmingham, Oct. 13th, 1902. ROBERT am, Sirs, yours faithfully, Birmingham, Oct. 13th, 1902. ROBERT SAUNDBY. power applies hospital. Regarding the sanitary state of Madras city the following recent remarks of the Government are to the point :- p One of the most unsatisfactory features disclosed by the report is the further increase in the already high death-rate of the city of Madras, where the mortality was no less than 55’5 per mille, the mean of the previous five years being 40’9. Colonel King has referred to the imperfections of the existing water-supply of the city, and the attention of the municipal commissioners will be drawn to his criticisms. The present drainage, too, is defective and inadequate; but work has already been commenced on a new system which is estimated to cost Rs.31,81,000. The Governor-in-Council fears, however, that even the improvement of the drainage and the water- supply will not be sufficient to place the health of the city in a satis- factory condition unless the congested areas in Black Town and Triplicane are thoroughly opened up by means of wide streets which will allow the prevailing winds to penetrate into quarters where there is now no proper ventilation or access of fresh air. The importance of an improvement scheme of this nature has long been recognised, but it would be impossible for the municipality to carry it out without a large measure of financial assistance from provincial ur Imperial revenues, or both. S t 19th NOTES FROM INDIA. (FROM OUR SPECIAL CORRESPONDENT,) (FROM OUR SPECIAL CORRESPONDENT,) The lTig7t Infantile Mortality in Bombay and Calcutta.- The Development of another Plague Outbreak.-The Supply of Nurses for the Calcutta Hospital. -Sanitation in -Madras. ALTHOUGH attention has been drawn to the high infantile mortality in the cities of Calcutta and Bombay it must not be supposed that these cities alone record a high death-rate. In Bombay the high infantile mortality is attributed by the health officer to the poverty of the people. Mothers are compelled to work in the mills and elsewhere and the infants are insufficiently fed or are, a.s too often happens, drugged so that they can be left alone. Again, the off- spring of immature mothers are mostly weaklings and having low vitality readily contract disease. Dr. Turner questions the explanation of the Government that a con- siderable proportion of the working-classes migrate to their homes in the country districts every year, causing the deaths of children born outside Bombay to be put down to the mortality of that city. Although social con- ditions are beyond the control of the municipality it is thought that something might be done to mitigate the evil. Free medical relief, the appointment of a nurse or medical woman for each ward, and the improvement of the milch-cattle stables are the measures suggested. The establishment of a ereehe is also under considera- tion. Although there are no large factories in Calcutta where women are employed in great numbers the infantile mortality is equally high, the deaths of infants within a few days of birth being excessively numerous. Ignorance and carelessness have probably as much to do with the high mortality as poverty, and one cause of a considerable number of deaths lies in the custom of rubbing earth and ashes daily into the navel, leading to tetanus. Premature births are numerous, but probably so- called debility at birth is the heading under which most deaths are recorded. In the city of Madras the infantile mortality is given as 295 per 1000-an estimate which possibly does not err on the side of excess. evenues, b Sept.19th. revenues, Sept.19th. 1080 1080 (FROM OUR OWN CORRESPONDENT.) Opening of the .Winter Session of, the Medical Faculty University College, Liverpool, by Sir Frederick 2’rev es, Bart., K. C. V. 0., C.B. , , , THE ceremony of opening the winter session of the medical faculty of University College, which took place on Oct. 10th at the Philharmonic Hall, was an event of more than ordinary interest by reason of the presence of Sir Frederick Treves who delivered the address to the students of the faculty. The hall was filled by a large and distinguished gathering. A feature of the proceedings was a procession through the main aisle to the platform of members of the governing body and teaching staff, former students of the medical faculty, and members of the staffs of the hospitals of the city. In one of the galleries a number of nurses belong- ing to the various hospitals occupied seats in the front row. With the exception of the governors of University College and a few guests, amongst whom was included the Lord Mayor, all wore academic costume. Sir Frederick Treves, in his address, described the symptoms of ailments within the human body as the physician’s best friends, as the patient’s most precious safeguards, and as the most beneficent efforts of nature to repair whatever might have gone wrong. The speaker illustrated his meaning by saying that when para- sites attacked the lungs there were no symptoms to give notice of the attack, but nature immediately endeavoured to expel possibly The mortality from plague throughout India is rising rapidly. For the week ending Sept. 13th it was 7544, as compared with 5550 for the previous seven days. For the corresponding week in 1901 the deaths from plague
https://openalex.org/W2801838739	https://research-information.bris.ac.uk/files/161870879/Seropian_et_al_2018_Journal_of_Geophysical_Research_Solid_Earth.pdf	English	null	The Gravitational Stability of Lenses in Magma Mushes: Confined Rayleigh‐Taylor Instabilities	Journal of geophysical research. Solid earth	2,018	cc-by	12,445	Seropian, G., Rust, A., & Sparks, S. (2018). The gravitational stability of lenses in magma mushes: confined Rayleigh-Taylor instabilities. Journal of Geophysical Research: Solid Earth, 123(5), 3593-3607. https://doi.org/10.1029/2018JB015523 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1029/2018JB015523 Link to publication record on the Bristol Research Po Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1029/2018JB015523 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via AGU at https://agupubs.onlinelibrary.wiley.com/doi/abs/10.1029/2018JB015523. Please refer to any applicable terms of use of the publisher. Seropian, G., Rust, A., & Sparks, S. (2018). The gravitational stability of lenses in magma mushes: confined Rayleigh-Taylor instabilities. Journal of Geophysical Research: Solid Earth, 123(5), 3593-3607. https://doi.org/10.1029/2018JB015523 Seropian, G., Rust, A., & Sparks, S. (2018). The gravitational stability of lenses in magma mushes: confined Rayleigh-Taylor instabilities. Journal of Geophysical Research: Solid Earth, 123(5), 3593-3607. https://doi.org/10.1029/2018JB015523 ©2018. The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. G. Seropian1,2 , A. C. Rust1, and R. S. J. Sparks1 G. Seropian1,2 , A. C. Rust1, and R. S. J. Sparks1 • Melt-mush Rayleigh-Taylor instabilities are generally laterally conﬁned, which reduces the growth rate 1School of Earth Sciences, University of Bristol, Bristol, UK, 2Now at Department of Geological Sciences, University of Canterbury, Christchurch, New Zealand • The conﬁned instability growth rate only depends on the mush viscosity, melt lens diameter, and density diﬀerence Abstract In the current paradigm, magma primarily exists in the crust as a crystalline mush containing distributed melt lenses. If a melt-rich (or ﬂuid) lens is less dense than the overlying mush, then Rayleigh-Taylor (RT) instabilities will develop and could evolve into spheroids of ascending melt. Due to contrasting melt-mush rheologies, the theoretical RT instability wavelength can be orders of magnitude larger than the magmatic system. We explored how this conﬁnement aﬀects the gravitational stability of melt lenses through laboratory experiments with pairs of liquids with one layer much thinner and up to 2.2 ⋅105 times less viscous than the other; we extended the viscosity ratio to 106 with linear stability analysis. We found the growth rate of a bounded RT instability is approximately Δ𝜌gD 6𝜋𝜇2 , where Δ𝜌is the diﬀerence in density between the ﬂuids, g is gravity, D is the container diameter, and 𝜇2 is the viscosity of the thicker viscous layer. This diﬀers from the unbounded case, where the growth rate also depends on the thickness and viscosity of the thin, low-viscosity layer. Applying the results to melt lenses in magmatic mushes, we ﬁnd that for the ranges of expected rheologies, the timescales for development of the instability, and the volumes of packets of rising melt generated span very wide ranges. They are comparable with the frequencies and sizes of volcanic eruptions and episodes of unrest and so suggest that RT instabilities in mush systems can cause episodic volcanism. • Mush rheology is a key control on size and frequency of eruptions related to buoyancy instabilities Correspondence to: G. Seropian, gs16066@my.bristol.ac.uk Received 22 JAN 2018 Accepted 14 APR 2018 Accepted article online 30 APR 2018 Published online 2 MAY 2018 University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Citation: Seropian, G., Rust, A. C., & Sparks, R. S. J. (2018). The gravitational stability of lenses in magma mushes: Conﬁned Rayleigh-Taylor instabilities. Journal of Geophysical Research: Solid Earth, 123, 3593–3607. https://doi.org/10.1029/2018JB015523 1. Introduction A major challenge of modern volcanology concerns subsurface magma transport and accumulation. Concep- tual models are emerging that depict subsurface systems as large uneruptible crystalline networks (mushes) containing heterogeneously distributed pockets of eruptible magma and exsolved volatiles that can extend deep in the crust and down to the mantle (e.g., Bachmann & Huber, 2016; Cashman et al., 2017). The dynamics of igneous mush systems has become a dominant theme in contemporary magma physics (e.g., Bergantz et al., 2017; Dufek & Bachmann, 2010; Parmigiani et al., 2014) and a key feature of interpretations of geophysical, geochemical, and petrological data (e.g., Jaxybulatov et al., 2014; Putirka, 2017). One aspect of igneous mush dynamics is the development of buoyancy instabilities related to intrusion of new magma or segregation of melt- or ﬂuid-rich layers within a mush, leading to magma or ﬂuid ascent through the mush. Magmas, melts, and ﬂuids are commonly less dense than the overlying mush; therefore, Rayleigh-Taylor (RT) instabilities develop naturally wherever buoyant layers form. However, for some condi- tions (e.g., suﬃciently high mush viscosity) the growth rate may be suﬃciently slow that other processes (e.g., solidiﬁcation due to cooling) dominate. The viscosity contrast between a buoyant layer and an igneous mush is typically very large. For example, if we consider mushes with eﬀective viscosities from 1013 Pa⋅s for melt-rich mush (∼40% melt) to 1017 Pa⋅s for melt-poor mush (<10% melt, Costa et al., 2009; Lejeune & Richet, 1995), and melt (magma) lenses with viscosities of 1−105 Pa⋅s (basalt to wet rhyolite), then the viscosity ratios are of order 108–1017. There is a the- ory for RT instabilities for inﬁnite horizontal layers with very high viscosity ratios (Whitehead & Luther, 1975), which has been veriﬁed in experiments with viscosity ratios of up to a few hundred. Although never tested experimentally, this theory should be applicable to the much higher viscosity ratios expected in lens-mush magmatic systems. However, a consequence of the high viscosity ratios is that the horizontal dimension of a magma reservoir is commonly much less than the theoretical fastest-growing wavelength assuming an inﬁ- nite horizontal layer (Figure 1). Thus, it is necessary to consider cases in which the buoyant layer is conﬁned by boundaries separated by lengths much less than the optimum wavelength. The purpose of this study ©2018. The Authors. RESEARCH ARTICLE G. Seropian1,2 , A. C. Rust1, and R. S. J. Sparks1 1. Introduction 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. 3593 2.1. RT Instabilities A RT instability occurs when a dense ﬂuid overlies a less dense ﬂuid. Such instabilities can arise in magmatic systems when a layer of buoyant melt is trapped within a denser crystalline mush. The full dynamics of RT instability are too complex to be described analytically. Nonetheless, linear stability analysis (LSA) provides good insight into the early stage, during which small initial perturbations of the interface grow exponentially (Waddell et al., 2001). Mush-melt RT instabilities involve very high viscosity ratios. The relevant low Reynolds number RT instability formalism for such high viscosity contrasts was developed by Whitehead and Luther (1975). They analyzed the case of a thin layer of light Newtonian ﬂuid (density 𝜌1, viscosity 𝜇1) below a deep layer of denser Newtonian ﬂuid (𝜌2, 𝜇2); both ﬂuids are horizontally inﬁnite (Figure 1). In this scenario, for a free slip boundary condition (BC) below the thin layer, and a large viscosity ratio (𝜀= 𝜇2 𝜇1 ≫1), there exists a fastest-growing mode, the critical mode, with wavelength 𝜆c and growth rate nc given by 𝜆c = 4𝜋 2.88h𝜀1∕3, (1) gΔ𝜌h 𝜆c = 4𝜋 2.88h𝜀1∕3, (1) nc = 0.232gΔ𝜌h 𝜇2 𝜀1∕3, (2) 𝜆c = 4𝜋 2.88h𝜀1∕3, (1) (1) nc = 0.232gΔ𝜌h 𝜇2 𝜀1∕3, (2) (2) where h is the thickness of the thin layer and Δ𝜌= \|𝜌2 −𝜌1\| is the density diﬀerence. It is also assumed that the wave-like perturbations are small, with amplitude less than ∼0.4𝜆c. These equations have been modiﬁed to investigate the thickening of a buoyant layer at a ﬁxed rate (i.e., constant dh dt ; de Bremond d’Ars et al., 1995). The theory has been tested and veriﬁed experimentally and numerically for viscosity ratios up to 𝜀= (102) (de Bremond d’Ars et al., 1995; Whitehead & Luther, 1975). where h is the thickness of the thin layer and Δ𝜌= \|𝜌2 −𝜌1\| is the density diﬀerence. It is also assumed that the wave-like perturbations are small, with amplitude less than ∼0.4𝜆c. These equations have been modiﬁed to investigate the thickening of a buoyant layer at a ﬁxed rate (i.e., constant dh dt ; de Bremond d’Ars et al., 1995). The theory has been tested and veriﬁed experimentally and numerically for viscosity ratios up to 𝜀= (102) (de Bremond d’Ars et al., 1995; Whitehead & Luther, 1975). Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 Figure 1. Sketch of an eruptible melt-rich layer within a much more viscous crystal mush. Rayleigh-Taylor instabilities arise due to density diﬀerence; however, the theoretical wavelength of instability 𝜆c may be much larger than the melt layer diameter D. Figure 1. Sketch of an eruptible melt-rich layer within a much more viscous crystal mush. Rayleigh-Taylor instabilities arise due to density diﬀerence; however, the theoretical wavelength of instability 𝜆c may be much larger than the me layer diameter D. is to address this issue through experimental and theoretical approaches and then to apply the results to understanding RT instabilities in igneous mushes. The suggestion that RT instabilities can control the frequency of volcanic eruptions is explored. is to address this issue through experimental and theoretical approaches and then to apply the results to understanding RT instabilities in igneous mushes. The suggestion that RT instabilities can control the frequency of volcanic eruptions is explored. SEROPIAN ET AL. SEROPIAN ET AL. Journal of Geophysical Research: Solid Earth 2.1. RT Instabilities The normalized function n(K) nc = 𝜀2∕3 0.232K [ cosh K−1+𝜀(sinh K−K) sinh K+K+2𝜀cosh K+𝜀2(sinh K−K) ] is plotted in Figure 2 for 𝜀= 106. It reaches a maximum at Kc = 4𝜋h 𝜆c , such that n(Kc) = nc (equation (2)). For K ≫Kc and 𝜀≫1, the growth rate decreases with increasing K, according to The normalized function n(K) nc = 𝜀2∕3 0.232K [ cosh K−1+𝜀(sinh K−K) sinh K+K+2𝜀cosh K+𝜀2(sinh K−K) ] is plotted in Figure 2 for 𝜀= 106. It reaches a maximum at Kc = 4𝜋h 𝜆c , such that n(Kc) = nc (equation (2)). For K ≫Kc and 𝜀≫1, the growth rate decreases with increasing K, according to n(K) nc ≈ 1 0.232𝜀1∕3 K−1. (4) (4) If the RT instability occurs in a container with horizontal dimension D < 𝜆c, the tank walls will prevent the growth of all modes with K < KD = 4𝜋h D . Graphically, any mode within the gray shaded area in Figure 2 is inaccessible. Thus, in the presence of lateral boundaries with KD > Kc (i.e., 𝜆c > D), the fastest allowed growth rate is n(KD). It is useful to deﬁne a conﬁnement parameter 𝛽= 𝜆c D , such that the system is conﬁned for 𝛽> 1. Using the deﬁnitions of 𝛽and 𝜆c, we can rewrite KD = 2.88𝛽𝜀−1∕3, therefore yielding If the RT instability occurs in a container with horizontal dimension D < 𝜆c, the tank walls will prevent the growth of all modes with K < KD = 4𝜋h D . Graphically, any mode within the gray shaded area in Figure 2 is inaccessible. Thus, in the presence of lateral boundaries with KD > Kc (i.e., 𝜆c > D), the fastest allowed growth rate is n(KD). It is useful to deﬁne a conﬁnement parameter 𝛽= 𝜆c D , such that the system is conﬁned for 𝛽> 1. Using the deﬁnitions of 𝛽and 𝜆c, we can rewrite KD = 2.88𝛽𝜀−1∕3, therefore yielding n(KD) = nc 0.668𝛽= Δ𝜌gD 4𝜋𝜇2 . (5) (5) This suggests that, in the case of a laterally conﬁned RT instability, the growth rate is independent of both the thin layer thickness h and the viscosity ratio 𝜀, though equation (5) requires experimental validation. 2.1. RT Instabilities This suggests that, in the case of a laterally conﬁned RT instability, the growth rate is independent of both the thin layer thickness h and the viscosity ratio 𝜀, though equation (5) requires experimental validation. 2.2. Are Mush-Melt Systems Conﬁned? 2.1. RT Instabilities Equations (1) and (2) assume that both layers are horizontally inﬁnite, but for a ﬁnite domain, for suﬃciently large h and 𝜀, the theoretical fastest-growing wavelength, 𝜆c, will be greater than the extent of the layers and so is not physically possible. Whether the domain is ﬁnite or inﬁnite, all perturbations of all wavelengths will grow but the instability will develop with the wavelength that grows fastest. So we need to consider the growth rate of all modes with wavelengths equal to or smaller than the horizontal extent of the layers. In general the growth rate of a mode with wavelength 𝜆and dimensionless wave number K = 4𝜋h 𝜆is (Whitehead & Luther, 1975): n = gΔ𝜌h K𝜇1 [ cosh K −1 + 𝜀(sinh K −K) sinh K + K + 2𝜀cosh K + 𝜀2(sinh K −K) ] . (3) (3) 3594 SEROPIAN ET AL. SEROPIAN ET AL. Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 Figure 2. Normalized growth rate n(K) nc as a function of dimensionless wave number K, for 𝜀= 106. Any mode with K < KD(gray area) cannot form because its wavelength is greater than the diameter of the container. As an example we illustrate this for KD = 0.13, which corresponds to h D = 0.01. For this case, the critical mode (Kc) cannot form. For large K, n(K) nc = 1 0.232 𝜀−1∕3K−1 = 0.0431K−1 (see text for details). Figure 2. Normalized growth rate n(K) nc as a function of dimensionless wave number K, for 𝜀= 106. Any mode with K < KD(gray area) cannot form because its wavelength is greater than the diameter of the container. As an example we illustrate this for KD = 0.13, which corresponds to h D = 0.01. For this case, the critical mode (Kc) cannot form. For large K, n(K) nc = 1 0.232 𝜀−1∕3K−1 = 0.0431K−1 (see text for details). Figure 2. Normalized growth rate n(K) nc as a function of dimensionless wave number K, for 𝜀= 106. Any mode with K < KD(gray area) cannot form because its wavelength is greater than the diameter of the container. As an example we illustrate this for KD = 0.13, which corresponds to h D = 0.01. For this case, the critical mode (Kc) cannot form. For large K, n(K) nc = 1 0.232 𝜀−1∕3K−1 = 0.0431K−1 (see text for details). Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 Table 1 Parameters Used for a Silicic, an Andesitic, and a Basaltic System Symbol Description Value used Unit − − Silicic Andesitic Basaltic − 𝜇1 Melt shear viscosity 105 3 ⋅102 10 Pa⋅s 𝜇2 Mush shear viscosity 1014 1014 1014 Pa⋅s 𝜀 Viscosity ratio 109 3 ⋅1011 1013 − 𝜌1 Melt density 2,300 2,500 2,800 kg/m3 Δ𝜌a Density diﬀerence 300 300 300 kg/m3 Note. 𝜇1, Scaillet et al. (1998) & Murase and McBirney (1973); 𝜇2, Lejeune and Richet (1995) & Caricchi et al. (2007); 𝜌1, Murase and McBirney (1973) & Annen et al. (2006); Δ𝜌, Whitney and Stormer (1985). aWe keep Δ𝜌= 300 kg/m3 constant as density variations are negligible compared to viscosity variations. Note. 𝜇1, Scaillet et al. (1998) & Murase and McBirney (1973); 𝜇2, Lejeune and Richet (1995) & Caricchi et al. (2007); 𝜌1, Murase and McBirney (1973) & Annen et al. (2006); Δ𝜌, Whitney and Stormer (1985). aWe keep Δ𝜌= 300 kg/m3 constant as density variations are negligible compared to viscosity variations. in magma mushes because the mechanical constraints from the side boundaries will prevent the growth of the critical mode with wavelength 𝜆c. Instead, we expect the dynamics of the conﬁned RT instability to be given by the fastest mode whose wavelength ﬁts within the layer width. Therefore, we need a modiﬁed theory for RT instability that accounts for wall eﬀects. in magma mushes because the mechanical constraints from the side boundaries will prevent the growth of the critical mode with wavelength 𝜆c. Instead, we expect the dynamics of the conﬁned RT instability to be given by the fastest mode whose wavelength ﬁts within the layer width. Therefore, we need a modiﬁed theory for RT instability that accounts for wall eﬀects. 2.2. Are Mush-Melt Systems Conﬁned? The application of equations (1) and (2) to magmatic systems requires that melt layers can be considered horizontally inﬁnite. To assess the condition for which this assumption is valid, we compute the conﬁnement parameter 𝛽= 𝜆c D for natural magmatic systems. A system will be conﬁned (or “clipped”; Burgisser & Bergantz, 2011) for 𝛽> 1, but it can be considered unconﬁned when 𝛽≤1. We examine the conﬁnement of three example scenarios: (a) a silicic, (b) an andesitic, and (c) a basaltic system, with viscosities and densities as indicated in Table 1. For each of the three systems, we calculate 𝛽for a range of melt layer widths (diameters if a circular lens) 500 ≤D ≤2⋅104 m and heights 0.1 ≤h ≤1, 000 m (Figure 1), whichencompasstypicalvaluesassumedintheoreticalmodels(e.g.,Annenetal.,2015;Bachmann&Bergantz, 2004) and consistent with estimates from geophysical surveys of natural systems (e.g., Lees, 1992; Tarasewicz et al., 2012). The majority of mush-melt systems are laterally conﬁned (Figure 3). In fact, for the rheologies explored (Table 1), even an extremely wide (D = 20 km) layer of silicic (viscous) melt would be conﬁned if thicker than 4 m. This result suggests that we should not simply apply equations (1) and (2) to typical lenses SEROPIAN ET AL. SEROPIAN ET AL. 3595 Journal of Geophysical Research: Solid Earth 3.2. Experimental Methods 3.2. Experimental Methods We use two tank sizes to investigate the eﬀect of lateral conﬁnement. All tanks are made of transparent Perspex and are cylindrical to avoid corner eﬀects. p We use two tank sizes to investigate the eﬀect of lateral conﬁnement. All tanks are made of transpar Perspex and are cylindrical to avoid corner eﬀects. The narrowest tanks have a diameter D = 5.00 ± 0.02 cm and height H = 40.00 ± 0.05 cm (Figure 4). Three tanks of this geometry were made in order to eﬃciently run concurrent experiments. The conﬁnement factors (𝛽= 𝜆c D ) for experiments in these tanks are 10 ≤𝛽≤210; hence, wall eﬀects are likely to be very important in all experiments with D = 5 cm. We conducted experiments in two temperature-controlled rooms, at 18∘C (laboratory) and 8∘C (cold room), to sample a wider range of viscosity ratios. We ﬁrst pour more than 30 cm of glucose syrup into the tank, cover the top of the tank, and leave it to rest until all the air bubbles have escaped. The uppermost part of the syrup usually dehydrates and stiﬀens. We thus remove this stiﬀlayer a few minutes before starting the experiment. This is long enough for the disturbed interface to ﬂatten but short enough to avoid signiﬁcant drying. The experiment is started by delicately pouring dyed K2CO3 solution on top of the glucose layer. Pouring takes a few seconds, which is much faster than the time for an RT instability to develop (always >3 min). This con- ﬁguration (viscous ﬂuid underneath) is ﬂipped compared to magmatic systems with a buoyant lens under a more viscous mush. Nonetheless, the dynamics of the instability will be identical because the driving force, buoyancy, is independent of which ﬂuid is on top of the other; only the density diﬀerence Δ𝜌is important (Whitehead & Luther, 1975). All experiments are recorded with a ﬁxed camera. In most experiments, there is air directly above the K2CO3 solution, forming a free-slip BC above. To check the inﬂuence of the BC, we repeated some experiments with a no-slip upper BC by placing a circular perspex lid on top of the K2CO3 layer just after pouring it. The lid diameter is 0.4 mm less than the tank to allow air escape during placement. 3. Materials and Methods To model mush-melt RT instabilities, we perform high viscosity contrast and laterally bounded experiments in cylindrical tanks, at low Reynolds number. We use glucose syrup (42DE-GL0106 from Ragus) as our viscous ﬂuid and aqueous potassium carbonate (K2CO3) solutions as our low-viscosity ﬂuids. Physical properties of these two ﬂuids are summarized in Table 2 at both 18 and 8∘C. Both ﬂuids are Newtonian, and viscosity ratios in our experiments are in the interval 4.7 ⋅104 ≤𝜀≤2.2 ⋅105, a range which extends previous experimental data to much higher ratios. Viscosity measurements were performed on a HAAKE RheoStress 1 rheometer (Thermo Fisher Scientiﬁc), with a concentric cylinders sensor system. Glucose viscosity at 8∘C was too high to be measured directly as the rheometer was not calibrated to such high values. Instead, we measured viscosity from 18 to 28∘C, and, assuming an Arrhenius model, we ﬁtted an exponential curve through these data and extrapolated it down to 8∘C. We measured glucose density via Archimedes’ principle, by weighing a calibrated 10-cm3 sinker (Mettler Toledo) in both air and glucose. The very low viscosity of the K2CO3 solutions allowed us to measure density by weighing 200 ml of solution in a volumetric ﬂask. Figure 3. Conﬁnement factor for a silicic, an andesitic, and a basaltic system. The red lines indicate where 𝛽= 1, whereas the black lines show 𝛽= 100. The systems are conﬁned for 𝛽> 1. Figure 3. Conﬁnement factor for a silicic, an andesitic, and a basaltic system. The red lines indicate where 𝛽= 1, whereas the black lines show 𝛽= 100. The systems are conﬁned for 𝛽> 1. 3596 SEROPIAN ET AL. Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth The experimental procedure is identical to the narrow tank case, with the exception that we pour the K2CO3 solution through a nozzle with seventy-two 1.5-mm-diameter holes to reduce disturbance of the interface. Again, we conduct experiments at both 18 and 8∘C. We, however, did not run any no-slip upper BC experiment with the wide tank. 3.3. Linear Stability Analysis To complement our experimental investigation, we perform an linear stability analy- sis (LSA), based on the work of Sweeney et al. (2013). They theoretically investigated RT instabilities in a narrow, ﬁnite cylinder (no-slip BC), but their published results cannot be directly applied to our scenario for two reasons. First, they only examined cases where the two ﬂuid layers have equal thicknesses, and second the viscosity ratiosconsideredaretoolow(𝜀≤102).Weusetheirnumericalroutinestoreproduce our experimental conditions and to extend results to higher 𝛽values than achieved in the laboratory. Full technical details can be found in Sweeney et al. (2013). T AL. 3597 3.2. Experimental Methods We perform similar experiments in a wide tank with diameter D = 28.70 ± 0.05 cm and height H = 39.20±0.05 cm, achieving 1.2 ≤𝛽≤6.9. Although 𝛽> 1, we expect wall eﬀects to be small and our results to approach the theoretical predictions of Whitehead and Luther (1975). The experimental procedure is identical to the narrow tank case, with the exception that we pour the K2CO3 solution through a nozzle with seventy-two 1.5-mm-diameter holes to reduce disturbance of the interface. Again, we conduct experiments at both 18 and 8∘C. We, however, did not run any no-slip upper BC experiment with the wide tank. Figure 4. Diagram of a narrow tank experimental setup. We perform similar experiments in a wide tank with diameter D = 28.70 ± 0.05 cm and height H = 39.20±0.05 cm, achieving 1.2 ≤𝛽≤6.9. Although 𝛽> 1, we expect wall eﬀects to be small and our results to approach the theoretical predictions of Whitehead and Luther (1975). The experimental procedure is identical to the narrow tank case, with the exception that we pour the K2CO3 solution through a nozzle with seventy-two 1.5-mm-diameter holes to reduce disturbance of the interface. Again, we conduct experiments at both 18 and 8∘C. We, however, did not run any no-slip upper BC experiment with the wide tank. 3.3. Linear Stability Analysis To complement our experimental investigation, we perform an linear stability analy- sis (LSA), based on the work of Sweeney et al. (2013). They theoretically investigated RT instabilities in a narrow, ﬁnite cylinder (no-slip BC), but their published results cannot be directly applied to our scenario for two reasons. First, they only examined cases where the two ﬂuid layers have equal thicknesses, and second the viscosity ratiosconsideredaretoolow(𝜀≤102).Weusetheirnumericalroutinestoreproduce our experimental conditions and to extend results to higher 𝛽values than achieved in the laboratory. Full technical details can be found in Sweeney et al. (2013). Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 Table 2 Density and Viscosity of the Glucose Syrup and the Potassium Carbonate Solutions Used at 8 and 18∘C Fluid Density (kg/m3) Viscosity (Pa⋅s) 8∘C 18∘C 8∘C 18∘C Glucose syrup 1,450 ± 5 1,435 ± 5 1,530–2,000 300–480 K2CO3 solution 1,440–1,550 ± 1 0.009 ± 0.001 0.007 ± 0.001 3.2. Experimental Methods We use two tank sizes to investigate the eﬀect of later Perspex and are cylindrical to avoid corner eﬀects. 3.2.1. Narrow Tank Experiments The narrowest tanks have a diameter D = 5.00 ± 0.02 cm tanks of this geometry were made in order to eﬃciently ru (𝛽= 𝜆c D ) for experiments in these tanks are 10 ≤𝛽≤210 all experiments with D = 5 cm. We conducted experiments in two temperature-control to sample a wider range of viscosity ratios. We ﬁrst pour m the top of the tank, and leave it to rest until all the air bub usually dehydrates and stiﬀens. We thus remove this stiﬀ This is long enough for the disturbed interface to ﬂatte experiment is started by delicately pouring dyed K2CO3 a few seconds, which is much faster than the time for an ﬁguration (viscous ﬂuid underneath) is ﬂipped compare more viscous mush. Nonetheless, the dynamics of the in buoyancy, is independent of which ﬂuid is on top of the (Whitehead & Luther, 1975). All experiments are recorded In most experiments, there is air directly above the K2CO inﬂuence of the BC, we repeated some experiments with on top of the K2CO3 layer just after pouring it. The lid diam during placement. Diagram of a narrow tank experimental setup. 3.2.2. Wide Tank Experiments We perform similar experiment and height H = 39.20±0.05 cm wall eﬀects to be small and ou Whitehead and Luther (1975). T tank case, with the exception th seventy-two 1.5-mm-diameter we conduct experiments at bo upper BC experiment with the w 3.3. Linear Stability Analysis To complement our experiment sis (LSA), based on the work of S RT instabilities in a narrow, ﬁn cannot be directly applied to ou cases where the two ﬂuid laye ratiosconsideredaretoolow(𝜀 our experimental conditions an in the laboratory. Full technical ET AL. 3.2. Experimental Methods We use two tank sizes to investigate the eﬀect of lateral conﬁnement. All tanks are made of transparent Perspex and are cylindrical to avoid corner eﬀects. 3.2.1. Journal of Geophysical Research: Solid Earth Narrow Tank Experiments The narrowest tanks have a diameter D = 5.00 ± 0.02 cm and height H = 40.00 ± 0.05 cm (Figure 4). Three tanks of this geometry were made in order to eﬃciently run concurrent experiments. The conﬁnement factors (𝛽= 𝜆c D ) for experiments in these tanks are 10 ≤𝛽≤210; hence, wall eﬀects are likely to be very important in all experiments with D = 5 cm. We conducted experiments in two temperature-controlled rooms, at 18∘C (laboratory) and 8∘C (cold room), to sample a wider range of viscosity ratios. We ﬁrst pour more than 30 cm of glucose syrup into the tank, cover the top of the tank, and leave it to rest until all the air bubbles have escaped. The uppermost part of the syrup usually dehydrates and stiﬀens. We thus remove this stiﬀlayer a few minutes before starting the experiment. This is long enough for the disturbed interface to ﬂatten but short enough to avoid signiﬁcant drying. The experiment is started by delicately pouring dyed K2CO3 solution on top of the glucose layer. Pouring takes a few seconds, which is much faster than the time for an RT instability to develop (always >3 min). This con- ﬁguration (viscous ﬂuid underneath) is ﬂipped compared to magmatic systems with a buoyant lens under a more viscous mush. Nonetheless, the dynamics of the instability will be identical because the driving force, buoyancy, is independent of which ﬂuid is on top of the other; only the density diﬀerence Δ𝜌is important (Whitehead & Luther, 1975). All experiments are recorded with a ﬁxed camera. In most experiments, there is air directly above the K2CO3 solution, forming a free-slip BC above. To check the inﬂuence of the BC, we repeated some experiments with a no-slip upper BC by placing a circular perspex lid on top of the K2CO3 layer just after pouring it. The lid diameter is 0.4 mm less than the tank to allow air escape during placement. gram of a narrow tank experimental setup. 3.2.2. Wide Tank Experiments We perform similar experiments in a wide tank with diameter D = 28.70 ± 0.05 cm and height H = 39.20±0.05 cm, achieving 1.2 ≤𝛽≤6.9. Although 𝛽> 1, we expect wall eﬀects to be small and our results to approach the theoretical predictions of Whitehead and Luther (1975). 3.3. Linear Stability Analysis To complement our experimental investigation, we perform an linear stability analy- sis (LSA), based on the work of Sweeney et al. (2013). They theoretically investigated RT instabilities in a narrow, ﬁnite cylinder (no-slip BC), but their published results cannot be directly applied to our scenario for two reasons. First, they only examined cases where the two ﬂuid layers have equal thicknesses, and second the viscosity ratiosconsideredaretoolow(𝜀≤102).Weusetheirnumericalroutinestoreproduce our experimental conditions and to extend results to higher 𝛽values than achieved in the laboratory. Full technical details can be found in Sweeney et al. (2013). Figure 4. Diagram of a narrow tank experimental setup. SEROPIAN ET AL. SEROPIAN ET AL. 3.3. Linear Stability Analysis 3597 Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 Table 3 Summary of Experiments Performed and Corresponding Linear Stability Analysis Results in Order of Increasing 𝛽 Exp Δ𝜌 𝜇2 𝜇1 h 𝛽 nc nobs Comment nc nobs − (±5 kg/m3) − (±0.05 cm) − (Exp) − (LSA) 46 6 66,000 ± 5,000 0.2 1.2 ± 0.3 1.2 ± 1.0 Wide Tank 1.8 45 22 220,000 ± 14,000 0.2 1.8 ± 0.4 1.9 ± 0.7 Wide Tank 2.2 41 12 55,000 ± 5,000 1.2 6.9 ± 0.4 4.0 ± 1.8 Wide Tank 7.7 4 50 53,000 ± 5,000 0.3 10 ± 2 15 ± 3 11 15 15 53,000 ± 5,000 0.3 10 ± 2 14 ± 6 11 39 67 66,000 ± 5,000 0.3 10 ± 2 10 ± 2 No slip 12 31 65 66,000 ± 5,000 0.5 18 ± 2 19 ± 3 20 14 35 53,000 ± 5,000 0.8 26 ± 2 28 ± 5 29 5 50 53,000 ± 5,000 0.8 26 ± 2 33 ± 5 29 20 50 53,000 ± 5,000 0.8 26 ± 2 30 ± 4 29 21 12 66,000 ± 5,000 0.8 26 ± 2 21 ± 9 29 38 44 66,000 ± 5,000 0.8 28 ± 2 36 ± 6 No slip 38 42 52 220,000 ± 14,000 0.6 31 ± 3 24 ± 3 No slip 35 25 50 66,000 ± 5,000 1 35 ± 2 46 ± 6 39 7 15 53,000 ± 5,000 1.4 46 ± 2 40 ± 14 51 8 15 53,000 ± 5,000 1.4 46 ± 2 43 ± 15 51 13 35 53,000 ± 5,000 1.4 46 ± 2 60 ± 9 51 22 50 66,000 ± 5,000 1.3 46 ± 2 62 ± 7 51 44 12 47,000 ± 5,000 1.6 50 ± 3 49 ± 21 No slip 56 27 67 66,000 ± 5,000 1.6 56 ± 2 68 ± 7 63 9 15 53,000 ± 5,000 2 65 ± 2 52 ± 18 73 3 50 53,000 ± 5,000 2 66 ± 3 49 ± 7 73 2 116 53,000 ± 5,000 2 66 ± 3 65 ± 8 73 12 17 166,000 ± 11,000 1.4 67 ± 3 61 ± 19 75 11 30 166,000 ± 11,000 1.6 77 ± 3 85 ± 15 86 30 60 66,000 ± 5,000 2.3 81 ± 3 120 ± 13 90 35 44 66,000 ± 5,000 2.3 81 ± 3 113 ± 15 No slip 90 40 52 220,000 ± 14,000 1.7 89 ± 3 65 ± 7 No slip 99 19 50 166,000 ± 11,000 1.9 91 ± 3 61 ± 8 102 28 65 220,000 ± 14,000 1.8 94 ± 3 88 ± 8 105 36 53 220,000 ± 14,000 2 105 ± 3 109 ± 15 117 18 12 75,000 ± 10,000 3.1 114 ± 6 100 ± 45 127 32 52 220,000 ± 14,000 2.3 121 ± 4 98 ± 11 134 43 52 220,000 ± 14,000 2.4 126 ± 4 110 ± 13 No slip 140 6 12 53,000 ± 5,000 4 131 ± 5 87 ± 38 146 37 51 220,000 ± 14,000 2.7 142 ± 4 121 ± 13 158 29 65 220,000 ± 14,000 2.9 152 ± 4 155 ± 14 170 33 52 220,000 ± 14,000 2.9 152 ± 4 164 ± 18 170 24 50 220,000 ± 14,000 3.4 178 ± 4 151 ± 16 199 26 59 220,000 ± 14,000 4 210 ± 5 278 ± 26 234 Note. Note. All symbols are deﬁned in the text. Unless indicated by “Wide Tank”, all experiments were performed in a narrow tank with D = 5.00 ± 0.02 cm. The ﬁnal column indicates nc nobs for nc calculated by equation (2) and nobs determined by linear stability analysis. Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 Table 4 Summary of Parameters Used in the Additional Linear Stability Analysis Performed and Resulting Growth Rates D (cm) h (cm) 𝜀 𝛽 nc nobs 100 2.5 104 2.3 3.05 100 3.75 104 3.5 4.34 100 5 104 4.7 5.67 100 6.25 104 5.9 7.01 100 7.5 104 7.0 8.35 2 2.3 106 500 560 2 4.6 106 1,000 1,180 1 4.6 106 2,000 2,230 1 9.2 106 4,000 4,470 1 11.5 106 5,000 5,590 1 18.5 106 8,000 9,120 1 23 106 10,000 11,280 Note. We kept Δ𝜌= 50 kg/m3 constant in all the runs. Results are plotted as “Complementary LSA” in Figure 7. Note. We kept Δ𝜌= 50 kg/m3 constant in all the runs. Results are plotted as “Complementary LSA” in Figure 7. 3.3. Linear Stability Analysis All symbols are deﬁned in the text. Unless indicated by “Wide Tank”, all experiments were performed in a narrow tank with D = 5.00 ± 0.02 cm. The ﬁnal column indicates nc nobs for nc calculated by equation (2) and nobs determined by SEROPIAN ET AL. SEROPIAN ET AL. 3598 4. Results We conducted a total of 40 experiments, sampling the range 1.2 ≤𝛽≤210. The viscosity ratios (4.7 ⋅104 ≤ 𝜀≤2.2 ⋅105) are more than 2 orders of magnitude higher than previous large viscosity contrast RT experi- ments (e.g., de Bremond d’Ars et al., 1995; Huppert et al., 1984). A summary of the experimental parameters and corresponding results is presented in Table 3. We performed an LSA for each set of experimental param- eters (Table 3). To complete and extend our data set, we also carried out LSA in the ranges 2.3 ≤𝛽≤7.0 and 500 ≤𝛽≤10, 000, with sets of parameters that have not been or could not be experimentally investigated. The conditions and results for these additional LSA runs are summarized in Table 4. We thus obtain theoret- ical growth rates for the conﬁned RT instabilities considered, which can be compared to our experimental observations and to unconﬁned theory. Figure 5 shows the time series of two typical experiments in a narrow tank: one at room temperature with air above (Figure 5a) and one at 8∘C with a no-slip upper BC (Figure 5b). Regardless of the tank size or the BC, the overall dynamics are similar. First, a single, small protrusion of K2CO3 solution forms at the interface between the two ﬂuids. The protrusion grows into an ellipsoidal to spheroidal pocket of solution. When all of the initial Figure 5. Time series of the initial stage of (a) experiment 25, at 18∘C with h = 1 cm, Δ𝜌= 50 kg/m3, 𝜀= 6.6 ⋅104, and 𝛽= 35 and (b) experiment 40, at 8∘C with h = 1.7 cm, Δ𝜌= 52 kg/m3, 𝜀= 2.2 ⋅105, 𝛽= 89 and a no-slip boundary condition. The initial interfaces are highlighted and an example of how amplitude s measured is provided. Figure 5. Time series of the initial stage of (a) experiment 25, at 18∘C with h = 1 cm, Δ𝜌= 50 kg/m3, 𝜀= 6.6 ⋅104, and 𝛽= 35 and (b) experiment 40, at 8∘C with h = 1.7 cm, Δ𝜌= 52 kg/m3, 𝜀= 2.2 ⋅105, 𝛽= 89 and a no-slip boundary condition. The initial interfaces are highlighted and an example of how amplitude is measured is provided. SEROPIAN ET AL. 3599 Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 Figure 6. Amplitude of the perturbation as a function of time for experiment 25 (Figure 5a). 4. Results An exponential curve is ﬁtted through the ﬁrst data points and a line is ﬁtted through the last ones. Equations of best ﬁt are provided. Figure 6. Amplitude of the perturbation as a function of time for experiment 25 (Figure 5a). An exponential curve is ﬁtted through the ﬁrst data points and a line is ﬁtted through the last ones. Equations of best ﬁt are provided. volume has been drawn into this spheroid, it sinks as a single pocket of ﬂuid. The dimensions of this poc of ﬂuid stay constant, until it reaches the bottom of the tank. The location of the initial protrusion is sensitive to asperities on the syrup surface or disturbance induced by pouring the K2CO3 solution; hence, the protrusion is sometimes initially slightly oﬀcentered (Figure 5b for instance). Replicate experiments show that these deviations do not signiﬁcantly aﬀect the results: The growth rate of the protrusion is similar, and the K2CO3 solution layer always becomes thickest near the center of the tank well before it evolves into a spheroid sinking through the syrup. The amplitude of the perturbation, deﬁned as the vertical distance from the initial liquid-liquid interface loca- tion to the bottom of the protrusion (see Figure 5a), was measured as a function of time for each experiment (Figure 6). For all experiments there is an initial exponential increase in amplitude before transitioning to linear growth. This transition corresponds to the stage at which linearized stability theory (e.g., Whitehead & Luther, 1975) is no longer valid. We therefore obtain an exponential ﬁt of the form y(t) = Aenobst for the initial growth of each experiment. The nobs parameter in the exponential then corresponds to the growth rate of our experimental RT instability and can be compared to theoretical unconﬁned predictions. Figure 7. Ratio of theoretical unconﬁned growth rates nc from Whitehead and Luther (1975) to our experimental and analytical conﬁned growth rates nobs as a function of conﬁnement parameter 𝛽. The red line represents nc nobs = 𝛽and the horizontal black line indicates nc = nobs. The symbol and color code is used to distinguish diﬀerent experimental conditions. The crosses represent linear stability analysis (LSA) results reproducing experimental conditions whereas the stars show complementary LSA runs which have no experimental counterpart (Table 4). Figure 7. Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 Figure 7 displays the ratio nc nobs as a function of the conﬁnement parameter 𝛽for both experiments and LSA, where nc is the theoretical unbounded critical growth rate from equation (2). The no-slip experiments are indistinguishable from the free-slip ones, indicating that the top BC only has a minor inﬂuence on the growth rate. Both the experimental and analytical results follow a linear trend of nc nobs ≈𝛽, although for 𝛽≲10, the slope of the LSA data trend decreases. The linear best ﬁt through the experimental data is nc nobs = (0.93 ± 0.03)𝛽+ (0.15 ± 0.07). Similarly, a ﬁtted line through the LSA points for 𝛽> 10 is nc nobs = (1.0009 ± 0.0002)𝛽+ (0.0454 ± 0.0005). Figure 7 displays the ratio nc nobs as a function of the conﬁnement parameter 𝛽for both experiments and LSA, where nc is the theoretical unbounded critical growth rate from equation (2). The no-slip experiments are indistinguishable from the free-slip ones, indicating that the top BC only has a minor inﬂuence on the growth rate. Both the experimental and analytical results follow a linear trend of nc nobs ≈𝛽, although for 𝛽≲10, the slope of the LSA data trend decreases. The linear best ﬁt through the experimental data is nc nobs = (0.93 ± 0.03)𝛽+ (0.15 ± 0.07). Similarly, a ﬁtted line through the LSA points for 𝛽> 10 is nc nobs = (1.0009 ± 0.0002)𝛽+ (0.0454 ± 0.0005). In summary, our results show that bounded growth rates (nobs) are reduced compared to the unbounded theoretical values (nc) by approximately a factor of 𝛽. For simplicity, in our applications to magmatic system we take nc nobs = 𝛽, which is within 10% of the lines of best ﬁt to our data (Figure 7) and introduces negligible error compared to the uncertainties on the magma and mush properties that aﬀect RT instabilities. 5.1. Conﬁned RT Instability Dynamics 5.1. Conﬁned RT Instability Dynamics y y Our experimental results with 1.2 ≤𝛽≤210 and 4.7 ⋅104 ≤𝜀≤2.2 ⋅105 demonstrate that the dynam- ics of a conﬁned RT instability is qualitatively similar to the unconﬁned case. Indeed, the instability starts with an exponential growth, followed by a linear growth (e.g., Figure 6), a phenomenon well documented for the unbounded case (e.g., Waddell et al., 2001). Moreover, the shape of the perturbations is similar to pre- vious experimental studies (e.g., Waddell et al., 2001; Whitehead & Luther, 1975; Wilkinson & Jacobs, 2007). Our instabilities diﬀer from the unbounded case because only a single protrusion forms, as opposed to mul- tiple, uniformly spaced bulges, as observed by Whitehead and Luther (1975) and de Bremond d’Ars et al. (1995) for instance. Our experimental and analytical results indicate that as 𝛽approaches unity, there is a transition in the instabil- ity growth rate from nc nobs = 𝛽when the system is bounded towards nc = nobs, characteristic of an unbounded (𝛽≪1) system (i.e., nobs matches the theoretically predicted value for a laterally inﬁnite system). Based on these results, we estimate a characteristic RT instability timescale, that is, the time for the instability amplitude to increase by a factor of e (≈2.72), by 𝜏RTI = { 1 nc = 𝜇2 0.232gΔ𝜌h𝜀−1∕3 if 𝛽≤1, 𝛽 nc = 6𝜋𝜇2 Δ𝜌gD if 𝛽> 1. (6) (6) Incontrasttotheunboundedcase,theconﬁnedtimescaledoesnotdependoneithertheinitiallayerthickness h or the viscosity ratio 𝜀because the instability wavelength is set by D. Rather, the controlling parameters are the diameter of the layer D, the viscosity of the upper layer 𝜇2, and the density contrast between the ﬂuids Δ𝜌. Comparing equations (5) and (6), we notice that our experimental growth rates are a factor of 3/2 slower than estimated with the linear stability theory of Whitehead and Luther (1975) for 𝜆= D. This is most likely because the tank walls add a no-slip BC (i.e., will exert signiﬁcant drag on the ﬂuids) that is not accounted for in equation (5). 4. Results Ratio of theoretical unconﬁned growth rates nc from Whitehead and Luther (1975) to our experimental and analytical conﬁned growth rates nobs as a function of conﬁnement parameter 𝛽. The red line represents nc nobs = 𝛽and the horizontal black line indicates nc = nobs. The symbol and color code is used to distinguish diﬀerent experimental conditions. The crosses represent linear stability analysis (LSA) results reproducing experimental conditions whereas the stars show complementary LSA runs which have no experimental counterpart (Table 4). SEROPIAN ET AL. 3600 Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 Figure 8. Conﬁned Rayleigh-Taylor instability timescale as a function of melt layer diameter and mush viscosity. Figure 8. Conﬁned Rayleigh-Taylor instability timescale as a function of melt layer diameter and mush viscosity. for melt- or ﬂuid-layer generation could be modeled individually and in detail, but this is beyond the scope of the current paper. Here we investigate the stability of buoyant magma layers of ﬁxed thickness or grow- ing at a ﬁxed rate to provide a ﬁrst-order understanding of the likely timescales and length scales that can be expected. Our analysis is restricted to RT instablities; we have not explored the potential importance of melt-mush ﬂow mechanisms and smaller-scale instabilities related to the porous or brittle nature of the mush, which could develop along with or instead of RT instabilities (e.g., Connolly & Podladchikov, 2013; Oppenheimer et al., 2015; Sandnes et al., 2011; Schmeling et al., 2017; Scott & Stevenson, 1984, 1986). Figure 8 presents estimates of conﬁned RT instability timescales for a range of mush viscosities 𝜇2 and melt layer diameters (or widths if not circular) D relevant to igneous systems. We use the instability timescale deﬁned in equation (6), with 𝛽calculated as 𝜆c∕D using 𝜆c deﬁned in equation (1), and physical parame- ter values listed in Table 1. The wide range of calculated timescales (101–104 years) match the variability of timescales observed for volcanic processes, from small and frequent events to large and rare events (e.g., caldera forming eruptions). On the other hand, the very long timescales involved with the slowest insta- bilities imply that other processes (e.g., solidifaction by cooling) could occur faster and inhibit the onset of RT instability. 5.2.1. Comparison to Cooling Timescale First, we consider a case where there is a strong temperature diﬀerence between the melt and the mush and compare our RT instability timescale to a characteristic cooling timescale. Such a scenario could occur when a cold mush system is replenished by hot magma from depth. In this case, if the cooling timescale is shorter than the RT instability timescale, the melt layer will freeze before an instability can develop. For simplicity, we assume cooling occurs via conduction only and deﬁne a characteristic cooling timescale 𝜏cool = h2 𝜅, where h is the melt layer thickness and 𝜅is the thermal diﬀusivity. 5.2. Application to Igneous Systems There are multiple mechanisms where buoyant layers can form within igneous mush systems. One mecha- nism is by replenishment with new magma spreading out within or at the base of a mush. Initially, the new magma layer may intrude as a denser layer at the base of the reservoir or at its neutral buoyancy level within the reservoir. In either case the magma layer may become less dense with time by, for example, crystalliza- tion and diﬀerentiation with dense components segregating to the base of the ﬂow. Volatile exsolution can also increase the buoyancy of the layer (Huppert et al., 1982). Additionally, the overlying mush may be heated from below (e.g., Burgisser & Bergantz, 2011; Couch et al., 2001) or be ﬂuxed by volatiles released from the replenishing magma layer (Bachmann & Bergantz, 2006) to develop a zone of reduced density. Another mech- anism of layer formation is through dynamic melt percolation due to mush compaction, which can result in formation of melt-rich regions. One-dimensional models of porous media ﬂow and compaction predict the development of multiple melt-rich regions (e.g., Jackson et al., 2003; Solano et al., 2012). Intrinsically, 1-D models cannot include RT instabilities of growing melt layers. Likewise, exsolved magmatic volatiles can migrate through mushes and accumulate as ﬂuid layers (Christopher et al., 2015). Each of the above scenarios SEROPIAN ET AL. 3601 SEROPIAN ET AL. 5.2.2. Comparison to Accumulation Timescale 5.2.2. Comparison to Accumulation Timescale The previous discussion assumes that melt accumulation into a layer occurs much faster than the insta- bility timescale, such that a melt lens of any thickness is a feasible starting condition for considering RT instabilities. Next, we relax this assumption and explore the relation between melt accumulation and RT instability timescales. DeBremondd’Arsetal.(1995)investigatedthecaseofahorizontallyextensivebuoyantlayerthatthickensand a constant rate, ̇h = dh dt . They showed that blobs of buoyant ﬂuid form and rise away from the base when the layer growth rate ̇h h and the instability growth rate 1 𝜏RTI are equal. For the formation of a new layer (i.e., starting withh = 0),thelayergrowthrate ̇h h isinitiallyverylargeandthenmonotonicallydecreasesashincreases.Thus, for any given melt input rate ̇h, there exists a maximum layer thickness beyond which melt will be removed faster via RT instabilities than it is added. We can estimate this thickness, the corresponding magma volume, and the time required to assemble it for a range of parameters. First, we calculate the time at which ̇h h = 1 𝜏RTI . We can then compute the layer thickness using the constant melt input rate and corresponding volume, assuming a cylindrical shape. We use the same criterion as de Bremond d’Ars et al. (1995) but account for lateral conﬁnement when relevant. Figure 10 shows the accumulation time and associated melt volume for a silicic melt under a melt-rich mush (𝜇2 = 1013 Pa⋅s; Figure 10a) and a near-solidus mush (𝜇2 = 1017 Pa⋅s; Figure 10b), as a function of the layer diameterDandthemeltinputrate ̇h.Thechosenrangeofinputrates ̇hspanvaluesfrommagmaaccumulation models and ﬁeld studies (e.g., Karakas et al., 2017; White et al., 2006). The kinks in the lines for melt input rates of ̇h = 0.1, 1, 10 m/year in Figure 10a correspond to the transition from conﬁned to unconﬁned instability regime with increasing D. For conditions where the RT instability is not conﬁned (i.e., 𝜆< D), the accumulation times are independent of D but depend on ̇h (forming horizontal lines in the upper panel of Figure 10a). In conﬁned scenarios (some combinations of ̇h and D in Figure 10a and all conditions plotted in Figure 10b), the accumulation times are independent of ̇h, hence the collapse into a single line for a given 𝜇2. The choice of mush viscosity has a major impact on the results and allows us to recover a large range of timescales (0.3–4 ⋅ 104 years). Journal of Geophysical Research: Solid Earth We use 𝜅= 6 ⋅10−7 m2/s (Annen et al., 2006; Romine et al., 2012; Whittington et al., 2009). Figure 9 shows the lines where 𝜏cool = 𝜏RTI as h and D are varied and for two mush viscosities. Above these lines, RT instabilities have time to develop, whereas under the lines, the melt layer will freeze before the instability develops signiﬁcantly. The results are dramatically diﬀerent depending on which mush viscosity is considered. For a melt-rich mush (𝜇2 = 1013 Pa⋅s), RT instabilities develop suﬃciently quickly that the required thickness for the instability to develop faster than conductive cooling is only 1.4–8.9 m, depending upon the layer diameter. This range, however, becomes 140–900 m for a near-solidus mush (𝜇2 = 1017 Pa⋅s). The mush rheology therefore exerts a critical control on the necessary thickness to develop instability and hence the volume of eruptible material ascending through the mush as a result of the instability. This simple analysis omits two potentially important but opposing mechanisms: (a) we only consider cool- ing via conduction and ignore convection, which can speed up cooling, and (b) the hot intrusion can reheat the mush, thus decreasing its viscosity and speeding up RT development. To get some insight about when convection may occur, we calculate the Rayleigh number Ra = g𝜌1𝛼ΔTh3 𝜅𝜇1 , where 𝛼is the thermal expansion of the melt and ΔT is the temperature diﬀerence between the melt and the mush. We use 𝛼= 5 ⋅10−5 K−1 and ΔT = 100 K and densities and viscosities for the three compositions considered in Table 1 (e.g., Bachmann & Bergantz, 2006; Degruyter & Huber, 2014; Huber et al., 2010; Jackson et al., 2003). Taking Ra = 27 𝜋4 4 ≈657.5 3602 SEROPIAN ET AL. SEROPIAN ET AL. Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 Figure 9. Isolines 𝜏cool = 𝜏RTI as a function of melt layer width and thickness and for two mush viscosities. Above these lines, there is suﬃcient time for the Rayleigh-Taylor instability (RTI) to grow, whereas under the lines, the melt layer will freeze before the instability develops signiﬁcantly. Figure 9. Isolines 𝜏cool = 𝜏RTI as a function of melt layer width and thickness and for two mush viscosities. Above these lines, there is suﬃcient time for the Rayleigh-Taylor instability (RTI) to grow, whereas under the lines, the melt layer will freeze before the instability develops signiﬁcantly. Journal of Geophysical Research: Solid Earth as the critical value for the onset of convection (Turcotte & Schubert, 1982), this calculation suggests that all the layers previously considered would convect, except for extremely thin silicic layers (h < 49 cm ). There- fore, the cooling timescale could be faster than assumed in Figure 9, which would raise the 𝜏cool = 𝜏RTI lines to greater h for a given D. Concerning reheating of the mush, Burgisser and Bergantz (2011) showed that RT instabilities could lead to signiﬁcant mush overturn (“unzipping”), yet their analysis is restricted to low mush viscosities (𝜇2 = 106 −1012 Pa⋅s) and considers high temperature diﬀerences (ΔT = 45–500 K). Therefore, the importance of this mechanism has yet to be tested for our scenario. 5.2.2. Comparison to Accumulation Timescale The corresponding volumes range from 105 up to 1012 m3. Our calculations suggest that RT instabilities could play an important role in controlling the size and fre- quency of volcanic events. A working hypothesis is that volcanic eruptions and episodes of volcanic unrest are the consequences of these instabilities. The calculated timescales and volumes are comparable with natural SEROPIAN ET AL. SEROPIAN ET AL. 3603 Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 Figure 10. Time before the onset of instability and accumulated volume in the case of a linearly growing buoyant layer of silicic melt under (a) a melt-rich mush and (b) a near-solidus mush. The kinks in the lines in (a) for ̇h = 0.1, 1, and 10 m/year correspond to a change from conﬁned to unconﬁned instability regime with increasing melt layer width. There are no kinks in (b) because for the higher mush viscosity, Rayleigh-Taylor instabilities are conﬁned for the full range of scenarios plotted. Figure 10. Time before the onset of instability and accumulated volume in the case of a linearly growing buoyant layer of silicic melt under (a) a melt-rich mush and (b) a near-solidus mush. The kinks in the lines in (a) for ̇h = 0.1, 1, and 10 m/year correspond to a change from conﬁned to unconﬁned instability regime with increasing melt layer width. There are no kinks in (b) because for the higher mush viscosity, Rayleigh-Taylor instabilities are conﬁned for the full range of scenarios plotted. volcanic values (e.g., Pyle, 2015) and span the full range between small maﬁc eruptions (e.g., Strombolian type) to the largest magnitude eruptions (e.g., caldera-forming eruptions and ﬂood basalts). The time needed for an instability to develop allows melt to accumulate in large layers and corresponds to a dormant period (e.g., Rougier et al., 2018; Sheldrake et al., 2016), whereas the instability may destabilize the system and pro- duce a period of unrest or an eruption. Additionally, successive instabilities without eruption could yield larger accumulated volumes (e.g., Sparks & Cashman, 2017). 6. Conclusion Rayleigh-Taylor instabilities occur naturally in magmatic systems when buoyant melt (or magmatic volatile phase) is trapped underneath a denser crystalline mush. For a wide range of expected viscosities, the large viscosity contrast between the mush and melt lens means that the theoretical fastest-growing wavelength is unfeasibly large and so the wavelength of the instability is the largest available: the diameter of the lens. This lateral conﬁnement means that the growth rate of the instability is reduced compared to the theoretical unconﬁned scenario. Importantly, if conﬁned, the instability growth rate no longer depends on the thickness or viscosity of the lens; rather, it depends only on the diameter of the lens, the viscosity of the mush and the density contrast between the lens and mush. The thickness of the lens will, however, play a role if the mush has anapparentyieldstrengthbecausetheinstabilitywillonlyinitiateifthebuoyancystress,whichisproportional to the lens thickness, is suﬃcient to overcome the yield strength. Thickness also matters in scenarios where the melt is hotter than the mush, as thinner lenses will cool more quickly, allowing less time for RT instability development before the magma is too crystalline to ﬂow. A fully developed conﬁned RT instability transforms the melt lens into a spheroid of melt, which ascends through the mush. A lens of melt can only exist if it thickens faster than melt is removed by a RT instability. So both the timescale for transforming a lens into a rising spheroid and the volume of the spheroid will depend on the rate of input of melt into the lens as well as its diameter, the viscosity of the mush, and the density contrast between the lens and mush. We postulate that RT instabilities may play a role in regulating the size and frequency of volcanic eruptions and volcanic unrest. Using feasible ranges of the relevant parameters, we calculate timescales and volumes that span small, frequent maﬁc eruptions to the largest and much rarer caldera-forming and ﬂood basalt eruptions. Also, multiple episodes of layer instability without eruption can lead to accumulations of larger magma volumes and provide one explanation of volcanic unrest. In a magmatic system composed of a vertically extensive mush containing multiple melt lenses, can the rise of ablobofmeltorvolatilestriggerafeedbacktocauselarge-scaledestabilitization?Thegrowthrateofalaterally conﬁned RT instability in a Newtonian mush does not depend on the thickness of the melt lens. Acknowledgments We thank Rich Kerswell for providing and helping with his numerical tools. We also thank the Editor A. Revil and reviewers J. de Bremond d’Ars and A. R. Cruden for their insightful comments that have improved the manuscript; some of their points are beyond the scope of this paper but will inﬂuence our further research. This work was supported by NERC grant NE/P017371/1. All the data from experiments and linear stability analysis are presented in Tables 3 and 4. 5.2.3. Crystal Mush Rheology A limitation in the application of our experimental and theoretical results to natural systems is the assumption of Newtonian mush rheology. Our experiments involved Newtonian ﬂuids, but natural crystal mushes are generally shear-thinning and may have a yield strength, that is, a minimum stress required for ﬂow to occur (e.g., Hoover et al., 2001; Kerr & Lister, 1991; Saar et al., 2001). With Newtonian ﬂuids, an RT instability will always develop in response to a denser ﬂuid overlying a less dense ﬂuid, although in some scenarios it will grow slowly enough that it can be neglected over the timescale of interest. The same is true of shear-thinning ﬂuids; here the relevant viscosity for the early stage of the instability is the (high) viscosity in the limit of the shear rate approaching 0. With a yield strength, however, the buoyant force from the melt layer Δ𝜌gh has to exceed the mush yield strength 𝜏0; otherwise, the mush will act as a solid (e.g., elastic) body, preventing the growth of the RT instability, akin to initiation of thermal convection of a ﬂuid with a yield strength (Balmforth & Rust, 2009). True yield strengths (i.e., a minimum stress for any ﬂow to occur) may not exist (e.g., Barnes, 1999); however, if the eﬀective viscosity of a mush at low stresses is extremely large such that it has an apparent yield strength, then the instability will grow so slowly as to be negligible unless Δ𝜌gh > 𝜏0. We can estimate the minimum thickness required for an RT instability to develop when the mush has a yield strength as hmin = 𝜏0 Δ𝜌g. For mush yield strengths in the range 𝜏0 = 105–106 Pa (Castruccio et al., 2013; Lejeune & Richet, 1995) and Δ𝜌= 300 kg/m3, we ﬁnd hmin = 34–340 m. This crude estimate suggests that the required thickness to overcome a yield strength could be of the same order of magnitude as typical melt layer thicknesses. Mush strength could thus facilitate the accumulation of melt lenses by impeding RT insta- bility development for the thinnest layers. This eﬀect could be enhanced if crystals have grown together and bonded in a stagnant mush, producing a yield strength greater than 106 Pa. SEROPIAN ET AL. SEROPIAN ET AL. 3604 Journal of Geophysical Research: Solid Earth 10.1029/2018JB015523 6. 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https://openalex.org/W4210620499	https://journals.iugaza.edu.ps/index.php/IUGJEPS/article/download/10356/4027	Arabic	null	أساليب التنشئة الأسرية وعلاقتها بضبط الذات من وجهة نظر الطلبة المراهقين	Maǧallaẗ al-ǧāmi'aẗ al-islāmiyyaẗ li-l-dirāsāt al-tarbawiyyaẗ wa-al-nafsiyyaẗ	2,021	cc-by	11,254	تاريخ اإلرسال( 12 - 04 - 2021 ،) تاريخ قبول النشر( 19 - 06 - 2021 ) أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر الطلبة املراهقني https://doi.org/10.33976/IUGJEPS.29.6/2021/21 امللخص: هدفت الدراسة الحالية إلى دراسة العالقة االرتباطية بين أساليب التنشئة األسرية وعالقتها بضبط الذات ، وكذلك التعرف إلى مستوى ،أساليب التنشئة األسرية وضبط الذات من وجهة نظر الطلبة المراهقين ولتحقيق أهداف الدراسة تم تطوير وتطبيق مقياسي أساليب التنشئة األسرية وضبط الذات( بعد التحقق من صدقهما وثباتهما على عينة مؤلفة من100 ) طالب من مرحلة المراهقه من مديرية تربية لواء الرصيفة في مد ينة الزرقاء خالل الفصل الدراسي الثاني من العام الدراسي2020 / 2021 . أظهرت نتائج الدراسة وجود مستوى متوسط في األبعاد (النمط التسلطي، والنمط المتساهل). بينما البعد الديمقراطي كانت ذات مستوى منخفض. كما بينت النتائج وجود مستوى منخفض في الدرجة الكلية لمقياس ضبط ال ذات. وقد أظهرت النتائج أيضا وجود عالقة ارتباطية إيجابية بين النمط الديمقراطي وضبط الذات، وإلى وجود عالقة سلبية بين النمط التسلطي والمتساهل وضبط الذات من وجهة نظر الطلبة المراهقين.، ويوصى الباحثان بتطوير برامج ارشاد جمعي تستند الى استخدام مفاهيم البحث Methods of Family Upbringing and its Relationship to Self-Control from the Viewpoint of Adolescent Students :كلمات مفتاحية أساليب .التنشئة األسرية، ضبط الذات، الطلبة المراهقين ethods of Family Upbringing and its Relationship to Self-Control from the Viewpoint of Adolesce Methods of Family Upbringing and its Relationship to Self-Control from the Viewpoint of Adolescent Students Abstract: The object of the current study was to study the correlation relationship between the methods of family upbringing and its relationship to self-control as well as to identify the level of methods of family upbringing and self-control from the viewpoint of adolescent students and to achieve the objectives of the study, the two measures of family upbringing and self-control methods were developed and applied after verifying their validity and reliability of (100) students from the Rusaifa District Education Directorate in Zarqa. The results of the study showed an average level in the dimensions (authoritarian and permissive patterns). While the democratic dimension was of a low level. The results also showed a low level in the total score of the Self-Control Scale. The results also showed a positive correlation between the democratic style and self-control, and there was a negative relationship between the authoritarian and permissive style and self-control from the viewpoint of adolescent students, The researchers suggest that a group counseling program should be developed based on the use of research concepts. Keywords: methods of family upbringing, self-control, adolescent students Keywords: methods of family upbringing, self-control, adolescent students Keywords: methods of family upbringing, self-control, adolescent students IUGJEPS Vol 29, No 6, 2021, pp 460-478 ISSN 2410-3152 مجلة الجامعة اإلسالمية للدراسات التربوية والنفسية تاريخ اإلرسال( 12 - 04 - 2021 ،) تاريخ قبول النشر( 19 - 06 - 2021 ) https://doi.org/10.33976/IUGJEPS.29.6/2021/21 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر الطلبة املراهقني 1 اسم الجامعة والبلد : * البريد االلكتروني للباحث ان : E-mail address: وزارة التربية والتعليم/ األردن ehab.asafrah@hotmail.com امللخص: هدفت الدراسة الحالية إلى دراسة العالقة االرتباطية بين أساليب التنشئة األسرية وعالقتها بضبط الذات ، وكذلك التعرف إلى مستوى ،أساليب التنشئة األسرية وضبط الذات من وجهة نظر الطلبة المراهقين ولتحقيق أهداف الدراسة تم تطوير وتطبيق مقياسي أساليب التنشئة األسرية وضبط الذات( بعد التحقق من صدقهما وثباتهما على عينة مؤلفة من100 ) طالب من مرحلة المراهقه من مديرية تربية لواء الرصيفة في مد ينة الزرقاء خالل الفصل الدراسي الثاني من العام الدراسي2020 / 2021 . أظهرت نتائج الدراسة وجود مستوى متوسط في األبعاد (النمط التسلطي، والنمط المتساهل). بينما البعد الديمقراطي كانت ذات مستوى منخفض. كما بينت النتائج وجود مستوى منخفض في الدرجة الكلية لمقياس ضبط ال ذات. وقد أظهرت النتائج أيضا وجود عالقة ارتباطية إيجابية بين النمط الديمقراطي وضبط الذات، وإلى وجود عالقة سلبية بين النمط التسلطي والمتساهل وضبط الذات من وجهة نظر الطلبة المراهقين.، ويوصى الباحثان بتطوير برامج ارشاد جمعي تستند الى استخدام مفاهيم البحث :اسم الباحث األول :اسم الباحث الثاني Methods of Family Upbringing and its Relationship to Self-Control from the Viewpoint of Adolescent Students :كلمات مفتاحية أساليب .التنشئة األسرية، ضبط الذات، الطلبة المراهقين تاال غالب العموش د.إيهاب علي العصافره IUGJEPS Vol 29, No 6, 2021, pp 460-478 ISSN 2410-3152 مجلة الجامعة اإلسالمية للدراسات التربوية والنفسية تاريخ اإلرسال( 12 - 04 - 2021 ،) تاريخ قبول النشر( 19 - 06 - 2021 ) https://doi.org/10.33976/IUGJEPS.29.6/2021/21 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر الطلبة املراهقني 1 اسم الجامعة والبلد : * البريد االلكتروني للباحث ان : E-mail address: وزارة التربية والتعليم/ األردن ehab.asafrah@hotmail.com امللخص: هدفت الدراسة الحالية إلى دراسة العالقة االرتباطية بين أساليب التنشئة األسرية وعالقتها بضبط الذات ، وكذلك التعرف إلى مستوى ،أساليب التنشئة األسرية وضبط الذات من وجهة نظر الطلبة المراهقين ولتحقيق أهداف الدراسة تم تطوير وتطبيق مقياسي أساليب التنشئة األسرية وضبط الذات( بعد التحقق من صدقهما وثباتهما على عينة مؤلفة من100 ) طالب من مرحلة المراهقه من مديرية تربية لواء الرصيفة في مد ينة الزرقاء خالل الفصل الدراسي الثاني من العام الدراسي2020 / 2021 . أظهرت نتائج الدراسة وجود مستوى متوسط في األبعاد (النمط التسلطي، والنمط المتساهل). بينما البعد الديمقراطي كانت ذات مستوى منخفض. كما بينت النتائج وجود مستوى منخفض في الدرجة الكلية لمقياس ضبط ال ذات. وقد أظهرت النتائج أيضا وجود عالقة ارتباطية إيجابية بين النمط الديمقراطي وضبط الذات، وإلى وجود عالقة سلبية بين النمط التسلطي والمتساهل وضبط الذات من وجهة نظر الطلبة المراهقين.، ويوصى الباحثان بتطوير برامج ارشاد جمعي تستند الى استخدام مفاهيم البحث :اسم الباحث األول :اسم الباحث الثاني Methods of Family Upbringing and its Relationship to Self-Control from the Viewpoint of Adolescent Students :كلمات مفتاحية أساليب .التنشئة األسرية، ضبط الذات، الطلبة المراهقين تاال غالب العموش د.إيهاب علي العصافره IUGJEPS Vol 29, No 6, 2021, pp 460-478 ISSN 2410-3152 مجلة الجامعة اإلسالمية للدراسات التربوية والنفسية تاريخ اإلرسال( 12 - 04 - 2021 ،) تاريخ قبول النشر( 19 - 06 - 2021 ) https://doi.org/10.33976/IUGJEPS.29.6/2021/21 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر الطلبة املراهقني 1 اسم الجامعة والبلد : * البريد االلكتروني للباحث ان : E-mail address: وزارة التربية والتعليم/ األردن ehab.asafrah@hotmail.com :اسم الباحث األول :اسم الباحث الثاني تاال غالب العموش د.إيهاب علي العصافره مجلة الجامعة اإلسالمية للدراسات التربوية والنفسية تاريخ اإلرسال( 12 - 04 - 2021 ،) تاريخ قبول النشر( 19 - 06 - 2021 ) https://doi.org/10.33976/IUGJEPS.29.6/2021/21 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر الطلبة املراهقني 1 اسم الجامعة والبلد : * البريد االلكتروني للباحث ان : E-mail address: وزارة التربية والتعليم/ األردن ehab.asafrah@hotmail.com :اسم الباحث األول :اسم الباحث الثاني تاال غالب العموش د.إيهاب علي العصافره Keywords: methods of family upbringing, self-control, adolescent students خلفية الدراسة وأهميتها ًتعتبر المراهقة من أكثر فترات العمر حرجاً، فهي مرحلة تتسم بالقلق والصراع والتمرد، وعادة ما توصف بعدم االستقرار تبعا للتغيرات السريعة في شتى مراحل النمو والتي تؤدي إلى ضعف التوازن، وحدوث ما يسمى بعدم االستقرار الجسمي والنفسي والعقلي، حيث تعرف المراهقة بأنها الفترة النمائية التي تبدأ مع بداية سن البلوغ وتنتهي بانتهاء النضج النفسي والجسمي. فالمراهقة من فترات النمو التي تعد حرجة جداً لكثرة ما فيها من اضطرابات وصراعات، حيث تبدأ فيها مشكلة الب حث عن الذات التي قد تعكس عدم قدرة المراهق على التعامل أو ضبط الذات لديه، فالنمو الجسمي يسبق بك ثير النمو العقلي والوجداني فينش أ صرع إشباع رغبات و حاجات ( 2006 Cormier & Cormier ). لذلك فالمراهقة تمثل فترة حرجة فيما يخص النضج وتطويرِ الحكم الذاتي واالستقاللية والهوية الذاتية التي تعتبر مكونات الكفاءة االجتماعية. وتتطلب الكفاءة االجتماعية موازنة المراهق بين األهداف الشخصية المتعلقة بالمطالب االجتماعية مع االحتفاظ باالرتباط اإليجابي م ع العائلة ،.وتتميز مرحلة المراهقة بالتغيرات االنفعالية واغلب هذه التغيرات من النوع العنيف الذي يجعل صورة المراهق غير صورة الطفل الهادئ الذي كان عليها في المراحل ،السابقة (محمود1981 ). ويتم من خالل مراقبه نشاطات المراهقين وصداقاتِهم م نح الحكم الذاتي ، وا لتحدّث م عهم، وتوفير عِالقات آمنة ومساعِدة ودافئة إلى تزويد المراهق بالسياق والبيئة األسرية الضرورية الزدهار قدرة وكفاءة المراهقين االجتماعية والنفسية(Markiewicz et al., 2001 ) . ويعـد تصـوّر المـراهقِين لحيـاتِهم العائليـة وفهمهـا واحـداً مـن األساليب الم .تبعة لفحص واستكشاف األداء والوظيفة العامة لألسرة وتدرك الوظيفة العائلية كمصفوفة من المهامِ (البيولوجية واالجتماعية واالقتصـادية والعاطفيــة والنفســية واألخالقيــة والدينيــة ) التــي تســهم بشــكل رئــيس فــي ت طـوير مهـاراتِ المـراهقين المتعلقـة بالمواجه ـة والتعامـل بكفـاءة مـع مواقـف اإلجهــاد اليــومي ( 1996 (Alnajja, . وتتطلب تربية المراهق الصحيحة أسرة سوية، يسودها االتزان والعالقات السليمة، التي توفر للمراهق اإلشباع العاطفي والشعور باالنتماء وتقدير الذات، وتتسع إلى الثقة بين المراهق ووالديه، فالثقة المتبادلة بين االثنين لها أثرها الهام في االستقرار االنفعالي والنضج االجتماعي للمراهق، ويساعد على بناء هذه الثقة اطمئنان المراهق لحب والديه ومساعدتهما إياه ، في متاعبه الخاصة مساعدة مترفة هادئة، وكذلك اعتزازه بشخصيتهما من حيث اتساع األفق الفكري والنجاح اال جتماعي والصفات ،السلوكية وغيرها من المقومات التي يتمناها المراهق في والديه(أحمد2009 ). IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 UG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 460 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين د. إيهاب العصافرة تاال العموش د. إيهاب العصافرة تاال العموش خلفية الدراسة وأهميتها 461 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4 0 وتتميز مرحلة المراهقة بالتغيرات االنفعالية، التي تتمثل في،البحث عن الذات التي قد تعكس عدم قدرة المراهق على ضبط ذاته فضبط الذات هو القدرة على استخدام الوعي الذاتي إلدر اك المشاعر في وقتها على نحو صحيح وفهم ميول وردود األفعال تجاه األشخاص واألحداث والمواقف، لقد ولدنا جميعا ولدينا القدرة على ضبط ذواتنا والسيطرة عليهـا لكن البعض يستخدمها أكثر من غيره فاألداء الناجح سـواء في المدرسـة أو في مكـان غيرهـا يعتمـد علـى القـدرة ع لـى ضبط الذات، فهو أمر مهم ال يسـمح للفـرد أن يتحـرك نحـو أهـداف بعيـدة وقـدرتهم علـى مواجهـة التحـديات فـالفرد الـذي لديـه ضـعف في القـدرة علـى السـيطرة علـى ذاتـه يـؤدي إلى عواقـب غـير مرغـوب فيهـا، إذن فـوعي الفرد بأهمية ضبط ذاته يجنبـه الفشـل في حيات ـه وتحقيـق النجـاح في العديـد مـن مجـاالت 461 د. إيهاب العصافرة تاال العموش الحيـاة وتحـدي الصـعاب وحـل المشـكالت، فـالفرد ال يعـيش منعـزال عـن اآلخـرين بـل إن وجـوده مـرتبط بـاآلخرين لـذلك فـإن الصـعوبات والمشـاكل الـتي تواجهـه تتطلب مشاركة اآلخـرين معـه إلحـداث حلـول وتغيـير في المواقف الـتي يتعـرض لهـا وتعـديل ،سـلوكياته(نسيمة2019 .) و يرتبط االتزان االنفعـالي وتحمـل المسـؤولية بشـكل وثيـق مـع العديـد مـن المتغـيرات الشخصـية، وخاصـة القـدرة علـى ضبط الذات فكلما تمتع الفـرد بقـدرة علـى ضـبط الـذات ّ اسـتطاع أن يسـيطر علـى انفعاالتـه ويتم تـع بـاتزان انفعـالي يمكنه من السيطرة على سلوكياته، بحيث يكون قادر على تحمل المسؤولية في جميع المواقف المختلفة ،(حسن1989 ). ويشـير االتـزان االنفعـالي إلى الفـرد الهـادئ الـذي يتسـم بالثبـات االنفعـالي وتظهـر عليـه عالمـات قليلـة مـن التهـيج االنفعالي إزاء أي ،نوع من المعارضة والغضب، ويكون واقعيا في الحياة منضبطا ذاتيا ومثابراً (سويف1996 ). و تعد األسرة بمثابة المكان االجتماعي الذي تنمو فيه بذور الشخصية اإلنسانية، وتترسخ أصول التطبيع االجتماعي والطبيعة اإلنسانية للفرد، وكما يتشكل الوجود البيولو جي للجنين في رحم األم، كذلك يتشكل الوجود االجتماعي للطفل في (األسرةMargorie,1993 ). خلفية الدراسة وأهميتها كما استخدمت الدراسة استبيان التنشئة والتكيف حيث تم التأكد من صدقها من خالل صدق االتساق الداخلي للبنود وأما الثبات من خالل معامل ألفا كرونباخ والتج زئة النصفية، حيث كانت معامالت الصدق والثبات جيدة، ثم طبقت األداة على عينة الدراسة والتي قدرها ( 140 .) طالبة من جامعة الوادي ولقد توصلنا في الدراسة الحالية إلى مجموعة من النتائج توجد عالقة ذات داللة إحصائية بين أساليب التنشئة األسرية وتكيف الطالبة في الوسط ا لجامعي. ( كما وأجرى عالء الدين والعلي2014 )) دراسة سـعت الستكشـاف العالقـات بـين األداء الـوظيفي األسـري (التماســك، التكيــف والممارســات الوالديــة والكفــاءة االجتماعيــة والتقــارير الذاتيــة للمــراهقين عــن درجــة القلــق لــديهم وجــنس المــراه ق ين. تألفــت عينــة =الدراســة مــن(ن378 ) طالباً وطالبة في الصفوفِ السابع والثامن والتاسع اختيروا عشوائياً مـن المــدارس الحكوميــة فــي محافظــة =الزرقــاء فــي األردن بمتوســط عمــري وانحــراف معياري بلغا (م13.6 =؛ ع1.06 ) أشارت نتائج الدراسة لوجود عالقة دالّة بـين الممارسـات الوالديـة اإليجابيـة وأداء األسـرة الصـحي لوظيفتهـا المتعلقـة بالتماسـك والتكيّف، كما أظهرت نتـائج تحلـيالت االنحـ دار المتـدرج أن عـاملي (أ) الممارسـات الوالديـة السـلبية لـألم و(ب) انخفـاض مسـتوى التماسـك األسـري أسـهما فـي التنبـؤ ب تقـديرات الطلبـة للقلـق، وأنّ عـامليْ (أ) الممارسـات الوالديـة اإليجابيـة لـألب و(ب)ارتفــاع مســتوى التماســك األســري، أســهما فــي التنبــؤ ِبتقــديرات الوالــدين للكفــاءة االجتماعيـة. وتبـين أيضـاً أن عامـل الممارسـة الوالديـة اإليجابيـة لـألب كـان ال عامـل الوحيد والفريد الذي أسهم في التنبؤ بتقـديرات المعلمـين للكفـاءة االجتماعيـة لـدى طالبهـم، وأخيـراً، أظهـرت النتـائج عـدم وجـود فـروق علـى مقـاييس الدراسـ ة تُعـزى لجنس المراهق باستثناء مقياس القلقِ. ت قترحُ الدراسة إمكانية اسْ تِعْمالِ هذه النتائج لت ص ِميم األهدافِ لتدخّالتِ العالجّ واإلرشاد .األسري وبرامج التعليم النفسـي لألسـرِ المختلــة وظيفيــاً وأطفالِهــا 463 IUG J l f Ed ti l d P h l S i (I l i U i it f G ) / CC BY 4 0 ( وقام الحربي2009 ) بدراسة أساليب التنشئة األسرية وعالقتها بكل من التفاؤل والتشاؤم لدى عينه من تالميذ المرحلة المتوسطة والثانوية بمنطقة جازا ن ومن أهداف الدراسة، تهدف الدراسة الحالية إلى معرفة العالقة بين أساليب التنشئة األسرية وكل من التفاؤل والتشاؤم لدى عينة الدراسة. إمكانية التنبؤ بكل من التفاؤل والتشاؤم من خالل أساليب التنشئة األسرية. واشتملت عينة ( الدراسة629) طالب وطالبة موزعة على النحو ا( لتالي273 طالب و356 طالبة ) من طالب الصف الثاني متوسط والصف الثاني ثانوي شرعي وطبيعي بمنطقة جازان. واستخدم األساليب اإلحصائية التالية، معامالت ارتباط بيرسون، اختبار ت، تحليل التباين األحادي االنوفا، اختبار شيفيه، االنحدار الخطي المتعدد. خلفية الدراسة وأهميتها وكلما زادت قدرة األسرة على رعاية أبنائها و توجيههم و تنشئتهم، دون أن يشعروا بالحرمان أو الضغط أو القسوة أو التساهل كان االبن سوياً قادرا على تحمل مسؤوليته، في إطار احترامه وتقديره لذاته، و ذوات ،اآلخرين في الوقت نفسه، أو القسوة أو التساهل ،كان في الوقت نفسه(قنديل و شلبي2006 .) و من أهداف التنشئة األسرية إتاحة الفرصة بالتعبير عن ذاته وتعويده على حل المشكالت وعلى اتخاذ القرار بنفسه وعدم اللجوء باستمرار أل بويه في كل صغير ة وكبيرة وتوجيه ه مهنيا للعمل فيما بعد حتى ال يكون عالة على أسرته أو مجتمعه، واالستقالل يجب أن يكون اجتماعيا ونفسيا وان تغرس المسؤولية في شتى مراحل النمو،(مصباح2003 ). و لقد تم مراجعة العديد من الدراسات السابقة التي تناولت متغير أساليب التنشئة األسرية وضبط الذات وعالقتها بالعديد من .المتغيرات االجتماعية والشخصية. ولكن لوحظ قلة الدراسات التي ربطت بين أساليب التنشئة األسرية وضبط الذات بشكل عام ( أجرى الداير2016 )درا سة هدفت إلى التعرف على طبيعة العالقة،بين أساليب التنشئة األسرية والتوافق النفسي من وجهة نظر طلبة دبلوم التعليم العام بمحافظة جنوب الباطنة خالل العام الدراسي2015 / 2016 ( . وقد تكونت عينة الدراسة من316 ) طالباً وطالبة، من طلبة دبلوم التعليم العام بمدارس واليت محافظة جنوب الباطنة في ثالث واليات من المحافظة وهي: والية المصنعة- والية بركاء- (والية وادي المعاول. منهم160 ( ) طالباً، و156 ) طالبة، وقد اختيرت عينة الدراسة األساسية بالطريقة العشوائية. ولتحقيق أغراض الدراسة قام الباحث بتطبيق مقياسين، األول مقياس أساليب التنشئة األسرية، والثاني مقياس التوافق النفسي للطلبة، وأظهرت الدراسة النتائج بأن أن أسلوب التقبل الوالدي أكثر أساليب التنشئة األسرية شيوعاً كما يدركها طلبة دبلوم التعليم العام في مدارس محافظة جنوب الباطنة. أن التوافق الصحي واألسري هما أكثر أساليب التوافق النفسي شيوعاً لدى طلبة دبلوم التعليم العام في مدارس محافظة جنوب الباطنة . IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 462 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين د. إيهاب العصافرة تاال العموش د. إيهاب العصافرة تاال العموش وأجر ى أيضاً كل من( العلجي وبلعربي2016 ) دراسة تهدف إلى معرفة وكشف العالقة بين أساليب التنشئة األسرية المتبعة من طرف األسرة والتكيف لدى الطالبة سنة أولى جامعي ولقد تم االعتماد على المنهج الوصفي اإلرتباطي وهذا لوصف الظاهرة المدروسة وألنه كان األنسب لدراسة الموضو ع وألننا نسعى إلى معرفة االرتباط بين التنشئة األسرية والتكيف طالبة جامعية وتعميم النتائج والتنبؤ بها مستقبال. خلفية الدراسة وأهميتها وكانت نتائج الدر اسة، وجود عالقة عكسية ذات دالله إحصائية ،بين التفاؤل ومعاملة األب واألم في األبعاد التالية (اإليذاء الجسدي، الحرمان، القسوة، اإلذالل، واإلشعار بالذنب، وتفضيل اإلخوة والتذليل) والرفض بالنسبة لمعاملة األم ، كما توجد عالقة ذات داللة إحصائية بين التفاؤل ومعامل ة األب واألم في األبعاد (التسامح، والتعاطف الوالدي، والتوجيه نحو األفضل، والتشجيع )، كما انه ال توجد عالقة ذات دالله إحصائية بين التفاؤل معاملة األب واألم في األبعاد (الحماية الزائدة ، التدخل الزائد ) والرفض بالنسبة لمعاملة األب، كما انه توجد عالقة ذات دال له إحصائية ،بين التشاؤم ومعاملة األب واألم في األبعاد التالية (اإليذاء الجسدي، الحرمان، القسوة، اإلذالل، واإلشعار بالذنب، وتفضيل اإلخوة IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 إاإإأأأ 463 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين د. إيهاب العصافرة تاال العموش د. إيهاب العصافرة تاال العموش د. إيهاب العصافرة تاال العموش والتذليل) والحماية الزائدة بالنسبة لمعاملة األب، كما توجد عالقة عكسية ذات داللة إحصائية بين التشاؤم ومعاملة األب واألم ف ي األبعاد التالية (تعاطف الوالدي، التوجيه نحو األفضل، والتشجيع) كما انه ال توجد عالقة ذات دالله إحصائية بين التشاؤم ومعاملة األب واألم في األبعاد التالية (التسامح، التدخل الزائد)، والحماية الزائدة بالنسبة لمعاملة األم. وجود فروق ذات دالله إحصائية بي ن الذكو ،ر واإلناث، في المجاالت التالية (اإليذاء الجسدي، والحرمان، والقسوة، واإلذالل، والرفض، والحماية الزائدة، والتدخل الزائد ،والتوجه لألفضل، اإلشعار بالذنب، والتشجيع، وتفضيل األخوة، والتدليل) لمعاملة األب، وفي المجاالت التالية (اإليذاء الجسدي ،والقسوة، واإلذالل واإلشعار بالذنب، وتفضيل اإلخوة، والتدليل) لمعاملة األم. كما انه ال توجد فروق ذات دالله إحصائية بين ،الذكور واإلناث في المجاالت التالية (التسامح والتعاطف الوالدي) لمعاملة األب، وفي المجاالت التالية (الحرمان، والرفض ،والتدخل الزائد، التسامح والتعاطف الوالدي . والتشجيع) لمعاملة األم كما قامDwairy,2009) ) دراسة هدفت إلى معرفة العالقة بين عوامل األسرة واألبوة واالضطرابات النفسية للمراهقين، جمعت ( حيث بلغت عينة الدراسة1358 ( ) مراهقا و1526) مراهقة ، جمعت من تسعة بلدان لمعرفة المستويات المختلفة من االضطرابات الن ،فسية للمراهقين، وأظهرت النتائج أن للعوامل األسرية واألبوية ترتبط ارتباطاً كبيرا باالضطرابات النفسية للمراهقين حيث تفسر37،2 من هذه االضطرابات% . IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 خلفية الدراسة وأهميتها ( كما أجرى العطوي2006) دراسة هدفت إلى التعرف على أثر أنماط التنشئة األسرية على تقدير الذات عند طلبة المرحلة ال ثانوية ( في مدينة تبوك بالمملكة العربية السعودية, وتكونت عينة الدراسة من660 ( ) طالبا وطالبة بينهم300 () طالبا و360 .) طالبة ،تم اختيـارهم بالطريقة العشوائية العنقودية, وقد تم استخدام مقياس التنشئة األسرية الـذي طـوره ( الهنداوي، والزغول ،والبكور2001) ومقي،اس تقدير الذات الذي طورته (قـسوس1985 ) وتوصلت الدراسة الى النتائج التالية، وجود أثر لنمط تنشئة األب (المتسلط، الديمقراطي، المهمل) في تقدير الذات، وجود أثر لنمط تنشئة األم (المتسلط، الديمقراطي، المهمل) في تقدير الذات، وجود فروق في نمط تنشئة األب المتسلط تبع ،ا للنوع االجتماعي, وعدم وجـود فروق في نمطي تنشئة األب (الديمقراطي والمهمل) تبعا للنوع االجتماعي، وجود فروق تبعاً للنوع االجتماعي في نمط تنشئة األم (المتسلط، المهمـل) فـي تقدير الذات، وعدم وجود فروق في نمط تنشئة األم (الديمقراطي ،)عدم وجود فروق ذات داللة إ حصائية في درجـة تقـدير الـذات تبعـا للنـوع االجتماعي . توصي الدراسة بإعداد البرامج اإلرشادية لألسر حول كيفية رعاية األبنـاء حسب األساليب التربوية الحديثة، وإجراء مزيد من الدراسات العلمية حول متغيرات الدراسة . ( وأجرى كيمبف (Kempf,2005 دراسة هدفت إلى معر فة مدى تأثير المتغيرات األسرية والوالدية المتعددة على االستقالل عند المراهق ضمن عينة من مراهقي منطقة األباالش بوالية كارولينا األمريكية. تكونت عينة ( الدراسة من707 ) طالباً من طالب المرحلة الثانوية، حيث أستخدم الباحث تحليل االنحدار المتعدد الهرمي لتحديد التأثير النسبي لمتغيرات السلوك الوالدي المتعدد والعالقات األسرية وتركيب األسرة على السلوك االستقاللي عند المراهقين، وتوصل الباحث إلى أن المتغيرات األساسية التي تشجع االستقالل عند المراهق تتضمن التقبل الوالدي واستخدام المنطق، أي استخدام الحجج والبراه ين، بينما تمنع القسوة الوالدية االستقالل عند المراهق. 464 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين د. إيهاب العصافرة تاال العموش د. إيهاب العصافرة تاال العموش وتتفق هذه الدراسة الحالي ة مع(دراسة الحربي2009 ()، ودراسة العطوي2006 ) (ودراسة الداير2016 ) و دراسة ( Dwairy,2009 ) إلى وجود عالقة ارتب اطيه بين السلوك الوالدي وتحقيق ا شباعات األبناء في الحياة، مثل الشعور بال رضا واالتزان االنفعالي والشعور (بالسعادة. وأن للعوامل األسرية واألبوية ترتبط ارتباطاً كبيرا باالضطرابات النفسية للمراهقين. وعند استعراض دراسة الحربي2009 ) يتبين أنه توجد عالقة بين التشاؤم ومعاملة األب واألم في األبعاد التالية (اإليذاء الجسدي، الحرمان، القسو ة، اإلذالل، واإلشعار (بالذنب، وتفضيل اإلخوة، والتذليل) والحماية الزائدة بالنسبة لمعاملة األب. خلفية الدراسة وأهميتها ويالحظ من دراسة العطوي2006 ) إلى أثر أنماط .التنشئة األسرية ( الديمقراطي، المهمل، المتسلط) على تقدير الذات عند طلبة المرحلة الثانوية وتختلف الدراسة الحالي ة عن الد راسات السابقة أنها توجه االهتمام إلى فئة طلبة المراهقين، ألن هذه الفئة من أكثر الفئات التي تحتاج إلى تنمية مهارات ضبط الذات ومعرفة أساليب التنشئة األسرية المتبعة من قبل الوالدين على النمو النفسي واالجتماعي .والتعامل مع المواقف المختلفة و يظهر تفرد الدراسة وأهميتها في كشفها عن عالقة بين أساليب التنشئة األسرية وضبط الذات لدى طلبة المرحلة الثانوية في مدينة الرصيفة، وأيضاً الكشف عن مستوى أساليب التنشئة األسرية وضبط الذات و هو لم تتطرق له الدراسات السابقة . مشكلة الدراسة وأسئلتها : من خالل عمل الباحث ان في ،الميدان التربوي وممارسة مهنة اإلرشاد لسنوات عدة حيث الحظ الباحثان بأن مرحلة المراهقة من المراحل التي يرافقها العديد من المشكالت النفسية واالجتماعية والتكيفية، لذلك تولد لدى الباحثان إحساس بضرورة معرفة أساليب التنشئة األسرية التي تمارس من قبل الوالدين وعال قتها بالقدرة على ضبط الذات لدى الطلبة، وذلك كون األسرة هي المكان األساسي إلشباع الحاجات البيولوجية والنفسية واالجتماعية لألبناء، فإذا حدث خلل في البناء األسري وأسلوب التنشئة األسرية أو الوالدية، فإن ذلك سيترتب عليه زيادة المشكالت النفسية واالجتماعية وعد م القدرة على ضبط الذات من خالل فقد أعصابهم واالنسياق وراء مشاعرهم، وفعل الكثير من األمور بدون تخطيط، وقلة الوعي الذاتي والمتمثل في إدراك المشاعر في وقتها وعلى نحو صحيح وفهم ميول وردود األفعال تجاه األشخاص واألحداث والمواقف، فاألساليب الوالدية المتبعة تنعك س إيجاباً وسلباً وفقا لنمط األسلوب المتبع. :وبناء على ما سبق فإن الدراسة الحالية تحاول اإلجابة على األسئلة التالية من خالل عمل الباحث ان في ،الميدان التربوي وممارسة مهنة اإلرشاد لسنوات عدة حيث الحظ الباحثان بأن مرحلة المراهقة من المراحل التي يرافقها العديد من المشكالت النفسية واالجتماعية والتكيفية، لذلك تولد لدى الباحثان إحساس بضرورة معرفة أساليب التنشئة األسرية التي تمارس من قبل الوالدين وعال قتها بالقدرة على ضبط الذات لدى الطلبة، وذلك كون األسرة هي المكان األساسي إلشباع الحاجات البيولوجية والنفسية واالجتماعية لألبناء، فإذا حدث خلل في البناء األسري وأسلوب التنشئة األسرية أو الوالدية، فإن ذلك سيترتب عليه زيادة المشكالت النفسية واالجتماعية وعد م القدرة على ضبط الذات من خالل فقد أعصابهم واالنسياق وراء مشاعرهم، وفعل الكثير من األمور بدون تخطيط، وقلة الوعي الذاتي والمتمثل في إدراك المشاعر في وقتها وعلى نحو صحيح وفهم ميول وردود األفعال تجاه األشخاص واألحداث والمواقف، فاألساليب الوالدية المتبعة تنعك س إيجاباً وسلباً وفقا لنمط األسلوب المتبع. :وبناء على ما سبق فإن الدراسة الحالية تحاول اإلجابة على األسئلة التالية 1 – ما مستوى أساليب التنشئة األسرية لدى الطلبة المراهقين؟ 2 -ما مستوى ضبط الذات لدى الطلبة المراهقين؟ 3 - هل توجد عالقة ارتباطية ذات داللة إحصائية بين أساليب التنشئة األسرية وضبط الذات لدى الطلبة المراهقين؟ :أهداف الدراسة 1 -التعرف على مستوى أساليب التنشئة األسرية لدى الطلبة المراهقين. أ 2 -ما مستوى ضبط الذات لدى الطلبة المراهقين؟ 3 - هل توجد عالقة ارتباطية ذات داللة إحصائية بين أساليب التنشئة األسرية وضبط الذات لدى الطلبة المراهقين؟ :أهداف الدراسة 1 -التعرف على مستوى أساليب التنشئة األسرية لدى الطلبة المراهقين. 2 -التعرف على مستوى ضبط الذات لدى الطلبة المراهقين. 3 - التعرف على العالقة بين التنشئة وضبط الذات لدى طلبة المراهقين. مشكلة الدراسة وأسئلتها : IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 465 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين د. إيهاب العصافرة تاال العموش 1 - ،قلة االهتمام الكافي لدى الباحثين في البيئة العربية بموضوع التنشئة األسرية وضبط الذات حيث أن الدراسات التي .أجريت في هذا المجال قليلة محليا 2 - تستمد أهميتها أيضاً في تحسين فهمنا للشخصية وباألخص شخصية الطلبة المراهقين في مديرية تربية لواء الرصيفة . :األهمية التطبيقية - توفر الدراسةُ عددًا من المقاييس التي يمكن استخدامها في المجاالت البحثية ذات الصلة بالموضوع. وتمهد هذه الدراسة الطريق نحو : 1 _ تطوير برامج إرشادية تتعلق بتحسين أساليب التنشئة األسرية وضبط الذات ، 2 _ التحقق من فعاليتها .في تحسين جوانب مختلفة في مجال صحة الفرد النفسية :التعريفات االصطالحية واإلجرائية :أساليب التنشئة األسرية هو األسلوب الذي يتلقاه االبن من والديه في المنزل وطبيعة عال قته بهما، فأسلوب التنشئة األسرية هو عملية تفاعل يٌع دل عن طريقها سلوك الفرد، وهو كل سلوك يصدر من الوالدين ويؤثر على األبناء وعلى ،شخصياتهم(دندي2010 .)وأنه يقاس بمجموع الدرجات التي يحصل عليها المستجيب لمقياس أساليب التنشئة األسرية المستخدم في .الدراسة ضبط الذات : هو اإلجراء الذي يتبعه الفرد من تلقاء نفسه في ضبط سلوكه وفق قوانين وقواعد بصيغها لنفسه من اجل تحقيق أهداف معينة، وقدرة الفرد على التحكم في انفعاالته وسلوكياته ومعرفته بها وكيفية التعبير عنها في ظل الضغوط التي يتعرض لها ،(نسيمة2019 ).وأنه يقاس .بمجموع الدرجات التي يحصل عليها المستجيب لمقياس ضبط الذات المستخدم في الدراسة المراهقه : وهي الفترة العمرية التي تمتد من عمر12 - 21 سنة، وفي هذا الدراسة، هم طالب المرحلة الثانوية في مدرسة الحسن .بن الهيثم في لواء الرصيفة محددات الدراسة : ستقتصر هذه :الدراسة على الحدود اآلتية - الحدود البشرية: تم إجراء الدراسة على المرحلة الثانوية في مديرية تربية لواء الرصيفة في مدينة الزرقاء . - الحدود الزمانية: تم إجراء الدراسة خالل الفصل الدراسي الثاني من العام الدراسي2020 / 2021 . - الحدود المكانية: تم تطبيق الدراسة .في مدرسة الحسن بن الهيثم الثانوية للبنين التابعة لمديرية لواء الرصيفة - :الحدود اإلجرائية تتحدد نتائج الدراسة الحالية باستجابة المفحوصين على مقاييس الدراسة التي تم تطويرها من قبل الباحث ان .والتي تستخدم لجمع البيانات (تم استخدام المنهج الوصفي االرتباطي) وهو أحد أنواع مناهج البحث العلمي التي تهتم ببيان العالقة بين متغيرين أو أكثر، كما يهتم بمعرفة نوع هذه العالقة سواء أكانت سالبة أم مو به ج.. مشكلة الدراسة وأسئلتها : IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 ا IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 ا 466 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين د. إيهاب العصافرة تاال العموش مجتمع :الدراسة تكون مجتمع الدراسة من الطلبة المراهقين في مدرسة الحسن بن الهيثم الثانوية في مديرية تربية لواء الرصيفة. وبلغ عددهم140 طالب من العام الدراسي2020 / 2021 . عينة الدراسة: تم اختيار العينة بالطريقة العشوائية البسيطة من المجتمع األصلي، حيث بلغ عدد أفراد ا( لعينة100 ) ،طالب من المرحلة الثانوية ( أما العينة االستطالعية فتكونت من(30 .طالب من خارج عينة الدراسة أدوات الدراسة: تم اختيار العينة بالطريقة العشوائية البسيطة من المجتمع األصلي، حيث بلغ عدد أفراد ا( لعينة100 ) ،طالب من المرحلة الثانوية ( أما العينة االستطالعية فتكونت من(30 .طالب من خارج عينة الدراسة أدوات الدراسة: تم اختيار العينة بالطريقة العشوائية البسيطة من المجتمع األصلي، حيث بلغ عدد أفراد ا( لعينة100 ) ،طالب من المرحلة الثانوية ( أما العينة االستطالعية فتكونت من(30 .طالب من خارج عينة الدراسة من أجل تحقيق أهداف الدراسة قام الباحث ان بتطوير مقياس أس اليب التنشئة األسرية من خالل اال طالع على األدب النظري :والرجوع إلى عدة مقاييس ومنها المقياس المستخدم في دراسة(العلجي وبلعربي2016 )، والمقياس المستخدم في دراسة الداير ( 2016 )، ( والمقياس المستخدم في دراسة العطوي2006 ). و أما مقياس ضبط الذات الذي تم تطويره من خالل اال طالع على األدب النظري، وا:لرجوع إلى عدة مقاييس ومنها(المقياس المستخدم بدراسة نسيمة2019 ،) والمقياس المستخدم في دراسة الربيع ( وعطية2016 ) . :أوال: مقياس أساليب التنشئة األسرية تألف المقياس من( 20 ) فقرة بحيث يجيب عنها الطلبة في ضوء مقياس ليكرت خماسي التدريج ،ً(دائماً، غالبا .)ًأحياناً، نادراً، أبدا :صدق المحتوى للمقياس( للتحقق من صدق المحتوى عُرض المقياس بصيغته األولية على10 ) من المحكمين من أعضاء هيئة التدريس المتخصصين في علم النفس التربوي ، واإلرشاد النفسي والتربوي، والقياس والتقويم للحكم على صالحية الفقرات ومدى دقتها ووضوحه ا ومناسبتها لغوياً، وقد اعتمد معيار80 فأكثر كنسبة اتفاق بين المحكين على الفقرة الواحدة، وقام الباحثان% إلى التحكيم نتائج وخلصت مالحظاتهم، ضوء في بالتعديل الفقرات لبعض الصياغة إعادة (وهي2،6،5،9،10،11،12،13،14،17 ( )، ليصبح المقياس بصورته النهائية يتكون من20 .) فقرة :صدق البناء للمقياس( لحساب صدق البناء تم تطبيق المقياس على عينة مكونة من30 ) طالبٍ من خارج عينة الدراسة، حيث .تم حساب قيم معامالت االرتباط بين الفقرة والبعد الذي تنتمي له وال يوجد درجة كلية للمقياس. 467 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaz مشكلة الدراسة وأسئلتها : وأشارت نتائج التحليل إلى أن جميع معامالت االرتباط بين كل فقرة مع البعد، والفقرة مع المقياس الكلي كانت دالة إحصائياً، وتراوحت قيم معامالت االرتباط بين ( درجة الفقرة والبعد0,57 - 0,82 ،)والجدول التالي يبين ذلك . 467 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4 0 (جدول1 ) معامالت االرتباط بين الفقرة و الدرجة الكلية للبعد التي تنتمي إليه رقم الفقرة معامل االرتباط رقم الفقرة معامل االرتباط رقم الفقرة معامل االرتباط 1 .64 8 .80 15 .60 2 .77 9 .76 16 .75 3 .57 10 .62 17 .82 4 .64 11 .77 18 .70 د. إيهاب العصافرة تاال العموش أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين رقم الفقرة معامل االرتباط رقم الفقرة معامل االرتباط رقم الفقرة معامل االرتباط 5 .70 12 .72 19 .73 6 .61 13 .60 20 .64 7 .68 14 .67 * ( دالة إحصائيا عند مستوى الداللة0.05 .) ( دالة إحصائيا عند مستوى الداللة0.01 .) وتجــدر اإلشــارة أن جميــع معــامالت االرتبــاط كانــت ذات درجــات مقبولــة ودالــة إحصــائيا ، ولــذلك لــم يــتم حــذف أي مــن هــذه .الفقرات ( دالة إحصائيا عند مستوى الداللة0.01 .) وتجــدر اإلشــارة أن جميــع معــامالت االرتبــاط كانــت ذات درجــات مقبولــة ودالــة إحصــائيا ، ولــذلك لــم يــتم حــذف أي مــن هــذه .الفقرات :ثبات المقياس تم التحقق من ثبات المقياس بحساب قيم معامالت االتساق الداخلي باستخدام معادلـة (كرونبـاخ ألفـا)، وبلغـت قيمـة االتسـاق الـداخلي للمقيــاس ككـل( 0,80 ) )، كمــا تـم حســاب أبعـاد المقيــاس: (الـنمط الــديمقراطي، والـنمط التســلطي، والـنمط المتســاهل باستخدام معادلـة (كرونبـاخ ألفـا)، حيـث بلغـت علـى التـوالي) 0, 77 ، 0,83 ، 0,80 ). و تشـير هـذه القـيم إلـى تمتـع المقيـاس بـدالالت ثبات مقبولة ألغراض الدراسة الحالية. كما تم التحقق من ثبات المقياس من خـالل طريقـة ثبـات اإلعـادة حيـث قـام الباحثـان بتطبيـق ( المقياس علـى عينـة اسـتطالعية مكونـة مـن30 ( طالـب مـن خـارج عينـة الدراسـة ، وبعـد مـرور أسـبوعين أعيـد تطبيـق ا لمقيـاس مـرة أخــرى علــى ذات المجموعــة، وبلــغ معامــل ثبــات المقيــاس الكلــي( 0,81 ). كمــا تــم حســاب أبعــاد ثبــات اإلعــادة ،(الــنمط الــديمقراطي والنمط التسلطي، والنمط المتساهل) حيث بلغت على التوالي ( 0, 90 ، 0,90 ، 0,85). IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 مشكلة الدراسة وأسئلتها : وتشير هذه القيم إلى تمتع المقياس بـدالالت ثبات مقبولة.ألغراض الدراسة الحالية أ طريقة التصحيح( : لإلجابة على فقرات المقياس، أُستخدم تدريج ليكرت الخماسي وبدرجات تتراوح بين5 - 1 ) تمثل اإلستجابة على ( كل فقرة، حيث أن الرقم5 ( ) يعني دائماً على الفقرة، والرقم4 ( ) يعني غالباً على الفقرة، والرقم3 ) يعني أحياناً على الفقر ة، والرقم ( 2 ) يعني نادراً على الفقرة، و( الرقم1) يعني أبداً على الفقرة ،وتم عكس الدرجات بالنسبة للفقرات السلبية وللحكم على مستوى أساليب التنشئة األسرية قد جرى استخدام التحليل االحصائي التالي لتوزيع المتوسطات الحسابية وذلك حسب المعيار الذي قاس :كل فقرة طول الفئة= الحد األعلى للبدائل- .الحد األدنى/ عدد المستويات = طول الفئة5 – 1 / 3 = 1.33 :وبذلك تكون حدود المستويات على النحو اآلتي ( 1 - 2.33 .) مستوى منخفض ( 2.34 – 3.67 .) مستوى متوسط ( 3.68 - 5 .) مستوى مرتفع :ثانياً: مقياس ضبط الذات = طول الفئة5 – 1 / 3 = 1.33 آ :ثانياً: مقياس ضبط الذات ( تألف المقياس من12 ) فقرة بحيث يجيب عنها الطلبة في ضوء مقياس ليكرت خماسي التدريج .)ً(دائماً، غالباً، أحياناً، نادراً، أبدا :صدق المحتوى للمقياس( للتحقق من صدق المحتوى عُرض المقياس بصيغته األولية على10 ) من المحكمين من أعضاء هيئة التدريس المتخصصين في علم النفس التربوي ، واإلرش اد النفسي والتربوي، والقياس والتقويم للحكم على صالحية الفقرات ومدى دقتها ووضوحها ومناسبتها لغوياً، وقد اعتمد معيار80 فأكثر كنسبة اتفاق بين المحكين على الفقرة الواحدة، وقام الباحثان% بالتعديل في ضوء مالحظاتهم، وخلصت نتائج التحكيم إعادة الصياغة لبعض الفقرات ( وهي5،8،11،1،7،6،9 )، ليصبح ( المقياس بصورته النهائية يتكون من12 .) فقرة IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 () IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 468 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين د. إيهاب العصافرة تاال العموش :صدق البناء للمقياس(لحساب صدق البناء تم تطبيق المقياس على عينة مكونة من30 ) طالب من خارج عينة الدراسة، حيث تـم حساب قيم معامالت االرتبـاط بـين الفقـرة والدرجـة الكليـة للمقيـاس. وأشـارت نتـائج التحليـل إلـى أن جميـع معـامالت االرتبـاط بـين كـل فقـــرة والدرجـــة الكليـــة للمقيـــاس كانـــت دالـــة إحصـــائياً، وتراوحـــت قـــيم معـــامالت االرتبـــاط بـــين درجـــات الفقـــرات والمقيـــاس الكلـــي بـــين ( 0.41 - 0.81 )، والجدول التالي يبين ذلك. مشكلة الدراسة وأسئلتها : إيهاب العصافرة تاال العموش أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين :السؤال األول ما مستوى أساليب التنشئة األسرية لدى الطلبة المراهقين؟ لإلجابة عن هذا السؤال تم استخراج المتوسطات الحسابية واالنحرافات المعيارية ل مستوى أساليب التنشئة األسرية لدى الطلبة المراهقين، و لقد تم حساب المتوسطات الحسابية واالنحرافات المعيارية لتقديرات أفراد عينة الدراسة على فقرات كل نمط على حدة ، .والجدول أدناه يوضح ذلك :أوال النمط الديمقراطي ( جدول3 ) المتوسطات الحسابية واالنحرافات المعيارية للفقرات المتعلقة بالنمط الديمقراطي مرتبة تنازلياً حسب المتوسطات الحسابية الرتبة الرقم الفقرات المتوسط الحسابي االنحراف المعياري المستوى 1 2 توجهني أسرتي إلى أن أعتمد على نفسي في تحقيق ما أريده. 2.52 1.022 متوسط 2 1 .أتبادل مع والدي في أمور األسرة 2.35 1.068 متوسط 3 7 تشجعني أسرتي على أن .أمارس هواياتي وميولي الخاصة 2.25 1.064 منخفض 4 4 تقدم لي أسرتي التعزيز عند تنظيم أوقاتي ويعاقبوني على .الفوضى 2.23 1.165 منخفض 4 5 اشعر بالرعاية واالهتمام من طرف أسرتي. 2.23 1.093 منخفض 6 3 .تؤكد أسرتي على التعاون والتضامن داخل األسرة 2.20 1.104 منخفض 7 6 تستشيرني أسرتي في القرارات التي تخصني. 2.07 1.103 منخفض النمط الديمقراطي 2.26 .738 منخفض أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين :السؤال األول ما مستوى أساليب التنشئة األسرية لدى الطلبة المراهقين؟ لإلجابة عن هذا السؤال تم استخراج المتوسطات الحسابية واالنحرافات المعيارية ل مستوى أساليب التنشئة األسرية لدى الطلبة المراهقين، و لقد تم حساب المتوسطات الحسابية واالنحرافات المعيارية لتقديرات أفراد عينة الدراسة على فقرات كل نمط على حدة ، .والجدول أدناه يوضح ذلك :أوال النمط الديمقراط ( جدول3 ) المتوسطات الحسابية واالنحرافات المعيارية للفقرات المتعلقة بالنمط الديمقراطي مرتبة تنازلياً حسب المتوسطات الحسابية الرتبة الرقم الفقرات المتوسط الحسابي االنحراف المعياري المستوى 1 2 توجهني أسرتي إلى أن أعتمد على نفسي في تحقيق ما أريده. 2.52 1.022 متوسط 2 1 .أتبادل مع والدي في أمور األسرة 2.35 1.068 متوسط 3 7 تشجعني أسرتي على أن .أمارس هواياتي وميولي الخاصة 2.25 1.064 منخفض 4 4 تقدم لي أسرتي التعزيز عند تنظيم أوقاتي ويعاقبوني على .الفوضى 2.23 1.165 منخفض 4 5 اشعر بالرعاية واالهتمام من طرف أسرتي. 2.23 1.093 منخفض 6 3 .تؤكد أسرتي على التعاون والتضامن داخل األسرة 2.20 1.104 منخفض 7 6 تستشيرني أسرتي في القرارات التي تخصني. مشكلة الدراسة وأسئلتها : ( )ي (جدول2 ) معامالت االرتباط بين الفقرة و الدرجة الكلية للمقياس رقم الفقرة معامل االرتباط رقم الفقرة معامل االرتباط رقم الفقرة معامل االرتباط 1 .66 5 .66 9 .50 2 .60 6 .49 ** 10 .41 * 3 .55 7 .56 11 .54 4 .62 8 .81 12 .60 * ( دالة إحصائيا عند مستوى الداللة0.05 .) دالة إحصائيا( عند مستوى الداللة0.01 .) وتجدر اإلشارة أن جميع معامالت االرتباط كانت ذات درجات مقبولة ودالة إحصائيا ، ولذلك لم يتم حذف أي من هذه .الفقرات :ثبات المقياس )تم التحقق من ثبات المقياس بحساب قيم معامالت االتساق الداخلي باستخدام معادلة (كرونباخ ألفا ، وبلغت قيمة االتساق الداخلي للمقياس ككل ( 0,87 ) . كما تم التحقق من ثبات المقياس من خالل طريقة ثبات اإلعادة حيث قام الباحث بتطبيق ( المقياس على عينة استطالعية مكونة من30 ( طالب من خارج عينة الدراسة، وبعد مرور أسبوعين أعيد تطبيق المقياس مرة أخرى على ذات المجمو عة، وبلغ معامل ثبات المقياس الكلي( 0,92 ). وتشير هذه القيمة إلى تمتع المقياس بدالالت ثبات مقبولة .ألغراض الدراسة الحالية مقياس التصحيح ( لإلجابة على فقرات المقياس، أُستخدم تدريج ليكرت الخماسي وبدرجات تتراوح بين5 - 1 ) تمثل االستجابة على ( كل فقرة، حيث أن الرقم5 ( ) يعني دائماً على الفقرة، والرقم4 ( ) يعني غالباً على الفقرة، والرقم3 ) يعني أحياناً على الفقرة، والرقم ( 2 ( ) يعني نادراً على الفقرة، والرقم1 ،) يعني أبداً على الفقرة و تم عكس الدرجات بالنسبة للفقرات السلبية وللحكم على مستوى ضبط الذات قد جرى استخدام:التحليل االحصائي التالي لتوزيع المتوسطات الحسابية وذلك حسب المعيار الذي قاس كل فقرة طول الفئة= الحد األعلى للبدائل- .الحد األدنى/ عدد المستويات = طول الفئة5 – 1 / 3 = 1.33 ( 2.34 – 3.67) م.ستوى متوسط ( 3.68 - 5 .) مستوى مرتفع 469 د. IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 مشكلة الدراسة وأسئلتها : 2.07 1.103 منخفض النمط الديمقراطي 2.26 .738 منخفض ( يبين الجـدول3 ) ان( المتوسـطات الحسـابية قـد تراوحـت مـابين2.07 - 2.52 ) ، حيـث جـاءت الفقـر ة ( رقـم2 ) والتـي تـنص "على توجهني أسرتي إلى أن أعتمد على نفسي في تحقيق ما أريده " ( في المرتبة األولى وبمتوسط حسابي بلغ2.52 ) ، و جاءت الفقـر ة ( رقم1 ) "والتي تنص على أتبادل مع والدي في أمـور األسـرة " ( فـي المرتبـة الثانيـة وبمتوسـط حسـابي بلـغ2.35 ) ، بينمـا جـاءت الفقـر ة رقــم( 6 ) ونصــها"تستشــيرني أســرتي فــي القــرارات التــي تخصــني "بالمرتبــة األ( خيــرة وبمتوســط حســابي بلــغ2.07 ). وبلــغ المتوســط الحسابي ل لنمط الديمقراطي ( ككل2.26 .) (وتنســـجم الدراســـة الحاليـــة مـــع دراســـة العطـــوي2006 ()، ودراســـة الـــداير2016 ) ويعـــزو الباحثـــان وجـــود درجـــة منخفضـــة مـــن الـــنمط ،الديمقراطي بأن المراهقين يرغبون من آبـائهم أن يحبـوهم، يقبلـوهم، وأن يتقبلـونهم علـى الـرغم مـن أخطـائهم. إن المـراهقين ال يريـدون ان يشعروا بـأنهم يجـب علـيهم أن يكونـوا مثـاليين قبـل أن يتلقـوا أي موافقـة مـن آبـائهم. ال يسـتطيع المراهقـون أن يكبـروا ويزدهـروا فـي بيئــة تتخللهــا انتقــادات منت ظمــة وثابتــة باإلضــافة إلــى بيئــة ال يشــعرون فيهــا بــالقبول. ومــع ذلــك، فــإن اآلبــاء ال يجــب علــيهم أن يقبلــوا جميع التصرفات التي تصدر من أبنائهم. إن المناخ األسري الذي يسوده الـدف والـدعم والـذي يعـزز النقـاش بشـكل نـاجح عنـد وجـود الخالفـات بــين اآلبــاء والمــراهقين فإنـه يســاعد علــى المحافظــة علــى الصـراع فــي أدنــى مســتوياته. وفــي ظـل ظــروف قســرية فــإن اآلبــاء IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 470 د. إيهاب العصافرة تاال العموش أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين والمراهقين في بعض األحيان ال يستطيعون حل خالفاتهم وصراعاتهم وهو األمر الذي يؤدي في النهايـة إلـى تصـاعد هـذه الخالفـات لتصل إلى المستوى غير الوظيفي :ثانيا النمط المتساهل ( جدول4 ) المتوسطات الحسابية واالنحرافات المعيارية للفقرات المتعلقة ب النمط المتساهل مرتبة تنازلياً حسب المتوسطات الحسابية الرتبة الرقم الفقرات المتوسط الحسابي االنحراف المعياري المستوى 1 8 اشعر إني ال أطيق الحياة بعيد عن والدي . 3.68 1.092 مرتفع 2 12 تترك أسرتي لي حرية الدخول والخروج من المنزل 3.66 1.089 متوسط 3 13 يتغاضى والدي عندما أتحدث ببعض كلمات غير مناسبة. مشكلة الدراسة وأسئلتها : 3.48 1.143 متوسط 4 9 يقوم أفراد أسرتي بمعظم الواجبات التي بإمكاني القيام بها .بنفسي 3.42 .935 متوسط 5 11 .تلبي أسرتي طلباتي مهما كانت كثيرة وكبيرة 3.41 .935 متوسط 6 10 ال يتدخل والدي في تحديد أوقات المذاكرة أو النوم 3.29 1.008 متوسط النمط المتساهل 3.49 .673 متوسط ( يبين الجدول4 ) ان المتوسطات الحسابية قد تراوحت ما ( بـين3.29 - 3.68 ) ، حيـث جـاءت الفقـر ة ( رقـم8 ) والتـي تـنص "ل ال ا ال أط إ اش ال" (لغ ا ط ت ال ت ة األ ل ف3 68 )الفق ا(ق12 )الت د. إيهاب العصافرة تاال العموش 471 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 لا هم و و أل ر ي يؤ ي ي ه ي إ ى ي ون ل ال هم و ر و ر ين ي ب ض أل ي ن ال ي لتصل إلى المستوى غير الوظيفي :ثانيا النمط المتساهل ( جدول4 ) المتوسطات الحسابية واالنحرافات المعيارية للفقرات المتعلقة ب النمط المتساهل مرتبة تنازلياً حسب المتوسطات الحسابية الرتبة الرقم الفقرات المتوسط الحسابي االنحراف المعياري المستوى 1 8 اشعر إني ال أطيق الحياة بعيد عن والدي . 3.68 1.092 مرتفع 2 12 تترك أسرتي لي حرية الدخول والخروج من المنزل 3.66 1.089 متوسط 3 13 يتغاضى والدي عندما أتحدث ببعض كلمات غير مناسبة. 3.48 1.143 متوسط 4 9 يقوم أفراد أسرتي بمعظم الواجبات التي بإمكاني القيام بها .بنفسي 3.42 .935 متوسط 5 11 .تلبي أسرتي طلباتي مهما كانت كثيرة وكبيرة 3.41 .935 متوسط 6 10 ال يتدخل والدي في تحديد أوقات المذاكرة أو النوم 3.29 1.008 متوسط النمط المتساهل 3.49 .673 متوسط ( يبين الجدول4 ) ان المتوسطات الحسابية قد تراوحت ما ( بـين3.29 - 3.68 ) ، حيـث جـاءت الفقـر ة ( رقـم8 ) والتـي تـنص "على اشعر إني ال أطيق الحياة بعيد عن والدي " ( في المرتبـة األولـى وبمتوسـط حسـابي بلـغ3.68 ) ، و جـاءت الفقـر ة ( رقـم12 ) والتـي "تنص على تترك أسرتي لي حرية الدخول والخروج من المنزل " ( في المرتبة الثانية وبمتوسط حسابي بلغ3.66 ) ، بينمـا جـاءت الفقـر ة رقـــم( 10 ) ونصـــها" ال يتـــدخل والـــدي فـــي تحديـــد أوقـــات المـــذاكر ة أو النـــوم " ( بالمرتبـــة األخيـــرة وبمتوســـط حســـابي بلـــغ3.29 ). مشكلة الدراسة وأسئلتها : وبلـــغ المتوسط الحسابي ل لنمط المتساهل ( ككل3.49 .) ويعزو الباحثان،وجود درجة متوسطة من الـنمط المتسـاهل ،بـأن بعـض اآلبـاء أقـل اهتمامـاً بأبنـائهم أو عـدم معرفـة الوالـدين بأسـاليب التنشئة األسرية الصحيحة، أو استخدام نفس النمط الذي تربى عليه. وذلك من خالل تـرك المراهـق يفعـل مـا يشـاء وبـدون أي توجيـ ه مـن قبـل الوالــدين، ويضـعوا القواعـد فــي البيـت ولكـن ال يطبقوهــا. وبالتـالي يخلـق منــه شخصـية غيـر قــادرة علـى تحمـل المســئولية، وال تتحلـــى الصـــبر. وهـــذ ه النتيجـــة انســـجمت مـــع دراســـة العطـــو ي( 2006 ) و النتيجـــة كانـــت لهـــذه الدراســـة( وجـــود أثـــر لـــنمط تنشـــئة األب (المتســلط، الــديمقراطي، المهمــل) فــ ،ي تقــدير الــذات، وجــود أثــر لــنمط تنشــئة األم (المتســلط، الــديمقراطي، المهمــل) فــي تقــدير الــذات )وجود فروق في نمط تنشئة األب المتسلط تبعا للنوع االجتماعي, وعـدم وجــود فـروق فـي نمطـي تنشـئة األب (الـديمقراطي، والمهمـل ( يبين الجدول4 ) ان المتوسطات الحسابية قد تراوحت ما ( بـين3.29 - 3.68 ) ، حيـث جـاءت الفقـر ة ( رقـم8 ) والتـي تـنص "على اشعر إني ال أطيق الحياة بعيد عن والدي " ( في المرتبـة األولـى وبمتوسـط حسـابي بلـغ3.68 ) ، و جـاءت الفقـر ة ( رقـم12 ) والتـي "تنص على تترك أسرتي لي حرية الدخول والخروج من المنزل " ( في المرتبة الثانية وبمتوسط حسابي بلغ3.66 ) ، بينمـا جـاءت الفقـر ة رقـــم( 10 ) ونصـــها" ال يتـــدخل والـــدي فـــي تحديـــد أوقـــات المـــذاكر ة أو النـــوم " ( بالمرتبـــة األخيـــرة وبمتوســـط حســـابي بلـــغ3.29 ). وبلـــغ المتوسط الحسابي ل لنمط المتساهل ( ككل3.49 .) ويعزو الباحثان،وجود درجة متوسطة من الـنمط المتسـاهل ،بـأن بعـض اآلبـاء أقـل اهتمامـاً بأبنـائهم أو عـدم معرفـة الوالـدين بأسـاليب التنشئة األسرية الصحيحة، أو استخدام نفس النمط الذي تربى عليه. وذلك من خالل تـرك المراهـق يفعـل مـا يشـاء وبـدون أي توجيـ ه مـن قبـل الوالــدين، ويضـعوا القواعـد فــي البيـت ولكـن ال يطبقوهــا. وبالتـالي يخلـق منــه شخصـية غيـر قــادرة علـى تحمـل المســئولية، وال تتحلـــى الصـــبر. وهـــذ ه النتيجـــة انســـجمت مـــع دراســـة العطـــو ي( 2006 ) و النتيجـــة كانـــت لهـــذه الدراســـة( وجـــود أثـــر لـــنمط تنشـــئة األب (المتســلط، الــديمقراطي، المهمــل) فــ ،ي تقــدير الــذات، وجــود أثــر لــنمط تنشــئة األم (المتســلط، الــديمقراطي، المهمــل) فــي تقــدير الــذات )وجود فروق في نمط تنشئة األب المتسلط تبعا للنوع االجتماعي, وعـدم وجــود فـروق فـي نمطـي تنشـئة األب (الـديمقراطي، والمهمـل IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 471 د. إيهاب العصافرة تاال العموش د. مشكلة الدراسة وأسئلتها : إيهاب العصافرة تاال العموش تاال العموش وجهة نظر المراهقين ا للنــوع االجتمــاعي، وجــود فــروق تبعــاً للنــوع االجتمــاعي فــ ي نمــط تنشــئة األم (المتســلط، المهمـــل) فـــي تقــدير الــذات، وعــدم وج وق في نمط تنشئة األم (الديمقراطي ،)عدم وجود فروق ذات داللة إحصائية في درجـة تقـدير الـذات تبعـا للنـوع االجتماعي). :ثا النمط التسلطي ( جدول5 ) المتوسطات الحسابية واالنحرافات المعيارية للفقرات المتعلقة بالنمط التسلطي مرتبة تنازلياً حسب المتوسطات الحسابية الرتبة الرقم الفقرات المتوسط الحسابي االنحراف المعياري المستوى 1 15 يحرص والدي على إتباع نظام دقيق وحازم في .المنزل 3.93 .931 مرتفع 2 17 ترفض أسرتي أن أشارك في مناقشة األمور التي .تخص األسرة 3.87 .980 مرتفع 3 18 توجه لي أسرتي الكثير من األوامر دون أن أبدي رأياً فيها. 3.59 1.013 متوسط 4 20 .يميز والدي بين أفراد األسرة في المعاملة 3.53 .957 متوسط 5 16 تمنعني أسرتي من ممارسة الهوايات والنشاطات التي ارغب القيام بها.داخل المنزل 3.50 .870 متوسط 6 14 .تتدخل أسرتي في اختياري ألصدقائي 3.49 1.168 متوسط 7 19 ترفض أسرتي أن أتحاور معها. 3.39 1.065 متوسط النمط التسلطي 3.61 .561 متوسط ( يبين الجدول5 ) ان المتوسطات الحسابية قد تراوحت ما ( بين3.39 - 3.93 ) ، حيث جاءت الفقر ة ( رقم15 ) والتي تنص "على يحرص والدي على إتباع نظام دقيق وحازم في المنـزل " ( فـي المرتبـة األولـى وبمتوسـط حسـابي بلـغ3.93 ) ، و جـاءت الفقـر ة رقـم ( 17 ) "والتـي تـنص على تــرفض أسـرتي أن أشـارك فــي مناقشـة األمـور التــي تخـص األسـرة " فــي المرتبـة الثانيـة وبمتوســط حسـابي بلــغ ( 3.87 )، بينمــا جــاءت الفقــر ة رقــم( 19 ) ونصــها"تــرفض أســرتي أن أتحــاور معهــا " ( بالمرتبــة األخيــرة وبمتوســط حســابي بلــغ3.39 ). وبلغ المتوسط الحسابي ل لنمط التسلطي ( ككل3.61 .) 472 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 ( يبين الجدول5 ) ان المتوسطات الحسابية قد تراوحت ما ( بين3.39 - 3.93 ) ، حيث جاءت الفقر ة ( رقم15 ) والتي تنص "على يحرص والدي على إتباع نظام دقيق وحازم في المنـزل " ( فـي المرتبـة األولـى وبمتوسـط حسـابي بلـغ3.93 ) ، و جـاءت الفقـر ة رقـم ( 17 ) "والتـي تـنص على تــرفض أسـرتي أن أشـارك فــي مناقشـة األمـور التــي تخـص األسـرة " فــي المرتبـة الثانيـة وبمتوســط حسـابي بلــغ ( 3.87 )، بينمــا جــاءت الفقــر ة رقــم( 19 ) ونصــها"تــرفض أســرتي أن أتحــاور معهــا " ( بالمرتبــة األخيــرة وبمتوســط حســابي بلــغ3.39 ). مشكلة الدراسة وأسئلتها : 1.44 .988 منخفض مقياس ضبط الذات 2.32 .231 منخفض أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين د. إيهاب العصافرة تاال العموش د. إيهاب العصافرة تاال العموش نشأ عليها الوالدان في صغرهما، ويعود كذلك الى كبر حجم االسرة بشـكل يحـد مـن قـدرة الوالـدين علـى متابعـة االبنـاء باإلضـافة الـى تدني المستوى االقتصادي لألسرة بشـكل يزيـد مـن ممارسـة الوالـدين للتسـلط. إن النظـام المتسـلط يـؤدي إلـى الكثيـر مـن الصـراعات مـع اآلباء من خالل أعمال ال .بيت، األنشطة خارج البيت، األصدقاء، وصرف النقود أكثر من الحد المطلوب :السؤال الثاني ما مستوى ضبط الذات لدى الطلبة المراهقين؟ لإلجابـــة عـــن هـــذا الســـؤال تـــم اســـتخراج المتوســـطات الحســـابية واالنحرافـــات المعياريـــة ل مســـتوى ضـــبط الـــذات لـــدى الطلبـــة المراهقين، .والجدول أدناه يوضح ذلك نشأ عليها الوالدان في صغرهما، ويعود كذلك الى كبر حجم االسرة بشـكل يحـد مـن قـدرة الوالـدين علـى متابعـة االبنـاء باإلضـافة الـى تدني المستوى االقتصادي لألسرة بشـكل يزيـد مـن ممارسـة الوالـدين للتسـلط. إن النظـام المتسـلط يـؤدي إلـى الكثيـر مـن الصـراعات مـع اآلباء من خالل أعمال ال .بيت، األنشطة خارج البيت، األصدقاء، وصرف النقود أكثر من الحد المطلوب :السؤال الثاني ما مستوى ضبط الذات لدى الطلبة المراهقين؟ 473 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC B ء من خالل أعمال ال .بيت، األنشطة خارج البيت، األصدقاء، وصرف النقود أكثر من الحد المطلوب :ؤال الثاني ما مستوى ضبط الذات لدى الطلبة المراهقين؟ لإلجابـــة عـــن هـــذا الســـؤال تـــم اســـتخراج المتوســـطات الحســـابية واالنحرافـــات المعياريـــة ل مســـتوى ضـــبط الـــذات لـــدى الط هقين، .والجدول أدناه يوضح ذلك ( جدول6 ) المتوسطات الحسابية واالنحرافات المعيارية ل مستوى ضبط الذات لدى الطلبة المراهقين مرتبة تنازلياً حسب المتوسطات الحسابية الرتبة الرقم الفقرات المتوسط الحسابي االنحراف المعياري المستوى 1 5 يسهل علي السيطرة على نفسي. 3.11 1.113 متوسط 2 3 لدي القدرة على العمل بفعالية لتحقيق األهداف طويلة األجل. 3.07 1.086 متوسط 3 4 .أنظم كل أموري بشكل جيد 3.01 1.125 متوسط 4 7 أستطيع التحكم بضبط انفعاالتي . 2.96 1.078 متوسط 5 1 أشعر بأنني شخص يمكن االعتماد عليه. 473 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC مشكلة الدراسة وأسئلتها : وبلغ المتوسط الحسابي ل لنمط التسلطي ( ككل3.61 .) (وتنســـجم هـــذه الدراســـة الحاليـــة مـــع دراســـة الحربـــي2009 )، (ودراســـة العطـــوي2006 ) مـــن خـــالل وجـــود درجـــة متوســـطة مـــن الـــنمط التسلطي اعتقاد بعض اآلباء بأن الدالل يفسد الولد، لذا يلجآن إلى القسوة عليه والتقليل من مدح سلوك ي اته. وإلى نوعية التنشئة التي IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 د. إيهاب العصافرة تاال العموش أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين شأ عليها الوالدان في صغرهما، ويعود كذلك الى كبر حجم االسرة بشـكل يحـد مـن قـدرة الوالـدين علـى متابعـة االبنـاء باإلضـافة الـى دني المستوى االقتصادي لألسرة بشـكل يزيـد مـن ممارسـة الوالـدين للتسـلط. إن النظـام المتسـلط يـؤدي إلـى الكثيـر مـن الصـراعات مـع لباء من خالل أعمال ال .بيت، األنشطة خارج البيت، األصدقاء، وصرف النقود أكثر من الحد المطلوب :لسؤال الثاني ما مستوى ضبط الذات لدى الطلبة المراهقين؟ لإلجابـــة عـــن هـــذا الســـؤال تـــم اســـتخراج المتوســـطات الحســـابية واالنحرافـــات المعياريـــة ل مســـتوى ضـــبط الـــذات لـــدى الطلبـــة لمراهقين، .والجدول أدناه يوضح ذلك ( جدول6 ) المتوسطات الحسابية واالنحرافات المعيارية ل مستوى ضبط الذات لدى الطلبة المراهقين مرتبة تنازلياً حسب المتوسطات الحسابية الرتبة الرقم الفقرات المتوسط الحسابي االنحراف المعياري المستوى 1 5 يسهل علي السيطرة على نفسي. 3.11 1.113 متوسط 2 3 لدي القدرة على العمل بفعالية لتحقيق األهداف طويلة األجل. 3.07 1.086 متوسط 3 4 .أنظم كل أموري بشكل جيد 3.01 1.125 متوسط 4 7 أستطيع التحكم بضبط انفعاالتي . 2.96 1.078 متوسط 5 1 أشعر بأنني شخص يمكن االعتماد عليه. 2.90 1.121 متوسط 6 8 معظم األحيان أشعر بأني قادر على تغيير الغد بما .أفعله اليوم 2.89 1.066 متوسط 7 6 حصولي على درجات عالية في المدرسة معظم الوقت .يعني لي شيء كبير 2.77 .892 متوسط 8 2 أؤمن بأن التمني لألشياء يؤدي إلى حدوث مواقف و أشياء سعيدة 2.57 1.114 متوسط 9 10 .أرفض االنتقادات التي توجه إلي 1.88 1.008 منخفض 10 11 .انفق الكثير من المال على أشياء غير مهمة 1.86 .926 منخفض 11 15 .أواجه صعوبة في معارضة اآلخرين 1.82 1.096 منخفض 12 12 يعيقني السعي وراء المتعة والمرح عن إنجاز العمل بشكل جيد. 1.81 .980 منخفض 13 9 .افقد أعصابي بسهولة 1.78 .999 منخفض 14 14 أؤجل دراستي أو أعمالي إلى آخر لحظة. 1.65 .985 منخفض 14 16 أجد صعوبة في التخلي عن العادات السيئة. 1.65 .829 منخفض 16 13 أ فعل الكثير من األمور دون تخطيط. مشكلة الدراسة وأسئلتها : 2.90 1.121 متوسط 6 8 معظم األحيان أشعر بأني قادر على تغيير الغد بما .أفعله اليوم 2.89 1.066 متوسط 7 6 حصولي على درجات عالية في المدرسة معظم الوقت .يعني لي شيء كبير 2.77 .892 متوسط 8 2 أؤمن بأن التمني لألشياء يؤدي إلى حدوث مواقف و أشياء سعيدة 2.57 1.114 متوسط 9 10 .أرفض االنتقادات التي توجه إلي 1.88 1.008 منخفض 10 11 .انفق الكثير من المال على أشياء غير مهمة 1.86 .926 منخفض 11 15 .أواجه صعوبة في معارضة اآلخرين 1.82 1.096 منخفض 12 12 يعيقني السعي وراء المتعة والمرح عن إنجاز العمل بشكل جيد. 1.81 .980 منخفض 13 9 .افقد أعصابي بسهولة 1.78 .999 منخفض 14 14 أؤجل دراستي أو أعمالي إلى آخر لحظة. 1.65 .985 منخفض 14 16 أجد صعوبة في التخلي عن العادات السيئة. 1.65 .829 منخفض 16 13 أ فعل الكثير من األمور دون تخطيط. 1.44 .988 منخفض مقياس ضبط الذات 2.32 .231 منخفض ( يبين الجـدول6 ) ان( المتوسـطات الحسـابية قـد تراوحـت مـابين1.44 - 3.11 ) ، حيـث جـاءت الفقـر ة ( رقـم5 )والتـي ت "يســهل علـــي الســيطرة علـــى نفســـي " ( فـــي المرتبــة األولـــى وبمتوســـط حســابي بلـــغ3.11 ) ، و جــاءت الفقـــر ة ( رقـــم3 )والتـــي تـ ( يبين الجـدول6 ) ان( المتوسـطات الحسـابية قـد تراوحـت مـابين1.44 - 3.11 ) ، حيـث جـاءت الفقـر ة ( رقـم5 )والتـي تـنص "على يســهل علـــي الســيطرة علـــى نفســـي " ( فـــي المرتبــة األولـــى وبمتوســـط حســابي بلـــغ3.11 ) ، و جــاءت الفقـــر ة ( رقـــم3 )والتـــي تـــنص 473 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 473 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين د. مشكلة الدراسة وأسئلتها : إيهاب العصافرة تاال العموش "على لدي القدرة على العمل بفعالية لتحقيق األهداف طويلة األجـل " ( فـي المرتبـة الثانيـة وبمتوسـط حسـابي بلـغ3.07 ) ، بينمـا جـاءت الفقـــر ة رقـــم( 13 ) ونصــها"أ فعـــل الكثيــر مـــن األمــور دون تخطـــيط " ( بالمرتبــة األخيـــرة وبمتوســـط حســـابي بلــغ1.44 ).وبلـــ غ المتوســـط الحسابي ل مقياس ضبط الذات( ككل2.32 .) "على لدي القدرة على العمل بفعالية لتحقيق األهداف طويلة األجـل " ( فـي المرتبـة الثانيـة وبمتوسـط حسـابي بلـغ3.07 ) ، بينمـا جـاءت الفقـــر ة رقـــم( 13 ) ونصــها"أ فعـــل الكثيــر مـــن األمــور دون تخطـــيط " ( بالمرتبــة األخيـــرة وبمتوســـط حســـابي بلــغ1.44 ).وبلـــ غ المتوســـط الحسابي ل مقياس ضبط الذات( ككل2.32 .) ويعزو الباحثان ،وجود درجة منخفضة من الضبط الذاتي بسبب الخصائص النمائية ألفراد عينة البحث، وهي مرحلـة المراهقـة والتـي يواجهوا فيها العديد من الصراعات النفسية والتكيفيـة وا ال،جتماعيـة وذلـك مـن خـالل شـعورهم باإلحبـاط ، وفقـدان الثقـة بـالنفس، وعـدم ،الرضــا ممــا يــؤدي إلــى ضــعف فــي ضــبط أنفســهم ، ويعــزى هــذه النتيجــة إلــى أســاليب التنشــئة األســرية التســلطية أو المتســاهلة التــي (يتبعها اآلباء مع األبناء، وتنسـجم هـذه الدراسـة الحاليـة مـع دراسـة عـالء الـين والعلـي2014 ) نتـائج الدراسـة لوجـود ع القـة دالـّة بــين الممارســـات الوالديـــة اإليجابيـــة وأداء األســـرة الصـــحي لوظيفتهـــا المتعلقـــة بالتماســـك والتكيــّف ، كمــا أظهــرت نتـــائج تحلـــيالت االنحـــدار المتـدرج أن عـاملي (أ) الممارسـات الوالديـة السـلبية لـألم و(ب) انخفـاض مسـتوى التماسـك األسـري أسـهما فـي التنبـؤ بتقـديرات الطلبـة للقلـــق، وأنّ عـــامليْ (أ) الممارســـات الوالديـــة اإليجابيـــة لـــألب و(ب)ارتفــــاع مســــتوى التماســــك األســــري، أســــهما فــــي التنبــــؤ ِبتقــــديرات الوالــدين للكفــاءة االجتماعيـة. وتبـين أيضــاً أن عامــل الممارســة الوال ديــة اإليجابيــة لــألب كــان العامــل الوحيـد والفريـد الـذي أسـهم فـي التنبؤ بتقـديرات المعلمـين للكفـاءة االجتماعيـة لـدى طالبهـم، وأخيـراً، أظهــرت النتــائج عــدم وجــود فــروق علــى مقــاييس الدراســ ة تُعــزى ِلجنس المراهق باستثناء مقياس القلق، (ودراسة علجي وبلعري2016 ) وكانـت النتـائج توجـد عالقـة ذات داللـة إحصـائية بـين أسـاليب التنشئة األسرية وتكيف الطالبة في الوسط الجامعي. . IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY مشكلة الدراسة وأسئلتها : أاا ا :لسؤال الثالث هل توجد عالقة ارتباطية ذات داللة إحصائية بين أساليب التنشئة األسرية وضبط الذات لدى الطلبة المراهقين؟ لإلجابــة عـــن هــذا الســـؤال تــم اســـتخراج معامــل ارتبـــاط بيرســون بـــين أســـاليب التنشـــئة األســـرية وضـــبط الــذات لـــدى الطلبـــة المراهقين( ، والجدول7 .) يوضح ذلك 474 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 (ح ) ( جدول7 ) معامل ارتباط بيرسون للعالقة بين أساليب التنشئة األسرية وضبط الذات لدى الطلبة المراهقين مقياس ضبط الذات النمط الديمقراطي معامل االرتباط ر .339 الداللة اإلحصائية .001 العدد 100 النمط المتساهل معامل االرتباط ر -.374 ** الداللة اإلحصائية .000 العدد 100 النمط التسلطي معامل االرتباط ر -.208 * الداللة اإلحصائية .038 العدد 100 * دالة إحصائيا ( عند مستوى الداللة0.05 .) دالة إحصائيا ( عند مستوى الداللة0.01 .) 474 IUG J l f Ed ti l d P h l S i (I l i U i it f G ) / CC BY 4 0 ( جدول7 ) معامل ارتباط بيرسون للعالقة بين أساليب التنشئة األسرية وضبط الذات لدى الطلبة المراهقين مقياس ضبط الذات النمط الديمقراطي معامل االرتباط ر .339 الداللة اإلحصائية .001 العدد 100 النمط المتساهل معامل االرتباط ر -.374 ** الداللة اإلحصائية .000 العدد 100 النمط التسلطي معامل االرتباط ر -.208 * الداللة اإلحصائية .038 العدد 100 * دالة إحصائيا ( عند مستوى الداللة0.05 .) ** دالة إحصائيا ( عند مستوى الداللة0.01 .) 474 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 ا 474 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين أساليب التنشئة األسرية وعالقتها بضبط الذات من وجهة نظر المراهقين د. إيهاب العصافرة تاال العموش يتبين من الجدول ( 7 ) :اآلتي وجود عالقة ايجابية دالة إحصائيا بين مقياس ضبط الذات وبين النمط الديمقراطي. وجود عالقة سلبية دالة إحصائيا بين مقياس ضبط الذات من جهة وبين كل من النمط المتساهل ، و النمط التسلطي من جهة اخرى. وتنسجم هذه الدراسة الحالي ة (ودراسة الحربي2009 ()، ودراسة العطوي2006 ). IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 مشكلة الدراسة وأسئلتها : من خالل وجود عالقة ارتباطيـة إيجابيـة بـين الـنمط الديمقراطي وضبط الذات، والى وجود عالقة سلبية بين النمط المتساهل والنمط التسلطي، بأن ه ذه النتيجة واقعية إلى حد كبيـر ألن أســاليب التنشــئة األســرية تعــد مــن العوامــل التــي تــؤثر فــي تكــوين شخصــية المراهــق، فاألســرة التــي تشــجع االســتقاللية، وتقــوم علــى المبادئ الديمقراطية تنتج أفراداً لديهم ضبط ذاتي مرتفـع، واألسـر التـي تقـوم علـى أسـلوب التسـلط والـنمط المتسـاهل تتـرك آثـاراً سـلبية كبيرة لدى المراهقين، وتفقدهم النظرة اإليجابية والمتفائلة للمستقبل، كما أن عدم الثقة بقدرات األبنـاء ومهـاراتهم، وميـولهم، واتجاهـ اتهم يؤدي إلى المشاكل النفسية وانخفاض ضبط الذات لدى المراهقين . :التوصيات 1 -إجراء برامج إرشادية من خالل نظريات األرشاد األسري .للتنشئة األسرية وضبط الذات لدى الطلبة المراهقين 2 - عقد حصص توجيه جمعي وارشاد جمعي ونشرات تربوية للطلبة المراهقين عن أساليب التنشئة األسرية الصحيحة وضبط .الذات 3 - .عقد برامج إرشادية ألولياء األمور للتعرف على أساليب التنشئة األسريه وانعكاسها على أبنائهم 4 - تنفيذ دراسات أخرى لفحص مستوى أساليب التنشئة األسرية وضبط الذات في ضوء متغيرات أخرى كالجنس، ومراحل عمرية .مختلفة 475 IUG Journal of Educational and Psychology Sciences (Islamic University of Gaza) / CC BY 4.0 د. إيهاب العصافرة تاال العموش ب عا ،أحمد، أماني ( 2009 . ) التنشئة األسرية وعالقتها بالتوافق النفسي للمراهق. رسالة ماجستير، كلية االداب، جامعة أم درمان اإلسالمية. اإلسالمية. ،الحربي، عبدهللا( 2009 .) أساليب التنشئة األسرية وعالقتها بكل من التفاؤل والتشاؤم لدى عينة من تالميذ المرحلة المتوسطة والثانوية بم نطقة جازان، رس الة ماجستير، جامعة ام القرى، مك ة المكرمة . الداير، سالم ، ( 2016 . ) أساليب التنشئة األسرية كما يدركها طلبة دبلوم التعليم العام وعالقتها بالتوافق النفسي لديهم في مدارس محافظة جنوب الباطنة، رسالة ماجستير، ج امعة نزوى. ، الربيع ،فيصل و عطية ، رمزي ( 2006 . ) االتزان االنفعالي وعالقته بضبط الذات لدى طلبة جامعة الير موك. العطوي، ضيف هللا ، ( 2006 .) أثر نمط التنشئة األسرية في تقدير الذات لدى طلبة المرحلة الثانوية في مدينة تبوك، رسالة .ماجستير، جامعة مؤتة ،الحربي، عبدهللا( 2009 .) أساليب التنشئة األسرية وعالقتها بكل من التفاؤل والتشاؤم لدى عينة من تالميذ المرحلة المتوسطة والثانوية بم نطقة جازان، رس الة ماجستير، جامعة ام القرى، مك ة المكرمة . ،الحربي، عبدهللا( 2009 .) أساليب التنشئة األسرية وعالقتها بكل من التفاؤل والتشاؤم لدى عينة من تالميذ المرحلة المتوسطة والثانوية بم نطقة جازان، رس الة ماجستير، جامعة ام القرى، مك ة المكرمة . الداير، سالم ، ( 2016 . مشكلة الدراسة وأسئلتها : ) أساليب التنشئة األسرية كما يدركها طلبة دبلوم التعليم العام وعالقتها بالتوافق النفسي لديهم في مدارس محافظة جنوب الباطنة، رسالة ماجستير، ج امعة نزوى. ، الربيع ،فيصل و عطية ، رمزي ( 2006 . ) االتزان االنفعالي وعالقته بضبط الذات لدى طلبة جامعة الير موك. للهأ الداير، سالم ، ( 2016 . ) أساليب التنشئة األسرية كما يدركها طلبة دبلوم التعليم العام وعالقتها بالتوافق النفسي لديهم في مدارس محافظة جنوب الباطنة، رسالة ماجستير، ج امعة نزوى. ، الربيع ،فيصل و عطية ، رمزي ( 2006 . ) االتزان االنفعالي وعالقته بضبط الذات لدى طلبة جامعة الير موك. العطوي، ضيف هللا ، ( 2006 .) أثر نمط التنشئة األسرية في تقدير الذات لدى طلبة المرحلة الثانوية في مدينة تبوك، رسالة الداير، سالم ، ( 2016 . ) أساليب التنشئة األسرية كما يدركها طلبة دبلوم التعليم العام وعالقتها بالتوافق النفسي لديهم في مدارس محافظة جنوب الباطنة، رسالة ماجستير، ج امعة نزوى. ال ط ة ل ف( 2006 )ا ة ال طل ة ل ط الذا الق اال ف ال اال ا ك ، الربيع ،فيصل و عطية ، رمزي ( 2006 . ) االتزان االنفعالي وعالقته بضبط الذات لدى طلبة جامعة الير موك. العطوي، ضيف هللا ، ( 2006 .) أثر نمط التنشئة األسرية في تقدير الذات لدى طلبة المرحلة الثانوية في مدينة تبوك، رسالة .ماجستير، جامعة مؤتة ،العلجي، عائشة و بلعربي، هاجر ( 2016 .) أساليب التنشئة األسرية وعالقتها بالتكيف لدى الطالبة في الوسط الجامعي، رسالة ماجستير، جامعة الشهيد حمه لخضر الوادي . حسن، عبد الحميد ، ( 1989 . )خصائص الشخصية المرتبطة بموازين النجاح لدى المدرسين في المرحلة اإلعداد ية، أطروحة دكتوراه، جامعة بغداد. دندي، إيمان (، 2010 .) أساليب التنشئة األسرية وعالقتها بمفهوم الذات والسلوك العدواني لدى طلبة الصف األول الثانوي العام بمدارس محافظة دمشق الرسمية، رسالة ماج.ستير، كلية التربية، جامعة دمشق سويف، مصطفى ، ( 1996 .) االتزان االنفعالي محور من محاور الشخصية، مجلة العربي. عالء الدين، جهاد وتغريد، العلي ( ، 2014 .) ،األداء الوظيفي األسري كما يدركه المراهقون وعالقته بالكفاءة االجتماعية والقلق الم جلة األردنية في العلوم التربوية ، 10 ( 1 ) ، 65 - 88 . أ قنديل، محمد متولي و شلبي، السعيد ، ( 2006 . ) مدخل إلى رعاية الطفل واألسرة، عمان : دار الفكر للنشر والتوزيع. محمود، إبراهيم ( ، 1981 .) المراهقة خصائصها ومشكالتها، دار المعارف. أ قنديل، محمد متولي و شلبي، السعيد ، ( 2006 . ) مدخل إلى رعاية الطفل واألسرة، عمان : دار الفكر للنشر والتوزيع. مشكلة الدراسة وأسئلتها : محمود، إبراهيم ( ، 1981 .) المراهقة خصائصها ومشكالتها، دار المعارف. مصباح، عامر( ، 2003 .) التنشئة االجتماعية والسلوك اإلنحرافي لتلميذ المدرسة الثانوي،ة الجزائر. نسيمة، بن عمر( ، 2009 .) ضبط الذات و عالقته بالقدرة على حل المشكالت لدى تالميذ المرحلة الثانوية دراسة ميدانية بثانوية عبد المجيد مزيان– المسيلة- رسالة ماجستير غير منشورة . :ًثانيا المراجع المرمونة واألجنبية مصباح، عامر( ، 2003 .) التنشئة االجتماعية والسلوك اإلنحرافي لتلميذ المدرسة الثانوي،ة الجزائر. نسيمة، بن عمر( ، 2009 .) ضبط الذات و عالقته بالقدرة على حل المشكالت لدى تالميذ المرحلة الثانوية دراسة ميدانية بثانوية عبد المجيد مزيان– المسيلة- رسالة ماجستير غير منشورة . :ًثان ا نة ال ا ع ال األ ن ة Ahmed, Amani, (2009). 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https://openalex.org/W4243459471	https://www.biodiversitylibrary.org/itempdf/114818	English	null	Agricultural resources and capabilities of Porto Rico : message from the President of the United States, transmitting a report on investigations of the agricultural resources and capabilities of Porto Rico with special reference to the establishment of an agricultural experiment station in that island	null	1,901	public-domain	21,959	wii wii Rew Dork Htate College of Agriculture At Cornell Aniversitp Ithaca, N. V. Librarp Rew Dork Htate College of Agriculture At Cornell Aniversitp Ithaca, N. V. TRANSMITTING Cornell University The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. COSI To the Senate and House of Representatives: I transmit herewith a report on investigati I transmit herewith a report on investigations of the agricultural resources and capabilities of Porto Rico with special reference to the establishment of an agricultural experiment station in that island, made in accordance with the act of Congress making appropriations for the Department of Agriculture for the fiscal year ending June 30, 1901. WILLIAM MCIXINLEY. WILLIAM MCIXINLEY. EXECUTIVE MANnsiIon, December 10, 1900, A REPORT ON INVESTIGATIONS OF THE AGRICULTURAL RE- SOURCES AND CAPABILITIES OF PORTO RICO WITH SPECIAL REFERENCE TO THE ESTABLISHMENT OF AN AGRICULTURAL EXPERIMENT STATION IN THAT ISLAND. DECEMBER 11, 1900,—Message and accompanying papers ordered printed and re- ferred to the Committee on Insular Affairs. To the Senate and House of Representatives: I transmit herewith a report on investigati MESSAGE FROM THE PRESIDENT OF THE UNITED STATES, TRANSMITTING A REPORT ON INVESTIGATIONS OF THE AGRICULTURAL RE- SOURCES AND CAPABILITIES OF PORTO RICO WITH SPECIAL REFERENCE TO THE ESTABLISHMENT OF AN AGRICULTURAL EXPERIMENT STATION IN THAT ISLAND. PRESIDENT OF THE UNITED STATES, COSI 56TH Coneress, \| HOUSE OF REPRESENTATIVES. \| Document 2d Session. No. 171. UNITED STATES DEPARTMENT OF AGRICULTURE, OFFICE OF THE SECRETARY Washington, D. C., December 5 Washington, D. C., December 5, 1900. transmit herewith a report on the agricul Washington, D. C., December 5, 1900. ransmit herewith a report on the agricul- Str: I have the honor to transmit herewith a report on the agricul- tural resources and capabilities of Porto Rico with special reference to the establishment of an agricultural experiment station in that island, made in compliance with the act of Congress making appropriations for this Department for the fiscal year ending June 30, 1901. The investigations have, in my judgment, shown the desirability and feasi- bility of maintaining an agricultural experiment station in Porto Rico, 2 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. and I earnestly recommend that provision be made by Congress for the establishment of such a station in the Territory on a permanent and efficient basis in accordance with the recommendations of the Director of the Office of Experiment Stations, herewith submitted, and that an appropriation be made for this purpose equal to that which is given for the maintenance of similar stations elsewhere in the United States. T have the honor to be, sir, your obedient servant, T have the honor to be, sir, your obedient servant, JAMES WILSON, T have the honor to be, sir, your obedient servant, JAMES WILSON, Secret JAMES WILSON, Secretary. The PRESIDENT. The PRESIDENT. UNITED STATES DEPARTMENT OF AGRICULTURE, OFFICE OF EXPERIMENT STATIONS, Washington, D. C., December 6, 1900. have the honor to transmit herewith a report on the agricul- Sir: I have the honor to transmit herewith a report on the agricul- tural conditions in Porto Rico with special reference to the establish- ment of an agricultural experiment station in thatisland. This inves- tigation was made in accordance with the terms of the appropriation act for this Department for the fiscal year ending June 30, 1901, which authorizes the Secretary of Agriculture to ‘investigate and report to Congress on the agricultural resources and capabilities of Porto Rico with special reference to the selection of locations for agricultural experiment stations and the determination of the character and extent of agricultural experiments immediately demanded by the condition of agriculture in that island.” As the agent to make this investiga- tion, Prof. 8. A. (5) The needs of the agricultural people of Porto Rico as regards information on agricultural subjects, and the best means for supplying them with this infor- mation by publications, oral instruction, and demonstration experiments, or otherwise. (6) The desirability and feasibility of securing the cooperation of the residents UNITED STATES DEPARTMENT OF AGRICULTURE, OFFICE OF THE SECRETARY Washington, D. C., December 5 Knapp, formerly of the Iowa Agricultural College and more recently engaged in agricultural enterprises in southern Louisiana, was appointed special agent in charge of agricultural investigations in Porto Rico and sent to that island about the middle of June, 1900, with the following instructions: The following subjects should be included in your investigations: (1) The general agricultural conditions existing in Porto Rico and The following subjects should be included in your investigations: (1) The general agricultural conditions existing in Porto Rico and the sary and feasible measures for the improvement of these conditions. The following subjects should be included in your investigations: (1) The general agricultural conditions existing in Porto Rico and The following subjects should be included in your investigations: (1) The general agricultural conditions existing in Porto Rico and the neces- sary and feasible measures for the improvement of these conditions. (2) The lines of experimental investigations which should be undertaken in (2) The lines of experimental investigations which should be undertaken in Porto Rico, and especially those which should he undertaken in the immediate future. As far as tog aoa the scope, extent, and cost of the experimental inquiries immediately demanded should be determined. (8) The locations suitable for agricultural experiment stations in Porto Rico, (2) The lines of experimental investigations which should be undertaken in Porto Rico, and especially those which should he undertaken in the immediate future. As far as tog aoa the scope, extent, and cost of the experimental inquiries immediately demanded should be determined. (8) The locations suitable for agricultural experiment stations in Porto Rico, including a main station with laboratories, farm buildings, and experimental fields, and outlying stations, whose work shall consist of field, garden, and orchard experiments and experiments with domestic animals. (4) The buildings, land, and equipment required for the proper maintenance (8) The locations suitable for agricultural experiment stations in Porto Rico, including a main station with laboratories, farm buildings, and experimental fields, and outlying stations, whose work shall consist of field, garden, and orchard experiments and experiments with domestic animals. (4) The buildings, land, and equipment required for the proper maintenance (4) The buildings, land, and equipment required for the proper maintenance of agricultural investigations in Porto Rico on the plan indicated in section 3. (6) The desirability and feasibility of securing the cooperation of the residents of Porto Rico in the conduct of experimental inquiries and the dissemination of agricultural information; the desirability and feasibility of including instruc- tion in agricultural subjects in the curriculum of the schools of Porto Rico. (7) The facilities for preparing, printing, publishing, and distributing in Porto (7) The facilities for preparing, printing, publishing, and distributing in Porto Rico circulars of inquiry and bulletins of information on agricultural subjects in (4) The buildings, land, and equipment required for the proper maintenance of agricultural investigations in Porto Rico on the plan indicated in section 3. Careful estimates should be made regarding the cost of the buildings, land, and equipment needed to inaugurate the work of the experiment station in the island, with special reference to the sum required for these purposes during the fiscal year ending June 30, 1902. The methods of acquiring real estate for the use of the station should also be investigated. (5) The needs of the agricultural people of Porto Rico as regards information The following subjects should be included in your investigations: (1) The general agricultural conditions existing in Porto Rico and the neces- sary and feasible measures for the improvement of these conditions. (2) The lines of experimental investigations which should be undertaken in Porto Rico, and especially those which should he undertaken in the immediate future. As far as tog aoa the scope, extent, and cost of the experimental inquiries immediately demanded should be determined. (8) The locations suitable for agricultural experiment stations in Porto Rico, including a main station with laboratories, farm buildings, and experimental fields, and outlying stations, whose work shall consist of field, garden, and orchard experiments and experiments with domestic animals. (4) The buildings, land, and equipment required for the proper maintenance of agricultural investigations in Porto Rico on the plan indicated in section 3. Careful estimates should be made regarding the cost of the buildings, land, and equipment needed to inaugurate the work of the experiment station in the island, with special reference to the sum required for these purposes during the fiscal year ending June 30, 1902. The methods of acquiring real estate for the use of the station should also be investigated. (5) The needs of the agricultural people of Porto Rico as regards information on agricultural subjects, and the best means for supplying them with this infor- mation by publications, oral instruction, and demonstration experiments, or otherwise. (6) The desirability and feasibility of securing the cooperation of the residents of Porto Rico in the conduct of experimental inquiries and the dissemination of agricultural information; the desirability and feasibility of including instruc- tion in agricultural subjects in the curriculum of the schools of Porto Rico. (7) The facilities for preparing, printing, publishing, and distributing in Porto Rico circulars of inquiry and bulletins of information on agricultural subjects in (8) The locations suitable for agricultural experiment stations in Porto Rico, including a main station with laboratories, farm buildings, and experimental fields, and outlying stations, whose work shall consist of field, garden, and orchard experiments and experiments with domestic animals. UNITED STATES DEPARTMENT OF AGRICULTURE, OFFICE OF THE SECRETARY Washington, D. C., December 5 This sta- tion should give immediate attention to promoting the production of larger and better crops of coffee, sugar, and tobacco, and of food products for home consumption. As soon as practicable it should undertake work in horticulture, forestry, animal husbandry, and dairying. Besides conducting experiments, it should give object les- sons in improved farming and should disseminate information by pub- lications and agricultural meetings. In these recommendations of Professor Knapp I heartily concur. In my judgment an agricul- tural experiment station should be immediately established in Porto Rico on the same general plan as that pursued elsewhere in the United States. Land should be obtained in the vicinity of San Juan on which to Land should be obtained in the vicinity of San Juan on which to erect office, laboratory, and farm buildings and to conduct experi- ments. A competent man should be appointed to act as the chief executive officer of the station, plan and supervise its operations, and begin the organization of astaff of scientific and practical men to con- duct investigations in various lines and instruct the people in improved methods of agriculture. It will be best to limit the work of the sta- tion at the outset to a few main lines, which will require the services of only asmall staff, and develop the organization of the working corps as the station becomes more fully established and the way is opened for the extension of its work. As the station will not have the aid of an agricultural college already equipped with buildings and land, as has been the case with most of the stations established in the United States, it will be necessary at the outset to devote a considerable amount of money to its equipment. Without doubt as much will be required for the current expenses involved in the proper maintenance of an experi- ment station in Porto Rico as is the case elsewhere in the United States. I can see no good reason why an appropriation of $15,000 a year should not be given to Porto Rico for the maintenance of-an experiment station as well as to the other Territories of the United States, and I hope that Congress will make the first appropriation of this kind during its present session. For the purchase of land and the erection of buildings the station should have in addition an initial fund of $15,000. UNITED STATES DEPARTMENT OF AGRICULTURE, OFFICE OF THE SECRETARY Washington, D. C., December 5 Careful estimates should be made regarding the cost of the buildings, land, and equipment needed to inaugurate the work of the experiment station in the island, with special reference to the sum required for these purposes during the fiscal year ending June 30, 1902. The methods of acquiring real estate for the use of the station should also be investigated. (5) The needs of the agricultural people of Porto Rico as regards information (5) The needs of the agricultural people of Porto Rico as regards information on agricultural subjects, and the best means for supplying them with this infor- mation by publications, oral instruction, and demonstration experiments, or otherwise. (6) The desirability and feasibility of securing the cooperation of the residents (6) The desirability and feasibility of securing the cooperation of the residents of Porto Rico in the conduct of experimental inquiries and the dissemination of agricultural information; the desirability and feasibility of including instruc- tion in agricultural subjects in the curriculum of the schools of Porto Rico. (7) The facilities for preparing, printing, publishing, and distributing in Porto (7) The facilities for preparing, printing, publishing, and distributing in Porto Rico circulars of inquiry and bulletins of information on agricultural subjects in 3 -AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. the English and Spanish languages, and the best ways of securing the preparation and dissemination of such information in printed form. rare (8) The cost of inaugurating and maintaining agricultural investigations and the English and Spanish languages, and the best ways of securing the preparation and dissemination of such information in printed form. rare (8) The cost of inaugurating and maintaining agricultural investigations and (8) The cost of inaugurating and maintaining agricultural investigations and disseminating information (exclusive of the buildings, land, and equipment) in a manner similar to that of an agricultural experiment station in one of the United States having an area approximating that of Porto ‘Rico. An estimate should be made with special reference to the cost of maintaining such work during the single fiscal year ending June 30, 1902. Professor Knapp’s report of his investigations is submitted here- with. In this report the need of experiment-station work in Porto Rico is plainly shown, and itis recommended that a station should be established with headquarters in the vicinity of San Juan. UNITED STATES DEPARTMENT OF AGRICULTURE, OFFICE OF THE SECRETARY Washington, D. C., December 5 In the case of the other Territories such expenses have been largely provided for by the local governments, and I think it would be well if this plan could be followed in Porto Rico, a portion of the revenues of the island being set aside for this purpose by the action of Congress or the Territorial legislature. Authority should be given the Secretary of Agriculture in the Authority should be given the Secretary of Agriculture in the appropriation act to establish and maintain an agricultural experi- ment station in Porto Rico, including the purchase of land, the erec- tion of buildings, the printing (in Porto Rico), illustration and distri- bution of reports and bulletins in the English and Spanish languages, 4 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. and all other expenses essential to the maintenance of said station. Half of the first appropriation should be made immediately available. Very respectfully, A. C. TRUE, Director. A. C. TRUE, Director. A. C. TRUE, Director. Hon. JAMES WILSON, Secretary of Agriculture. Hon. JAMES WILSON, Secretary of Agriculture. LAKE CHARLES, La., September 22, 1900. to submit herewith my report on the investi- LAKE CHARLES, La., September 22, 1900. to submit herewith my report on the investi- LAKE CHARLES, La., September 22, 1900. to submit herewith my report on the investi- Sir: I have the honor to submit herewith my report on the investi- gations regarding the agricultural conditions existing in Porto Rico, with special reference to the establishment of an agricultural experi- ment station in that island, made under your instructions dated June 11, 1900. Tarrived at San Juan June 19, 1900, and immediately com- menced my observations on the agricultural conditions and capabili- ties of the island. I traveled by private conveyance over 350 miles in the rural districts, and was everywhere cordially met by the farmers and given every facility for obtaining the information desired. Respectfully, Respectfully, SEAMAN A. KNAPP, Special Agent in Charge of Agricultural Investigations in Porto Rico. Investigatio Dr. A. C. TRUE, Director of Office of Experiment Stations. GENERAL STATEMENT. The island of Porto Rico is situated in latitude 18° north and lies in the direct line of trade between New York and South America. In a general way it may be described as about 100 miles long and 36 miles wide, and has an area, including its dependencies—the islands of Vieques, Culebra, and Mona—of 3,530 to 3,860 square miles. The whole island may be classed as mountainous except a border on the seacoast and numerous interior valleys. The moun- tains are not in bold and forbidding ranges, but consist of an endless variety of immense segregated and fertile hills, with interspersed valleys, in an ascending series, but without special order, from the north coast to two-thirds the distance across the island, where the hills attain an elevation of 1,500 to 2,500 feet, and the valleys, many of which are of considerable extent, are from 500 to 1,500 feet above the sea. 1 Water Supply and Irrig. Papers, U. 8. Geol. Survey, No. 32, p. 28. 5 CLIMATE. 80.9 .30 Canovanas 5 82.9 1.48 RS RTE AUD sae cps asa cts scl meena ieiasicrecaucic ee eae Saeed Hama cleseens Semiercemsee SSSR ae 81.4 1.33 The above being a report for one week does not indicate the relative rainfall for the year at the different points, but it shows how unequally the rainfall is liable to be distributed in a given period. It is said that the rainfall was much more equal when the summits of the mountains were well wooded. An article by Prof. Mark W. Harring- ton, of the United States Weather Bureau (in U. 8. Treasury Doc. 2118), is instructive: The published observations of Porto Rico are very scanty, consisting of a total of about nine years at San Juan only, and these are fragmentary, being scattered through twenty years. They show a true tropical climate, with a high mean temperature (78.9° F.) and very little difference in season, except in rainfall. The coldest month on the average is February (75.7°) and the hottest, June (81.5'), but December to March are very much alike in temperature, and so are the months from June to September. The very coldest month on record is January, 1895 (70°), and the very warmest is June, 1878 (86°). The average change from the coldest to the hottest is only 6”, but this is very appreciable to one who has lived long in the.Tropics. The cool months really seem to the natives to be decidedly cold, requiring additional covering on the bed and heavier clothing. The coldest temperature on record in San Juan is 57.2° on a day in January, 1894. The very hottest on record is 108° on a day in May, 1878. The absolute range of tempera- ture observed is therefore between 43° and 44°. The {ormer temperature is far above frost, but would seem to the natives very cold and would check the growth of tropical plants. The latter would seem very hot, for the air of San Juan is very moist and the evaporation of perspiration is slow. The comfort of San Juan as a place of residence, not to mention its healthful- ness, is very much increased by the ‘‘briza,” which is not given in the public reports. It is the northeast trade wind which has been turned toward the west, until the ‘‘ briza” comes quite regularly from the east. CLIMATE. The temperature of the island is tropical, but is so modified by alti- tude and ocean winds that extreme heat or cold is never experienced. Cold never reaches the frost line and rarely drops below 65° F., while 91° is usually the extreme of heat in a season, and that only fora short period. As the temperature is largely modified by the winds from the ocean, and especially by the trade winds, considerable varia- tion is found in different portions of the island, it being warmer where the trade winds are shut off by mountains. A much greater differ- ence is observablein the rainfall. Somesections are ordinarily deficient in rainfall; in others it is very heavy. Ina recent report on the water resources of Porto Rico, H. M. Wilson? Ina recent report on the water resources of Porto Rico, H. M. Wilson? states ‘‘that all the crops which the soil will produce can be grown over three-fourths of the extent of the island with the aid of the abun- dant rainfall alone. The other one-fourth, including all the region near the coast and from Cabo Rojo on the extreme west to beyond Guayama on the east, must be irrigated if the soil is to produce the full measure of crops of which it is capable. The total area of these irrigable lands is, however, relatively small.” The weekly crop bulletin issued by the Weather Bureau of this The weekly crop bulletin issued by the Weather Bureau of this Department, San Juan, P. R., June 18, 1900, confirms the above statement. 5 6 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. Temperature and rainfall for the week ending June 18, 1900. AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. City. Temper’ Rainfall. OF, Inches. BAO CLI ee os a Noh ahah cA Pellerin A ay al a) Plc ia geet eth eek erase anlar 78.4 1.65 Manati.. 80.4 58 Isabela. - 80.0 1.22 Baynas - : 79.2 1.90 San Lorenzo 79.8 4.37 OUT A 2 ate hata ass co Sadocnany lables x ole cnn aelnheone dgeiciuinie Spel nasa ey aS SEIS ee anieases 77.2 3.40 IMP NTA Oo oo a eiaie Spnsc a crop tonern oie ae oe euseeawicla xb Sa ny melange sone Re Rac Eee SESS - 80.8 3.19 FUUIMA CAO 222585 accis midioteaubondenneste ooeamsasaysocasemennce = 80.2 6.52 Juana Diaz... za 80.4 4,72 Lajas -......- 53 80.3 4.20 Bayamon .... AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. Judging by Jamaica, of which the climate has been carefully studied, the heaviest rainfall is in the northeast, and it may here in places amount to 100 inches annually or more. In Jamaica it is known to surpass 200 inches in some places, and El Yunque, as seen from San Juan, is very generally capped by a rain cloud. The interior valleys of the island are relatively dry, while the northern and eastern mountain slopes are wet. The following summary, taken from the United States Weather Bureau report for Porto Rico, shows the temperature, rainfall, cloudy and clear days, and wind velocity at San Juan for one year: Summary Jor twelve months. Highest Lowest Greatest Month. tempera-\| Date. \|tempera-\| Date. Mean. daily ture. ture. range. °F, OR, °F 2 Rs November, 1898 .....--.-2+=-s-sss0s---<- 88 1 65 9 17.2 18 December, 1898 _-.... _ 85 12 66 118 75.9 M7 January, 1899__.. Ace 82 28 66 19 74.6 ey February, 1899_--.- a 85 8 66 220 75.2 16 March, 1899_......--.-- 82 35 66 8 74.7 15 April, 1899. ..........-- 90 21 66 4 76.6 16 ay, 1899... = 2 89 43 68 1 79 17 June, 1899 -......--..-. 91 22 al 6 79.4 17 July, 1899.... pela = 87 2 70 54 16 August, 1899 --........-- ne 88 29 a 20 80 16 September, 1899. .__.-. ee 91 11 71 30 81 14 October, 1899 2 xcssncwendeeseceneeeesccees 89 10 68 1 80 17 Least \| Gionay \| Partly \| clear Maxi- Month. daily eae cloudy ae a Rain. aren Nes range. Re ays ve ocity of wind.8 oF. Inches. November, 1808 222222020; avxwecasecess- “4 84 612 PTZ OB! \| isis fon December, 1898 -- — “ 8 9 22 Dedd [ew coca os January, 1899 -_.. 8 \|. 9 Be 2.92 22 February, 1899. a Becomes 9 19 80 20 March, 1899 .-.. 10 1 9 21 2.29 21 April, 1899 -. 8 2 8 20 6.09 24 May, 1899 - 10 2 18 11 2.59 19 June, 1899 - 9 6 17 7.23 24 July, 1899 - 7 4 16 11 7.53 19 August, 1899. 7 5 12 10.38 766 September, 1 i 6 11 13 13. 66 31 October, 1899 -. eexbes — i 13 12 10,21, 38 \| ' Also 19, 22. 3 Also 7, 19, 20, 29, 30. 5 Also 8, 27, 28. 7 Righth, east. AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. ning. Rainy days arerare, but rainy afternoons or evenings—for an hour or two— common. The rain begins suddenly, falls heavily,and ends soon. There is no aa of rainy climate, except that everything seems constantly fresh and clean. The healthfulness of San Juan is the greatest of any city in the West Indies. The healthfulness of San Juan is the greatest of any city in the West Indies. Yellow fever is never at home here, and when imported it rarely, if ever, spreads. * * ° The climate of the rest of the island is much like that of San Juan, with modifi- The climate of the rest of the island is much like that of San Juan, with modifi- cations due to elevation above the sea and to changes in the ‘‘ briza” due to the topography. The change of the temperature with elevation is relatively rapid here, being approximately 4° of temperature to every 1,0U0 feet. Now, Mount Yunque, at the northeastern part of the island, is, according to the Now, Mount Yunque, at the northeastern part of the island, is, according to the chief of the department of engineers of the isiand, about 6,000 feet high, and its summit would have a mean temperature as low as that of many places in the States. Besides, elevations of 2,000 feet are not unusual for towns; snow apparently never falls upon the islands, but hoarfrosts are reported as occasional in high places. Several towns of some size in the interior have a popular reputation as being cold— Cayey, Adjuntas, and Utuado. That black frosts do not occur, however, is evi- dent from the fact that the banana grows freely up to at least 2,000 feet, and is very sensitive to frost. There appear to be three mountain ridges running from end to end in the island, There appear to be three mountain ridges running from end to end in the island, but the central is the commanding one, and the elevations are, on the whole, high- est toward the eastern end, and especially at the northeastern angle. The result is that the ‘‘ briza” most wets and refreshes the eastern end of the island, and the rainfall changes greatly from point to point. CLIMATE. It is not felt much during the day, but springs up late in the afternoon and lasts through the evening. Itis asoft, gentle breeze, laving the body and giving an effect which is most fresh and delightful. It has a regularity approaching that of the sun, and Santurce and Catafio, two suburbs of the capital, get it both more strongly and through a larger part of the twenty-four hours. At Catafio it may be felt until the middle of the forenoon, and begins again in the mid afternoon. At Santurce it makes the nights positively cool. The year at San Juan is divided into the dry season and the wet season; but the The year at San Juan is divided into the dry season and the wet season; but the dry season has about as much rainfall as the Northeastern States, and the wet season more than twice as much. The dry season embraces the months from December to March, with a rainfall of 10 or 11 inches. It is the most attractive season of the year, relatively dry and cool. It is the proper season for the visits of Northerners to San Juan, and winter residents would find its climate very gentle, mild, and safe. The wet season embraces the other eight months in the year, and has a rainfall of 48 to 49 inches, or more than the whole of the year for the most of the United States. The total rainfall at San Juan is nearly 60 inches, and the culmination is in November, when an average of nearly 8 inches falls. The rainfallis not excessive. Itisequaledin many places in the Southern States and in the northern part of the Pacific coast, andis surpassed in many places. It is less significant from the ease with which the rain comes down. There are no threatenings of storms for days beforehand. There is little wind and little light- 7 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. 2 Also 23. 4 Also 4, 22, 27. ° Beginning Noy. 13. ® Miles per hour. Summary Jor twelve months. Summary Jor twelve months. 8 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. SOILs. Near the ocean the soil is quite sandy, merging into a sandy loam a short distance inland and gradually changing into a clay loam as the hills are approached. This is generally but not universally the case. Sometimes a spur of the mountain projects into the ocean and carries its soil conditions to the water’s edge. Occasionally there are tracts of gravelly soil, but all very fertile as far as observed. The soil along the larger streams is a deep, rich, sandy loam, merging into a clay loam near the hills and frequently extends to the top of the hills and minor mountains, rendering them so fertile that they produce excellent crops of sugar cane. The soil of the mountains is a peculiar ferruginous clay, which readily disintegrates when exposed to the air, and under the influence of tropical heat and moisture furnishes an abundant supply of plant food. Bananas and coffee grow luxuriantly on the mountain sides, more than 2,000 feet above the sea level. Limestone is abundant in all portions of the island and more or less affects the soils, producing on the mountains a slightly caleareous soil. A green- sand marl, similar in appearance to that of New Jersey and rich in phosphates, is, according to Wilson,! ‘‘ found extensively in the north- western portion of the island, between Lares and San Sebastian, and also in the neighborhood of Carolina, at the northeastern extremity of the island.” The division of soils for taxation by General Henry in his Order No. 6, dated January 19, 1899, throws some light on the way soils are classified. It is as follows: ' Loe. cit., p. 33. LANDS AS CLASSIFIED FOR TAXATION, (1) The assessment of taxes upon iands will hereafter be made in accordance wae various cultivations existing in the island and the quality of the lund taxed. (2) In accordance with the various cultivations there will be taxes on cane (2) In accordance with the various cultivations there will be taxes on cane ene coffee lands. tobacco lands, pasture lands, minor-produce lands, and forest ands, (3) In accordance with the quality of the land there will be taxes of the first, second, and third classes—the first class comprising the best lands, the second class the next best, and the third class the poorest. (4) On all lands of the first class there will be a tax of 1 peso per cuerda (acre); (4) On all lands of the first class there will be a tax of 1 peso per cuerda (acre); on all lands of the second class a tax of 0.50 peso per cuerda; on all lands of the third class a tax of 0.25 peso per cuerda. (5) Each municipal corporation will appoint a classifying commission, which (5) Each municipal corporation will appoint a classifying commission, which will select commissioners in the different districts of each township, the subcom- missioners to report to the classifying commission on the class of lands in their respective districts. (6) These commissioners will be guided by the following instructions: First-class cane lands are plains and valleys and other alluvial lands lyin (6) These commissioners will be guided by the following instructions: First-class cane lands are plains and valleys and other alluvial lands lying near settled communities, highways, railroads, and seaports, and the lands of drained lagoons and mangrove marshes. Second-class cane lands are the highland plains, generally surcharged with oxids Second-class cane lands are the highland plains, generally surcharged with oxids iron, and known in the country as clayish lands. First-class coffee lands are valley lands and hills abounding in organic detritus. First-class coffee lands are valley lands and hills abounding in organic detritus. Second-class coffee lands are highlands having a calcareous or limy formation. Second-class coffee lands are highlands having a calcareous or First-class tobacco lands are valley lands watered by rivers. First-class tobacco lands are valley lands watered by rivers. Second-class tobacco lands are loamy highlands mixed with clay and sand. . Third-class tobacco lands are sandy lands along the coast and calcareous la _. RIVERS AND DRAINAGE. Few countries are better drained than Porto Rico. It is claimed that there are over 1,200 streams in the island, of which 50 are rivers of considerable size. Springs are abundant in the mountain district and the water is pure. There are several mineral springs of value, such as the sulphur There are several mineral springs of value, such as the sulphur baths near Ponce, the mineral springs near Coamo and at San Sebas- tian and San Lorenzo, and the hot springs of Caguitas. AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. Third-class pasture lands are those along the coast and lime hills, where grow only brush, ‘‘ rat-tail,” sweet grass, etc. First-class minor-produce lands are valley lands. First-class minor-produce lands are valley lands. Second-class minor-produce lands are highlands. Second-class minor-produce lands are highlands. Third-class minor-produce lands are sandy and limy lands. Second-class minor-produce lands are highlands. Third-class minor-produce lands are sandy and limy lands. First-class forest lands are those growing virgin forest whose Third-class minor-produce lands are sandy and limy lands. First-class forest lands are those growing virgin forest whose First-class forest lands are those growing virgin forest whose timber can supply building and cabinet woods, e. g., ‘‘aceitillo,” ‘ cedar,” ‘‘capa,” ‘‘ausubo,” ete. Second-class forest lands are lands with a rocky and calcareous soil growing Second-class forest lands are lands with a rocky and calcareous soil growing only bushes available for fuel. RENEWAL OF SOIL. While the soils of Porto Rico retain their fertility to a remarkable extent, they have deteriorated in a measure under the effects of con- stant cropping without the use of manure. Like most tropical soils, they are deficient in humus, and this is particularly noticeable in old fields ‘‘turned out to rest.” The Porto Ricans say that the land is “‘tired.” It is further noticeable in the fact that the soil is too adhe- sive in wet periods, and too dry, with a tendency to ‘‘bake,” in dry weather. That there is no deficiency of elements in the soil is shown by the large grass crops these old fields will produce. Cowpeas, velvet beans, and other renovating crops have enormously vigorous growth and furnish a ready means of supplying humus. In all parts of the island are caves with vast quantities of bat guano, which is a cheap and ready source of nitrogen. The large number of cattle and horses furnish considerable manure, easily obtained, because many cattle are herded on account of the absence of fences. LANDS AS CLASSIFIED FOR TAXATION, Third-class tobacco lands are sandy lands along the coast and calcareous lands among the hills. Fi First-class pasture lands are valleys, lagoons, and glens, where grow ‘‘malojila” First-class pasture lands are valleys, lagoons, and glens, where grow ‘‘malojila” and guinea grass. Second-class pasture lands are those on the hills and those on the coast, where Second-class pasture lands are those on the hills and those on the coast, where row guinea and dog grass, ' Loe. cit., p. 33. 9 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. FORESTRY. One of the most serious obstacles confronting the agriculturist in Porto Rico is the scarcity of timber and wood. There is practically no accessible building timber. Occasionally a small log is brought from the interior at great expense and sawed into boards by hand. There is not a power sawmill in the interior of the island. There are afew sawmills in the coast cities, mainly for sawing imported tim- ber. At Mayaguez one of these mills had a small stock of native logs. They were from 6 to 12 inches in diameter and 8 to 12 feet long, crooked and knotty. The mill had astock of four or five thousand feet of native lumber, in which were a few tropical cedar boards, short but of superior quality. The principal lumber sold in the coast cities is hard pine, which The principal lumber sold in the coast cities is hard pine, which retails at $30 to $50 per 1,000, depending upon the quality. This searcity of timber accounts for many things. It renders it almost impossible to erect or maintain creditable farm buildings in the inte- rior, especially where there are no good roads. It renders it necessary to construct the cottages of the laborers of bark and poles. It precludes the fencing of the farms into suitable fields for keeping the variety of 10 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. stock best adapted to the conditions and yielding the largest profits to the farmer. It accounts in part for only one-eighth of the arable land of the island being under cultivation. The absence of timber on the mountains is believed to affect in a large degree the amount and regularity of the rainfall. The records of history and the samples of the woods preserved prove that Porto Rico at the time of its discov- ery had a wealth of timber unsurpassed by any island in the world. In the United States quartermaster’s department at San Juan is the relic of an old Spanish military museum, which contains samples of Porto Rican woods selected over fifty years since, from which Mr. Paul L. Hardy, engineer, made me a list of 150 varieties, with the uses for which they are valuable. Among them are found cabinet woods, as mahogany, satinwood, cedar, walnut, etc.; other woods that are almost indestructible in air or water, especially valuable for wharves. FARMHOUSES. The huts in which farm laborers live are either in tent form, 10 by 14 feet on the ground and 6 feet high at the ridge, made of poles and covered with the bark of the royal palm or with grass, with the ground for floor; or they are made house fashion, 12 by 16 feet base, 5 to 6 feet high at the eaves, sides and roof covered with palm bark, with which the so-called windows are closed at night; rarely the roof is thatched with grass. In the vicinity of towns the sides are frequently made of boxes. Most of the huts built in house form stand on short posts and have a rough floor made of palm boards sawed by hand. Many of them have a small room, possibly 6 feet square, for cooking. In these small houses large families areraised. Sometimes more than one family reside in one house. There are rarely any gardens, flowers, or fruit, except the banana. The water is not always the best, and few make any effort to improve it. Under such circumstances it is impossible to have comfort or good morals. The houses occupied by the proprie- tors or farm managers are generally fairly comfortable dwellings, occa- sionally of a superior type, according to the taste and wealth of the owner. FORESTRY. Many varieties are suitable for posts, fencing, and building purposes. That immediate steps should be taken to reforest the island to the extent of its own uses is self-evident, especially as there is plenty of idle land for this purpose. There is much government land in the mountains that can be profitably used for woodland, on which could be planted hard-wood trees of quick maturity for fuel and more durable woods for posts and building purposes. Lack OF MANUFACTURES. There are very few manufactures in Porto Rico, except such as are the necessary adjuncts of a farm crop, to fit it for market—uas coffee and sugar mills. Manufactures other than these are not sufficient to produce any material effect upon the general industrial situation. At the time of the American occupation they were limited to cigars, ciga- rettes, macaroni, chocolate, ice, matches, and rum. The cost for the initiative, or the privilege of starting the enterprise, was high; the permit was slowin coming, and sometimes never granted; an enormous tax was placed upon the importation of all kinds of machinery, and, finally, the laws were so framed as to favor the Spanish merchant. For example, wheat and wheat flour had to pay a high duty when imported directly from the United States into Porto Rico, but they could be shipped to Spain and thence to Porto Rico free or at a nomi- nal duty. This benefited the millers of Spain and the manufacturers of biscuits, soup paste, and other products of flour. The following statement from the report of Dr. Henry K. Carroll (U. 8. Treasury Dept., Doc. 2118), is in point: As between Spanish and Porto Rican producers and manufacturers the latter had no chances. Nor were the needs of Porto Rican consumers, however urgent they might appear from the insular point of view, treated as worthy of serious attention. Indispensable articles of food not produced in the island had to come in a roundabout way through the hands of the merchants in Spain or pay enor- mous duties if imported direct from other countries. ‘lhe Porto Ricans thought that some of the many streams of the island might well furnish power to mills to grind wheat from the United States or Canada into flour, but the Government at Madrid punished these aspirations by making the duty on wheat almost as high as that on flour. Flour paid $4 per sack of 92 kilos (about 200 pounds) and wheat $3.15, and flour paid also, for municipal purposes, a consumption tax of $2.30. There were mills in Spain, and by importing wheat for them from the United States they could be kept going. The millers of Spain profited; the people of Porto Rico suffered. PUBLIC ROADS. In a country like Porto Rico there are no medium roads. They are either good or so bad as to be impassable with a wagon. Unless a road is constructed of stone, thoroughly ditched and bridged, the tor- rents in the rainy season will wreck it. There are about 150 miles of first-class road, which were constructed by the Spaniards at an aver- age cost of over $12,000 per mile (gold). These roads are a monu- ment to the science and thorough work of the Spanish engineers. Outside of these military highways transportation must mainly be by pack train. The cost of this in some seasons of the year operates as a complete embargo on marketing farm products. Landslides cover the trail, bridges have been swept away, and the swollen streams are impassable at the fords. One person reported that he sometimes paid 11 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. $12.80 (gold) for an ox load—distance, 21 miles. To relieve the most important rural sections and to furnish labor to the peasants, deprived of labor by the destruction of the coffee plantations, the insular gov- ernment has undertaken to construct about 125 miles of first-class roadsin theisland. (See map.) The construction of these roads will cost about $1,000,000. In addition, large sums have been expended in repairing the old military roads injured by the floods at the time of the hurricane. When these are completed and dirt roads con- structed in sections not liable to torrents, nearly all portions of the island will be accessible. Lack OF MANUFACTURES. Attempts were made in the island to manufacture soup paste and crackers, Attempts were made in the island to manufacture soup paste and crackers, The result is graphically described in the report of the manulacturers of Ponce, drawn up in 1898 for the use of the colonia] minister at Madrid and presented to the commissioner of the United States without change, as the best statement pos- sible of the needs of the island. The cracker manufacturers had to pay the high duty on flour and compete with crackers from the Peninsula entered free of all duty. Those who invested largely in the manufacture of soup paste saw their business killed in the same way. Their petition to the liberal minister, from which they hoped so much, is pathetic in its pleadings for simple justice. Appeal after appeal was made, they say, but all ‘‘ slept the sleep of the just” (are pigeon- holed), for if ever a minister intended to cast a pitying glance upon such injus- tice and relieve so much misfortune by some saving measure this intention never materialized, but was strangled in its birth by the influences brought to bear by Spanish manufacturers. The shoe manufacturers have the same story to tell; shoes imported free from Spain, shoes of the poorest quality—-' pasteboard soles, badly made, unsightly, coarse, and without durability "—while Porto Rican manufacturers were heavily 12 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. taxed for the raw materials. Of course shoes are costly, and 700,000 out of the 900,000 population go bare-footed. It was the opinion of the industrialists that they could make better shoes and furnish them more cheaply than the Balearic Island manufacturers, but they were not given the chance. They believed that the result of home manufacture would be to lower the prices, as in other instances, but com- petition with Spanish producers, when the latter had both the home and insular markets, was impossible. There are salt:mines at Cabo Rojo, but salt from Spain is free, and vessels loading with salt had to clear from Mayaguez, increasing the expenses of shipments, because the port of Cabo Rojo had been closed, so the salt industry was crippled. Lack OF MANUFACTURES. Those interested, or who would be glad to be interested, in the manufacture of Those interested, or who would be glad to be interested, in the manufacture of soap show that while soap from Barcelona paid only the transitory duty of ‘10 per cent at the ports of the island, amounting to $15 for every hundred boxes of a hundredweight each, the insular industry is compelled to pay $32.82 in duties for the raw materials to make that quantity of soap. No wonder they ask, in despair, What business can succeed under such circumstances? Classification of rural lands. Classification of rural lands. Classification of rural lands. Cuerdos (acres). MPODACGCG: joc cezacauee tae oo oo woe Rene Ree e eee eee eeensesemse 4, 264. 07 DUCATI CAN a, ceoysco = sty eytets cad ta Cc Se RLS etcne BEES EeS 61, 558. 43 COMCO yah atee chet enc eas awenletaen Lem aie aneaea nen mrameeoiacecse 122, 359. 76 MAING? Chops enn es ea eaten et hs bs eens teh eed Het eee eee 93, 511. 08 Cultivated: Brassesis. <c.cnccucniane She 3 An Se ERERESE RE Ss co eReRREAS ene 16, 277. 23 Natural, pasture. 22 cccccc cen. cee 5ce & Sone aceademesace mnie ss 1, 127, 087. 55 Mountain lands and forests _..._-.--.......------------ ----25----- 664, 273.37 Total area of island..._____......02-- 2-2-2. 22 pe eee ee eee 2, 089, 381, 49 AGRICULTURE ON NARROW LINES. Agriculture in Porto Rico is conducted on narrow lines. Sugar, coffee, tobacco, and cattle constitute almost the entire exports. In 1897 the total agricultural exports amounted to 18,352,541 pesos (peso valued at 60 cents gold), and of this total, sugar, coffee, tobacco, and cattle furnished 18,133,682 pesos. Rural lands are classified, as declared by their owners for assessment, Rural lands are classified, as declared by their owners for assessment, as follows: SUGAR-CANE LANDS. Cane lands may be divided into three divisions, according to quality required for this purpose: First. The rich alluvial bottoms along’the rivers. First. The rich alluvial bottoms along’the rivers. Second. Second bottoms somewhat remote from the Second. Second bottoms somewhat remote from the rivers. Third. Fertile hill lands. Third. Fertile hill lands. The best cane lands produ The best cane lands produce 50 to 60 tons of cane per acre when virgin, and one planting will last ten to twelve years. After the land has been in cane four or five years the annual crop falls to 30 and 35 tons, and finally to 20 tons. The productive power of the soil gradu- ally declines under the system of cultivation at present pursued. Twenty to 25 tons of cane per acre is now regarded as a fair crop Twenty to 25 tons of cane per acre is now regarded as a fair crop on old land, and one planting will not continue profitable more than three years on an average. Second-class land yields on virgin soil 30 to 35 tons of cane per acre Second-class land yields on virgin soil 30 to 35 tons of cane per acre and must be replanted after three crops. When somewhat worn, 15 to 20 tons of cane per acre is an average crop, and must be replanted the third season. Third-class lands produce about 20 to 25 tons of cane on virgin soil, Third-class lands produce about 20 to 25 tons of cane on virgin soil, and deterioriate within a few years below the point of profitable cul- tivation. The extent of first-class sugar lands is quite limited in Porto Rico, and nearly all of it has been farmed for many years. The proprietor of one tract stated that it had been in cane continuously for ninety 13 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. years without fertilization of the crop, a proof of the marvelous pro- ductive capacity of the soil. The growing crop of cane looked as if it would yield 20 tons per acre. years without fertilization of the crop, a proof of the marvelous pro- ductive capacity of the soil. The growing crop of cane looked as if it would yield 20 tons per acre. CANE CULTURE. Instead of planting in drills as in the United States, with the middle upon each side deeply furrowed for drainage, the Porto Rican method is to plant in double rows of hills, allowing the rubbish from the canes of the previous crops to be windrowed between the rows of cane and alternating with the deeply plowed middles, thus placing the drainage middles about 10 to 12 feetapart. The drainage middles are 5 to 6 feet wide, but those for piling the rubbish are narrower. This has some advantage over our plan and some disadvantages. It is of great bene- fit to the soil to allow the rubbish to decay upon it. It acts as a mulch and as a positive fertilizer. On the other hand, while our method of burning the rubbish is wasteful of the fertilizer, it gives better drain- age by allowing a deeply plowed middle on each side of a row of cane. Cane planting is almost a continuous operation from December till July following, as canes are cut for the mill. Onthe2d of July, near Arecibo, I saw trains of carts carrying cane to the sugar factory, and as late as the 7th of July a factory south of Mayaguez was still grind- ing cane. In both cases I was informed that the saccharine contents were very low—reduced by the rains—and gave only about 130 pounds of sugar per ton of cane. The average period for maturing cane is one year, dating from the The average period for maturing cane is one year, dating from the cutting of the previous crop. Where the planting for some excep- tional reason must be done as late as July, generally white cane is planted and allowed to stand till a year from the following December before cutting, allowing eighteen months in which to mature. Ribbon cane, if allowed to stand so long, matures and cracks open, souring the juice. This long period is given to avoid cutting in the rainy season. Factories with steam vacuum pans Factories with open steam evaporators.... Factories using ox power and open kettles GENERAL COMMENTS ON THE SUGAR INDUSTRY. With United States markets and under Spanish conditions of labor in semiservitude, Porto Rican planters could make money rapidly even with existing methods; but under American conditions of labor— i. e., labor under American laws—it is an even thing between the pro- duction of cane sugar in Porto Rico and cane sugar in the United States. The sugar estates in Porto Rico pay lower wages than those in the United States. They have a longer period in which to mature and manufacture their crop, with no danger of frost. The cane hasa full year for growth and five months in which it can be ground under favorable conditions, as against nine months for growth and seventy- five days for manufacture in the United States. Labor, however, owing mainly to the primitive methods employed, Labor, however, owing mainly to the primitive methods employed, accomplishes very little in a day in Porto Rico, and notwithstanding that men were paid only 50 cents a day, silver (worth 30 cents gold), it was expensive labor. It required 6 yoke of oxen and 3 men to plow three-fourths of an acre ina day. The oxen subsisted on grass with- out grain, could plow only half a day at a time, consequently 3 yoke were used in the forenoon and 3 in the afternoon. One of the most thorough planters in the island stated to me that he used 420 oxen to cullivate and harvest his cane crop—a little over 800 acres. He thought it would be economical to substitute mules. The farm work is universally done with oxen. No labor-saving machinery is employed in the cane shed or in the field. MOLASSES AND RUM. Inferior machinery used in such a large number of sugar factories leaves the molasses rich in sugar. Part of this is exported for reboil- ing in the United States; the remainder is manufactured into rum. In 1897 the number of distilleries of rum was.._...-.-----.------------ 198 In 1897 the number of distilleries of bay rum was...-_.---..-----.----- 28 Gallons of rum distilled in 1897. ......... 022-2222. ee eceeeeeeee eee e+ 1,615, 075 Gallons of rum exported in 1897 .._... .....------------------ ---- ---- 85, 252 Gallons of rum consumed at home__..._..-....--.-------.--.--------- 1,529, 823 Gallons of bay rum manufactured ...._.......---...-------------+----- 15, 143 Gallons of bay rum exported... ._.....--.--------- +--+ 2-2-2 - 2 ee eee 18, 843 Gallons of bay rum consumed at home -._..__--------- .--------------- 1,300 Total export of molasses in 1897: Number of gallons _..__....-.--.--2 2-22-22 ee ee ee eee ------- 8, 548, 830 EMC on ternal coped wee te aR ie tes BAN area ee niger engin Mae te ase (POOLE Waltie pot? Salons ccc ceciccns saacwake cised ant aicnngeaaicwenees cents. - 8.2 MANUFACTURE OF SUGAR. There are but few modern sugar mills in Porto Rico. Most of them have a single three-roller mill and are strictly upon the old open- kettle or Jamaica plan, using the dried bagasse under the kettles for fuel, and completing the reduction in an open steam evaporator. Draining is accomplished in the usual way, producing a low to extra grade of muscovado sugar, according to the care taken. Little fuel in addition to the bagasse is required. Large planters have introduced steam train vacuum pans and cen- Large planters have introduced steam train vacuum pans and cen- trifugals; a few have double mills, triple effects, vacuum pans, ete. All defecate the juice with lime, but I found no filter presses, though I was informed that some were in use. Factories make from 140 to 190 pounds of sugar per ton of cane, according to process pursued, Only one factory was reported as making 200 pounds. The progress of the sugar industry may be noted by the following The progress of the sugar industry may be noted by the followin statistics: Equipment of sugar factories, 1888 and 1898. Equipment of sugar factories, 1888 and 1898. Equipment of sugar factories, 1888 and 1898. Equipment of sugar factories, 1888 and 1898. Equipment of sugar factories, 1888 and 1898. Factory equipment. 1888. 1898. Factories with steam vacuum pans 20 50 Factories with open steam evaporators....-- 140 100 Factories using ox power and open kettles - .- 286 100 Factory equipment. 14 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. THE COFEEE INDUSTRY. The coffee plantisstrictly a tropical evergreen, and, if left unpruned, attains a height of 20 to 30 feet. It should be headed low; so as to be more productive, and to make all parts easily accessible to the picker. The leaves are 4 or 5 inches long. oblong-ovate, dark green in color. The whole plant is fresh and attractive in appearance. The fruit is much like a cherry in form. Each berry contains two seeds, surrounded by a thin, sweet pulp. The flat surfaces of the two seeds are facing and are separated by a thin layer of pulp, and also by a tough membrane or skin which envelopseach. Asthefruit approaches maturity the berry turns red, and Jater a dark purple. The plant requires good drainage. The taproot penetrates to a depth of 3 to 5 feet, and is injured if it reaches standing water. RENOVATING CROPS. Sugar planters appear to have little knowledge of the importance and value of renovating crops. When a field has been in cane so long that it is exhausted, the Porto Rican planter says ‘‘it is sick,” or “it is tired,” and forthwith turns it out to common till it is recuperated. A system of rotating cane with corn and the free use of cowpeas (which grow luxuriantly) would soon restore the cane fields. One planter near Bayamon putsome manure on his old field, and he reported a crop of 50 tons per acre last year. Improved implements, modern methods in handling cane fields, the substitution of mules for oxen, 15 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. and better sugar machinery will enable the Porto Rican farmer to compete with any country in the world in the sugar industry. The available lands for sugar are too limited in Porto Rico, however suc- cessfully managed, to materially affect the markets of the United States. Wages are certain to advance. In fact, since the substitu- tion of the gold standard, August 1, 1900, laborers are demanding 50 cents per day in gold instead of silver, and some sugar planters have granted the advance. With better wages more sugar will be con- sumed in the island. If the Porto Rican laborer used as much sugar as the American, nearly the entire crop would be consumed at home and there would be little sugar for export. PRESENT CONDITION OF THE SUGAR INDUSTRY. Prior to the American occupation of the island the sugar industry was ina bankrupt,condition, owing to the low prices of sugar and other causes. Many planters allowed their sugarhouses to go to, decay, and turned their cane fields into pastures. Of the 26 sugar estates in the district of Farjardo, 14 were idle and turned out for grazing in 1898—the year of our occupancy. Had they been prosperous they would have beenin canethat year. Further statistics show that about two-sevenths of the larger sugar estates of the island had gone out of business. A few sugar estates, with capital and close management, were able to pay expenses and continue the business. For the ten years prior to 1898 Porto Rico had marketed in the United States an average of over 80,000,000 pounds of sugar annually, at prices ranging from 1.7 cents to 2 cents per pound. Thecrop of 1899, though injured about 33 per cent by the hurricane, netted the planters more money than any crop for several years. They stated that they realized from 33 to 4 cents per pound, according to quality. It is estimated that the present crop will furnish about 80,000,000 pounds for export. The sugar industry is therefore in a flourishing condition. COFFEE CULTURE IN PORTO RICO. Notoriously, however, the number of acres given for assessment was below the real number in crop, and consequently is not an exact guide. : A coffee plantation should improve every year with increase in age A coffee plantation should improve every year with increase in age for twenty-five to thirty years, and the annual product should average 1,000 pounds per acre under cultivation. When it falls to 250 pounds, it shows faulty cultivation. ; The crop commences to ripen in the early fall, and is picked by hand The crop commences to ripen in the early fall, and is picked by hand at an average cost of half a cent (gold) per pound for the green fruit. There are three processes of curing. One exposes the ripe berry to the sun till the pulp is desiccated, which requires several weeks. In the second process the berries are passed between the rollers of a “‘pulper,” and reduced to a pulp, which is dried in the sun before removing the grain. By the third method, and the one generally used, the pulping process is followed by washing, to free the grains from the pulp. The grains are then dried and sent tothe merchant’s warehouse, or to the port, where the skin enveloping the grain is removed by machinery and the grain polished. Mocha and Old Gov- ernment Java are prepared by the first process, which is considered to givea coffee of higher flavor. The third process enables the planter to market his crop much earlier and with less labor. COFFEE CULTURE IN PORTO RICO. The favorite places selected for coffee plantations in Porto Rico are narrow ravines on the sides of mountains, where on three sides the plant will be protected from the wind. Plants are started in a seed bed and when 1 year old are transplanted into the field and set in rows 4 to 5 feet apart. In Porto Rico little attention is given the preparation of the soil, and no fertilizer is used. Very little pruning 16 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. is done. Shade for the first five years is furnished by planting bananas between the coffee plants. Later guava trees. furnish shade. On the coffee plantations I visited little attention had been paid to setting in rows to any exact distance apart. In many instances two plants had been set together, on the theory that one might die. Sufficient care was not taken to thin the bananas, so as to furnish the requisite amount of sunshine. The most reliable information I could obtain placed the average annual yield of coffee per acre at 100 to 400 pounds; but if the total product of coffee any year be divided by the number of acres given by the planters for assessment, it shows a product of 430 pounds per acre for the entire island. Notoriously, however, the number of acres given for assessment was below the real number in crop, and consequently is not an exact guide. : A coffee plantation should improve every year with increase in age is done. Shade for the first five years is furnished by planting bananas between the coffee plants. Later guava trees. furnish shade. On the coffee plantations I visited little attention had been paid to setting in rows to any exact distance apart. In many instances two plants had been set together, on the theory that one might die. Sufficient care was not taken to thin the bananas, so as to furnish the requisite amount of sunshine. The most reliable information I could obtain placed the average annual yield of coffee per acre at 100 to 400 pounds; but if the total product of coffee any year be divided by the number of acres given by the planters for assessment, it shows a product of 430 pounds per acre for the entire island. FINANCIAL CONDITION OF THE COFFEE PLANTERS. Sasteria Francisca, before United States Com missioner Carroll, November 1, 1898, is in point: Importations formerly were made on a half scale in Porto Rico—that is to say, were imported over and above the needs of the island, because the importers could get a year’s credit from Paris, London, or Hamburg commission houses. These merchants or importers, when they sold tosmaller houses, charged them from the date of invoice one-half per cent interest [monthly] outside of their commission on the merchandise shipped, while they only paid their bankers at the rate of 4 per cent per year. Moreover, these importers sold that very merchandise on long terms to merchants in the interior, these terms extending as long as one year and a haif in some cases, and generally sold at wholesale at higher prices than were paid by retail at the rates prevailing in the capital. These merchants of the interior would do exactly the same things in turn with the smaller merchants of the country, selling to them on long terms, and charging them at least 1 per cent a month on the invoiced values, and often from 14 to 24 per cent. This class of smaller merchants in the interior consists for the most part of cul- This class of smaller merchants in the interior consists for the most part of cul- tivators, and it is a very important matter to be considered that these small culti- vatorsare charged at least 35 per cent per annum over and above any profit realized in any country in the world. The results of that system have been that at least one-quarter of the small proprietors in the island, buying in that way, in the period of five years have all lost their estates, the estates going into the hands of Spanish merchants who commenced selling goods on credit without any capital to speak of, and who after five or ten years have become worth $20,000 and even $50,000. The estates on which they held mortgagés were unable to produce sufficient to pay back at the half rates that were collected. The evidence is to the effect that planters with plenty of capital could make money in the coffee industry, but those who were obliged to borrow lost money. FINANCIAL CONDITION OF THE COFFEE PLANTERS. All careful investigations show that when Porto Rico came under American control the financial condition of the coffee planters, in the aggregate, was about as bad as it could be. Of course there were some exceptions. A few wealthy merchants owned plantations, and some planters were very thrifty; but about 70 to 75 per cent of the coffee plantations were heavily mortgaged, 30 per cent or more being mortgaged to the full value of the plantations. To make clear how this occurred, the financial system under which coffee plantations were managed should be explained. It is well known in Louisiana as the system of ‘‘advances.” At the commencement of the fiscal year the Porto Rican-planter arranged with some city merchant (provided the planter was not a merchant or a capitalist) to advance him from time to time money and supplies sufficient to make his crop. He pledged his plantation and crop by mortgage, agreed to pay 12 to 18 per cent interest, as the merchant might demand, and to turn over his crop, when harvested, to be sold at the option of the merchant. The planter received very little money and was charged a profit of 50 to 75 per cent on all supplies. The merchant, if he found it necessary, could buy his goods in foreign ports on a year’s time. The coffee planter opened a small store on his plantation and paid his laborers in orders on this store or gave duebills on-some branch store of his merchant. Under this system it cost the planter 30 to 40 per cent for funds to make his crop, and at the end of the season he had no option as to price or time in disposing of his crop; the merchant was the sole judge. Frequently the crop did not pay expenses; then a mortgage was retained, which increased from year to year till it absorbed the property. The testimony of Mr. Sasteria Francisca, before United States Com- The testimony of Mr. Sasteria Francisca, before United States Com- missioner Carroll, November 1, 1898, is in point: The testimony of Mr. MARKETING THE CROP. Before placing coffee upon the genéral market it is graded by selec- tion into six classes, known in the island by the following names, in order of quality: First, caracolillo; second, hacienda; third, pueblo; fourth, cubano; fifth, merinda; sixth, frilla. The quality of the better grades of Porto Rican coffee is excellent, and compares favorably with the best coffees of the world. Till the American occupation it was chiefly marketed in Europe, as shown by the following table for the year 1896: Exportation of coffee from Porto Rico for the year 1896. Exportation of coffee from Porto Rico for the year 1896. Exportation of coffee from Porto Rico for the year 1896. Countries to which exported: Pounds. DAI cece ssa Se sete Sk ES Bek Ses lye dae eg erences telesales atie tale 16, 405, 900 PTANCG a cccecwass 2 iehiet Abad aeetee ase asses sere esee sete ee ss 24 11, 306, 689 GOMMaDY no. 222s ond as eomese nnennee caeemaesebesa nectoneies +2 bs epee 8, 120, 409 TAY. 2c cceccamiees res. 205 bomew andere are e eerceseekeeoneen ge oe 4, 388, 819 Oba: cegenssccasaccmenepe ares eect: «agen eEsee reer ees 15, 577, 710 United Kingdon, ..20c0022 0c: cncens awensscesececasceeeresseeneecs 304, 119 Austria-Hungaryencensooceseess Geeta Gecassdws Seyesecewen eu 2, 280, 221 United States 2.) c2o:.22 aconcssacsieseeces metne esa eeeus cous 822, 591 Daniah: possessions:. 22. c.scwssaesk Sosa mood ieee ee cease Ses s 19, 595 British: possessions... 2.2 sisco asaciadvier ieee secoehen cam'see? Ses 452 Santo Domingo... +0. 2--- 22262: esscsesaeceoge westeeaege des 22, 501 Total .wcssscesuesed: eegersecensnpels Bede aceiccece uses dss 58, 780, 606 17 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. FINANCIAL CONDITION OF THE COFFEE PLANTERS. By careful com- parison of this with statements by bankers, it appears that the mort- gage indebtedness of coffee estates at that time was about $8,000,000. The total acreage of coffee reported for assessment was 122,389.76 acres, which would make the indebtedness on coffee lands equal to $65.46 on every acre in cultivation. If 30 per cent of the estates were not mortgaged, as appeared evident, but were owned by capitalists, then the mortgage indebtedness on the remainder must have been $93.51 per acre, an amount altogether too large for a farmer to safely carry. The mortgage is purposely attached to the lands in coffee, for in coffee estates they are the only income-producing property. Under these conditions coffee planters were compelled to face a great decline in prices, owing to increased competition in the markets of the world and special changes in the Porto Rican market arising from change of flag. Under such burdens the coffee planters entered upon the season Under such burdens the coffee planters entered upon the season of 1899 and secured such advances as they could to make the crop. August 8, 1899, a destructive hurricane visited all portions of the island, sweeping away many buildings and a large portion of the coffee crop, tearing up the trees used for shade for the coffee, and greatly damaging the coffee plants. This left the coffee planters with- out means to continue work, because it destroyed credit as well as property. Had the planter possessed the capital to go immediately after the storm and clear away the débris, restore the buildings, and straighten up the coffee plants, the damage would largely have been limited to the partial loss of a crop; but not being able to do this, the loss in most cases amounted to the practical destruction of the planta- tion, which it will require five years to restore. In tropical countries the growth of grass, weeds, and vines is very rapid, converting the uncultivated coffee plantations in a few months into a jungle and. destroying the trees. From 70 to 75 per cent of the coffee plantations are now seriously From 70 to 75 per cent of the coffee plantations are now seriously injured, and the value of all coffee lands has declined. FINANCIAL CONDITION OF THE COFFEE PLANTERS. The Porto Rican coffee crop of the years 1892 to 1896, inclusive, sold in foreign markets in cargo lots at over 14 cents per pound (gold). This high price stimulated speculation in coffee plantations till they were rated at fabulous prices. In some cases H. Doe. 171——2 H. Doe. 171——2 18 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. they were quoted at $600 (gold) per acre for all the land in coffee- bearing plants. Probably $150 to $200 would be nearer a true aver- age. Many coffee estates were sold on long time at these high prices to planters. Small payments were made at the time of the purchase, and the unpaid amounts were secured by mortgage at 12 per cent interest. The records of mortgages in Porto Rico show $15,664,971.69 uncanceled mortgages on country property in 1898. By careful com- parison of this with statements by bankers, it appears that the mort- gage indebtedness of coffee estates at that time was about $8,000,000. The total acreage of coffee reported for assessment was 122,389.76 acres, which would make the indebtedness on coffee lands equal to $65.46 on every acre in cultivation. If 30 per cent of the estates were not mortgaged, as appeared evident, but were owned by capitalists, then the mortgage indebtedness on the remainder must have been $93.51 per acre, an amount altogether too large for a farmer to safely carry. The mortgage is purposely attached to the lands in coffee, for in coffee estates they are the only income-producing property. Under these conditions coffee planters were compelled to face a great decline in prices, owing to increased competition in the markets of the world and special changes in the Porto Rican market arising from change of flag. Under such burdens the coffee planters entered upon the season they were quoted at $600 (gold) per acre for all the land in coffee- bearing plants. Probably $150 to $200 would be nearer a true aver- age. Many coffee estates were sold on long time at these high prices to planters. Small payments were made at the time of the purchase, and the unpaid amounts were secured by mortgage at 12 per cent interest. The records of mortgages in Porto Rico show $15,664,971.69 uncanceled mortgages on country property in 1898. THE TOBACCO INDUSTRY. Tobacco, the third chief product of Porto Rico, does not labor under any such disadvantages as coffee, as the loss of a crop can be repaired in one year. Aside from the temporary loss by the hurricane, the chief difficulty of the tobacco industry is the change of markets. Formerly a large percentage of the tobacco of Porto Rico was shipped to Cuba. This consisted of the better grade of ‘‘fillers and wrap- pers.” In 1895 the amount shipped to Cuba was 2,160,347 pounds, or nearly two-thirds of the entire crop exported. In Cuba it was manufactured and sold as Cuban product. Of the remainder, about 1,000,000 pounds were manufactured and consumed at home. Of the balance, mainly low-grade tobacco, 1,375,751 pounds were sent to Spain and smaller lots to Germany, France, and other countries. In January, 1899, the Cuban tariff, imposing prohibitive dutes ($5 In January, 1899, the Cuban tariff, imposing prohibitive dutes ($5 per pound) on the Porto Rican product, went into effect, and the market for two-thirds of the crop was at once lost. The Spanish market was lost by change of flag, and tobacco was compelled to seek new markets. In this emergency the tobacco dealers met the difficul- ties by studying the markets of the United States and producing the qualities here demanded. Formerly they assorted their tobacco into three grades. Recently some have employed Cuban experts and are making fifteen or sixteen grades. Rorto Rican tobacco produced near the coast is of a low grade, but Rorto Rican tobacco produced near the coast is of a low grade, but in the interior valleys the tobacco is very superior and ranks with Cuban where the seed, care, and curing have been similar. The hills near Cayey are dotted with immense sheds for curing tobacco. For- merly tobacco was grown in the valley only; later it was discovered that the hills produced a better grade than the valleys. The plants are set in August or September, and the crop is harvested in April and May. At the proper time the stem of the tobacco plant is cut on the two-leaf plan and hung in the sheds for about fifty days. It is then taken down, stem removed, and packed in a warehouse. The average production is 400 to 600 pounds per acre. Afterwards it is assorted, which gives the country people employment. FINANCIAL CONDITION OF THE COFFEE PLANTERS. In case of the abandonment of this large percentage of coffee plantations these lands will become nonproductive, except the slight value in grasses and brush. This will reduce the export products of the island, as coffee constituted about seven-tenths of the exports of the island, during 1895, 1896, and 1897. In 1896 itamounted to 58,780,000 pounds and brought $8,505,665. Abandonment would mean death to many laborers, who could find no other immediate employment. If not to be abandoned, vigoroussteps should be taken to restore old plantations and setnewones. Can this be done profitably? Planters claim that itnow costs 9 cents a Can this be done profitably? Planters claim that itnow costs 9 cents a pound on an averageto make and marketa crop of coffee; that if former methods are to be continued and Porto Rican coffee is to be sold in the United States in competition with the Rio, it is better to abandon coffee planting in Porto Rico. But it is not necessary to continue former methods. The average product per acre should be increased three or fourfold, which would reduce the cost of production below 5 cents per pound. The best coffee in the world should be pro- duced. Such coffees ought to average 14 to 20 cents per pound. This result could be accomplished at a nominal expense by the United States 19 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. Department of Agriculture through an experiment Station, experi- menting in, and disseminating knowledge of, coffee production. From this standpoint coffee production in Porto Rico has a hopeful future. THE TOBACCO INDUSTRY. RICE. In former years Porto Rico produced considerable rice, and the only reason why it might not again is that there is very little level land easily flooded except near the coast, and this can be more prof- itably devoted to sugar cane. On the mountain summits, where there is a surplus of rain during the rainy season, small fields are raised without flooding. Here, however, the mole cricket (Gryllotalpa) attacks it and does considerable damage. It is probable that Porto Rico will continue to import the rice required by her people. The advance in rice at this time came inopportunely for Porto Rico, but it has increased the consumption of flour and corn meal. The annual importation of rice into Porto Rico will be seen from the following statistics: Imports of rice. Pounds. statistics: Imports of rice. Pounds. 1894 cutee ¢ Ge eemnmacaesen ce pee een neRie cee! de eek EE tel eeees 72, 674, 540 18065 02 sie we aueat Soeeinteci pina Sh ese VERE eee Vee eee ceemios oodeeie 74, 145, 046 ROG sir saee de iseecee neo esa sans gaeai eee ceecee Riis ctme Jen 102708, 240 NOT oc caizced sercet.ae a selonguenes itiichoncheieuicreicies dais pepsin Ny OO, Lee Imports of rice. ce pee een neRie cee! THE TOBACCO INDUSTRY. <A majority of the persons employed in the assorting were women, girls, and boys. Labor in the tobacco districts is noticeably better paid than in the coffee districts. As soon as it has been ascertained that Porto Rican tobacco and cigars can be profitably sold in the United States, there will be a large increase in the annual output, as coffee planters will engage in the industry to provide an income while they are restoring their coffee plantations. The following Sable shows the quantity and value of the tobacco exports for the eleven years preceding American occupation: Tobacco exports. Sah sae ees ase eed occupation: Tobacco exports. Pounds. WSS ooo ce noe Sa ee ees Sah sae ees ase eed eek ews abes pone eses 7, 633, 000 WS88 o crscc doo cca ae nels ansee casks soem ceceaed dee 2 eee cemeaers 3, 347, 000 1889). 3 one desa- Gia tecouoseeciseeees ua modded wed See le cee Wf, FO8, 000 1800 cei) Gaed SoS L SoS Maen Jgvaeindnies ocinitoe aeeases Gomi tinne Bs 984,000 1891 Steteer sea weer saa encaher Adteeis Liajedamnads oeie = ae sine DCO Ly OOO 1802 sc ce hk wooe: ocean Aue seaman Ree eater eaees aeeeee--.----- 4,207, 000 1898) nen kee eeeenies = Saat aoc aeescine eee ee --- 4,208, 000 DS a ASI a ste i ye gay pe ete nr fin Spa ley ato ues aytialy® Bae aah als 3, 370, 000 FTO By aes ies eto SSeat A oh rcienns eke ahaa nee tea alae Sota AS de Si eae ee eee, (000,000 1896 wscctie = xis ge = calste'sis oxic oeen Auch asices aware eins Go See aoe caine: (eye, 000 189% ssose cence reer ge a4 ogo seg ylege seat ekepieeee ei wee eae ei te 0,208,953 20 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. This table shows that the tobacco industry had been on the decline for nearly a decade. In 1897 it showed sudden increase, due doubt- less to the condition of the sugar and coffee industries. CORN. The climate, rainfall, and soil of Porto Ricv indicate, upon the whole, a good country for corn. Farmers report a crop of 60 bushels upon virgin land of good quality and 20 to 25 bushels on old land. This is equal to the average crop in the corn belt of the United States. A further evidence of the corn-producing capacity of Porto Rico is shown by the exports. In 1897 Porto Rico exported to Cuba 4,246,776 pounds of corn and 7,946 pounds of corn meal, and to Spain 30,133 pounds of corn. No corn was imported that year, thus showing that Porto Rico produced more than sufficient for home consumption. It should also be taken into account that the interior of the island could furnish no portion of this surplus, because, with the exception of one military road, there were no roads in the interior on which it could be transported to the coast. With a better knowledge of the corn plant and the conditions requisite for its best yield we may expect large corn products in Porto Rico. MINOR CROPS. Corn, rice, beans, potatoes, bananas, peanuts, citrus fruits, pine- apples, cocoanuts, vegetables, etc., are classed as minor crops. According to records, 93,511 acres are assessed as devoted to these products, the chief of which is corn. VEGETABLES. A visit to the markets does not give a favorable impression of the capacity of Porto Rico to produce vegetables. The cabbages for sale are very inferior in size and quality. Many are not more than 4 inches in diameter, and not solid heads. The tomatoes usually range from 1 to 2 inches in diameter. Root crops are in proportion. Watermelons offered are inferior in size and quality; the lettuce is small and bitter. An inspection of the gardens or fields where these vegetables are produced affords an entirely different view of the gar- den problem. Vegetables are planted on land long in use, imperfectly tiled, without fertilizer, and turned over for further care to Provi- dence. The seeds in many instances are sown broadeast. Gardeners from south Florida, located near San Juan, showed good results. Their vegetables were not only of good size, but of excellent quality. One gardener near Rio Piedras. delivered in San Juan several loads of watermelons each weighing 50 pounds or over. His produce brought high prices. The following vegetables can be successfully raised: Vegetables which can be raised in Porto Rico. Vegetables which can be raised in Porto Rico. Beans. Kenep. Peppers (green). Beets. Lettuce. Peas. Cabbage. Lentils. Pumpkins. Cassava. Lechosa (species of musk- Radishes. Carrots. melon). Sesame. Celery. Muskmelons. Squash. Corn (sweet). Okra. String beans. Corn (field). Onions. Tallote. Cucumbers. Potatoes (sweet). Turnips. Eggplant. Potatoes (Irish). Tomatoes. Garlic. Peanuts. Watermelon. Gourds. Peppers (tabasco). Yams. Beans. Beets. Cabbage. Cassava. Carrots. Celery. Corn (sweet). Corn (field). Cucumbers. Eggplant. Garlic. Gourds. Kenep. Lettuce. Lettuce. Lentils. Pumpkins. Radishes. Lentils. Lechosa Lechosa (species of musk- Radishes. melon). Sesame. Sesame. Squash. melon). uskmelons Muskmelons. Okra. Squash. String be Okra. Onions. String beans. Tallote. Tallote. Turnips. Onions. Potatoes Turnips. Tomatoes. Potatoes (sweet). Potatoes (Irish). Tomatoes. Watermelon Potatoes (Irish). Peanuts. Watermelon. Yams. Peanuts. Peppers ( Yams. Peppers (tabasco). BEANS, SWEET POTATOES, AND BANANAS. Beans are planted on farms for home consumption. The quality and yield are good. Iwas unable to obtain any data showing the amounts produced annually, but from the crops observed the supply appeared entirely inadequate to the necessities of the people. The nutritious sweet potato flourishes in all portions of the island, but, like beans, the supply produced for home consumption is less than it 21 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. should be. Itis amuch more healthful article of food than unripe bananas, and if supplied in abundance to the peasants of the coffee district there would be greater health and vigor. Bananas are ordinarily produced in great abundance, because they should be. Itis amuch more healthful article of food than unripe bananas, and if supplied in abundance to the peasants of the coffee district there would be greater health and vigor. Bananas are ordinarily produced in great abundance, because they Bananas are ordinarily produced in great abundance, because they furnish the shade of the young coffee plants. They are also grown for food without reference to coffee shade. They constitute the principal vegetable food of the laboring classes. The banana pos- sesses some advantages. The product per acre on rich land is enor- mous, yielding in some cases as much as 60,000 pounds of fruit. In well-cultivated orchards it is possible to have fruit most of the year, though it is more abundant atcertain seasons. The fruit should be cut before itis ripe and hung in a dark place till mellow. Some place it in a dirt house till mature; others simply cover it with dirt. In most tropical countries the ripening is neglected and the fruit is eaten in an immature state. It is usually cooked. A dwarf variety, known as Guineo fruit, about 4 inches long, is greatly prized. The skin is very thin, meat yellow and highly flavored. I observed a good many stems that had failed to mature more than half a bunch of bananas, probably the result of a deficiency in fertility or moisture, or both. A little knowledge and care would easily remedy this defect. When a line of fruit steamers is established to Porto Rico bananas will constitute an important part of the exports. MANGO. The mango is a luscious semiacid fruit, greatly prized in the Trop- ics. Its reliability as an annual product is one of its chief merits. It is valuable simply for home consumption. ORANGES, LIMES, AND LEMONS. Porto Rico is well adapted to the growth of the orange. Here the tree is hardy, healthy, a rapid grower, a marvelous producer, and 22 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. retains its vigor for many years. It develops its fruit perfectly, even in the wild state in the forest. In all parts of the island it is possible to find many excellent locations for orange orchards. The planter ean select such a conformation of hills as to protect his orchard from prevailing winds. In soils he can take his choice between the rich, sandy loam, the fertile gravel (6 feet deep), and the porous clay loams. Lands for such purposes can be purchased at from $5 to $20 per acre nearagood port. At Mayaguez I visited the fruit farm of F. G. Molina on the bay and photographed an orange tree 3 years old from setting. It was 6 inches in diameter at the base and about 18 feet high with branches 5 or 6 feet long, full of fruit. I also photographed a tree 5 years old, which bore 5,000 oranges last crop, and another 7 years old, which bore 7,000 oranges. Mr. Molina sold his oranges at $3 per thou- sand f. 0. b. ship for New York. What has been said of oranges applies equally to limes and lemons, What has been said of oranges applies equally to limes and lemons, except that lemons are produced solely by cultivation, and very little attention has been paid to them. . SHADDOCK, GRAPE FRUIT, OR POMELO. The shaddock is in habit like the orange, grows 30 to 40 feet high, ornamental, globose or pyriform in shape, pale yellow in color, and produces a large subacid fruit, rather coarse, with thick rind. Speci- mens of this fruit sometimes weigh 10 pounds each and in rare cases 15. Itis a native of the Polynesian Islands, and was early brought to the West Indies. It is hardy in Porto Rico. Grape fruit, the best variety of the shaddock family, attains perfec- Grape fruit, the best variety of the shaddock family, attains perfec- tionin Porto Rico. Conditions of growth are like those of the orange, except that it is more sensitive to frost, which makes it an unsafe product in the United States, only in the extreme south of Florida. This large, luscious fruit would be greatly appreciated in all portions of the United States if it could be placed on the market at a moderate price. With the low freight rates to New York, Porto Rico should supply this demand. MANGO. ; PINEAPPLES. Pineapples are produced in all parts of Porto Rico with great suc- cess at a trifling cost of labor. Eight to ten thousand plants are set on an acre, and after the sets are well established no further care is taken. Asa rule, after the plants are set they are neither hoed nor shaded, asin Florida. The ripe fruit weighs from 2 to 16 pounds per plant, and may be averaged at 6 pounds. At 3 cents apiece the pine- apple crop is very profitable. It is one of the most luscious of fruits when ripe, and if sold at reasonable rates the United States will prove an unlimited market for it. In time pineapples should become as abundant and as cheap as apples in our market. At Fajardo, San Juan, and Mayaguez small farmers are prepared to make large con- tracts for delivery to anyone establishing a cannery. GUAVA. The variety cultivated for the fruit is a small tree from 9 to 15 feet high, with angular branches and an abundance of pointed, elliptical leaves. The fruit is about 1 inch in diameter, apple or pear-shaped, yellow, very fragrant, and filled with a yellowish or reddish pulp, sub- acidin flavor. It isan abundant bearer, and furnishes one of the best fruits known for the manufacture of jelly. AGRICULTURAL RESOURCES, ETC,, OF PORTO RICO. AGRICULTURAL RESOURCES, ETC,, OF PORTO RICO. COCOANUTS. The cocoa palm is one of the most valuable trees produced in any country. It thrives on the sand dunes, valuable for little else, near the seashore. The trees are from 50 to 90 feet high, and 100 to 160 can stand upon an acre. When mature the trees produce an average of 150 nuts each annually. With due allowance the crop of an acre may be estimated at 10,000 nuts annually, worth $150 on shipboard. To gather the nuts, remove the fibrous shuck, and load costs $2.40 per 1,000 nuts, or $24 total cost of crop per acre. The shuck is sold for matting material. Trees begin to bear at 5 years old and continue for about one hundred years. Every partof the treeis valuable. The hard portion of the trunk makes the celebrated porcupine wood; the leaves are used for baskets and thatching; the fibrous husk of the nuts is made into mats; the shell of the nuts is used for drinking cups, frequently beautifully carved; the meat is used for confec- tionery, and the water or liquor is an agreeable and healthful drink. 23 Japanese persimmon. Leconte pear. Walnut. STocK RAISING. Five conditions are requisite to make an excellent stock country: (1) Abundant grasses, of superior quality, and pasturage as nearly continuous as possible. 2) Pure and abundant water. 2) Pure and abundant water. 3) Equable climate. 2) Pure and abundant water. 3) Equable climate. 3) Equable climate. 4) Freedom from ann 3) Equable climate. 4) Freedom from annoying inse 3) Equable climate. 4) Freedom from ann 4) Freedom from annoying insects. 5) Good markets. 4) Freedom from annoying insects. 5) Good markets. 5) Good markets. Porto Rico possesses 5) Good markets. Porto Rico possesses Porto Rico possesses all of these advantages in an eminent degree. As a grass-producing country it is unsurpassed. Its hills are covered with grama grass, corresponding for pasture to the mesquite grass and the buffalo grass of the United States. The cultivated grasses, guinea grass (Panicum maximum) and malojilla, are very nutritious, and produce a large amount of forage per acre. Dr. Don Augustin Stahl states that 50 square meters of these grasses will sustain an ox or a horse, which would be equal to 81 horses or oxen per acre. This could scarcely hold good for the entire year. Twelve animals are probably nearer the average. Authentic cases are given where land set in guinea grass has produced 130 tons of green fodder per acre, equal to 32.5 tons of hay. Cattle and horses in the working season rarely receive any other food than these grasses, cut and fed in a manger under shelter. The creeks and rivers have sufficient current to keep the water pure, and springs are plentiful in the mountains. The weather is always cool, mornings and evenings, when the stock is grazing. Flies and mosquitoes are about the same as in the higher portions of the South- ern States. Cuba and St. Thomas take all surplus stock at good prices. The horses are the exact type of those descended from Span- ish stock in the prairie sections of Louisiana, very small but full of energy. The foundation was evidently of the best Arabian stock, but now greatly deteriorated. The cattle are also of Spanish stock, but have been crossed more or The cattle are also of Spanish stock, but have been crossed more or less with Senegambian males, adding something to the size and fat- tening properties, but reducing their value for milk. STocK RAISING. Some sheep are kept, but the industry has not been a great success. Some sheep are kept, but the industry has not been a great success. There is too much rain in the highlands. Goats are common in all parts of the island, and with a better stock might be made the basis of an industry of much profit. Comparatively few swine are raised. Possibly the absence of fences Comparatively few swine are raised. Possibly the absence of fences is the cause. With small fields of cassava, sweet potatoes, peanuts, and sorghum it ought to be possible to raise hogs at a nominal cost, and the market is excellent. The small amount of poultry kept is a perpetual surprise. One may The small amount of poultry kept is a perpetual surprise. One may travel for miles in the country without finding a good flock of chickens. Most of those raised are inferior in size and quality. The eggs are small and lack in flavor, but the price is large enough—36 cents per dozen. Chickens sell at 40 to 75 cents each. Investigations show that there is little reason why chickens should not be produced in great abundance. FaRM WAGES. PEACHES, PEARS, FIGS, AND GRAPES. These fruits have not yet been produced to any extent. Sample trees of the peach, of the Leconte and Keiffer pear, planted near Baya- mon, show excellent growth and vigor. There is apparently no rea- son why they should not become staple products. Figs are yet on trial. They do fairly well in some parts of the island. It is a ques- tion whether they have been given a fair trial. Few grapes are produced. Under Spanish rule grape enltare was Few grapes are produced. Under Spanish rule grape enltare was discouraged, because it might interfere with the wine industry of Spain. Wine is commonly used as a table drink in Porto Rico. In 1897 the import of common wine was 4,314,473 ae (a liter is about a quart), at a cost of about 52 cents per liter. . George Bird, of Fajardo, stated to me that his father, on his ee ‘from Spain some years since, brought from Malaga a few of the best varieties of grapes produced there, and on trial they did better in Porto Rico than in Spain. By pruning he had produced in some years four crops. List of fruits and nuts produced in Porto Rico. Alligator pear. Grosella. Orange. Almond. Grape fruit. Pajuil. Bread fruit. Grapes. Pineapple. Banana. Guava. Plantain. Cactus pear. Ivory nut. Raspberry. Cocoanut. Lemon. Rose apple. Cherry (tropical). Lime. Russet fruit. Cocoa plum. Mamee sapota. Shaddock. Chocolate bean. Mango. Soursap. Cayoll (palin fruit). Mangosteen. Strawberry (wild). Coffee. Mulberry. Tamarind fruit. Custard apple. Nutmeg (spice). Fruits and nuts that may safely be introduced. Blackberry. Strawberry. Japanese persimmon. Japanese plum. Keitfer pear. Leconte pear. Olives. Pecan nut. Walnut. Alligator pear. Almond. Bread fruit. Banana. Cactus pear. Cocoanut. Cherry (tropical). Cocoa plum. Chocolate bean. Cayoll (palin fruit). Coffee. Custard apple. Fruit Blackberry. Japanese plum. Olives. Grosella. Grape frui Grape fruit. Grapes. Pineapple. Plantain. Grapes. Guava. Plantain. Raspberry. Guava. Ivory nu Raspberry. Rose apple. Ivory nut. Lemon. Rose apple. Russet fruit. Lemon. Lime. Russet fruit. Shaddock. Lime. Mamee Shaddock. Soursap. Mamee sapota. Mango. Soursap. Strawberr Mango. Mangoste Mangosteen. Mulberry. Tamarind fruit. Mulberry. Nutmeg (s Nutmeg (spice). Fruits and nuts that may safely be introduced. Japanese persimmon. Leconte pear. Walnut. Strawberry. Keitfer pear. Keitfer pear. Pecan nut. Pecan nut. 24 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. STocK RAISING. FaRM WAGES. {3} The oppressive consumers’ taxes on food and articles of neces- sity have been abolished. (3) The change of the money standard, which took effect August 1, {3} The oppressive consumers’ taxes on food and articles of neces- sity have been abolished. (3) The change of the money standard, which took effect August 1, (3) The change of the money standard, which took effect August 1, 1900, has benefited labor. In many cases laborers are now receiving in gold what they formerly received in silver. (4) Thousands of laborers in the coffee districts have been given (3) The change of the money standard, which took effect August 1, 1900, has benefited labor. In many cases laborers are now receiving in gold what they formerly received in silver. (4) Thousands of laborers in the coffee districts have been given (4) Thousands of laborers in the coffee districts have been given employment on the public roads now in process of construction by the insular government. (5) The Porto Rican tariff on flour, corn, and rice was formerly very (5) The Porto Rican tariff on flour, corn, and rice was formerly very high; now they are admitted free. (6) Indirectly labor has been benefited by the removalof the export (5) The Porto Rican tariff on flour, corn, and rice was formerly very high; now they are admitted free. (6) Indirectly labor has been benefited by the removalof the export (6) Indirectly labor has been benefited by the removalof the export taxes on sugar, coffee, and tobacco, and by the repeal of the law levy- ing royal dues and taxes on importing agricultural implements. (6) Indirectly labor has been benefited by the removalof the export taxes on sugar, coffee, and tobacco, and by the repeal of the law levy- ing royal dues and taxes on importing agricultural implements. FaRM WAGES. The usual hours of work in the field are from 6 to 6. A majority of the field hands commence work in the morning without having eaten anything. A few take early coffee. At 11 o’clock half an hour is given for breakfast. Breakfast consists of rice and beans, bread and 25 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. cheese, or sweet potatoes and fish. Where the plantation boards the hands, as sometimes occurs in the sugar-harvest season, board con- sists in furnishing daily one-half pound of rice and one-fourth pound of beans, or 3 pounds of sweet potatoes and half a pound of fish (dried), or 1 pound of bread and one-fourth of a pound of cheese. The laborers on coffee estates rarely eat meat, except on Sunday. Many laborers eat nothing till the close of the day. The customary wages were 30 cents (gold) per day prior to the The customary wages were 30 cents (gold) per day prior to the changes in currency, but, instead of money, in most cases the pay- ment was made by giving an order on the store, which reduced the value at least 33 per cent. In coffee-picking time the-women were able to earn 15 to 25 cents per day. Coffee planters are generally lib- eral in allowing their laborers to use the bananas on the farm, and in giving them theuse of small tracts for vegetables if they will use them. The laborers complained that they had no time to make a garden. Many laborers were unemployed in the coffee districts because the plantations were uncultivated and had remained so since the hurri- cane of last year. In the tobacco and sugar districts labor was in a much better condition. Tobacco culture gives more employment to women and children. On the sugar plantations higher wages have usually been paid during sugar making, which continues for four or five months. This better condition is shown more by the personal appearance of the laborers than by improvement in their houses and home surroundings. The constant efforts of the insular government since the island came under American control have heen directed to the relief of the labor situation. 1) All personal taxes have been removed from laborers. 1) All personal taxes have been removed from laborers. {3} The oppressive consumers’ taxes on food and articles o 1) All personal taxes have been removed from laborers. DEPRESSION IN AGRICULTURE. Many persons are under the impression that the agricultural indus- tries of Porto Rico were prosperous under Spain, and that their decline is due to bad management upon our part and to the hurricane of August 8,1899. The American flag was not formally raised over the island till October, 1898. Almostimmediately evidence was taken set- ting forth the condition of agriculture. This evidence conclusively establishes the fact that, while the income of the coffee farms was con- siderable, the profits did not go into the hands of the tillers of the soil, but went to enrich the capitalist, who in many cases returned to Spain in afew years toenjoy his wealth. Blocks of stately buildings in Bar- celona and other Spanish cities are shown the traveler which were built with the profits obtained from the products of Porto Rican farms. The conditions upon which the coffee planters secured loans to make The conditions upon which the coffee planters secured loans to make their crops is proof that the coffee industry had not been prospering as it should. But there is plenty of direct proof taken prior to the hurricane, and all to the same effect. Sefior Luis Cenal, of Fajardo, stated on November 6, 1898: Sefior Luis Cenal, of Fajardo, stated on November 6, 1898: Sefior Luis Cenal, of Fajardo, stated on November 6, 1898: The abandoned cane estates are run to pasture, but as this is not making proper use of the land it can be calculated that 75 per cent of the district is, properly speaking, unproductive. November 7, 1898, Mayor Eustaquio Torres, of Guyanilla stated: November 7, 1898, Mayor Eustaquio Torres, of Guyanilla stated: Agriculture, which has been languishing and has been impoverished, is over- Agriculture, which has been languishing and has been impoverished, is over- whelmed by enormous tributes, wanting facilities afforded by an agricultural bank, and fighting an unequal fight with the merchants, owing to the fact that the difficulties of the money system clozes to it foreign and national markets. From this cause originates the general depression of the country, especially of the laboring class. This class does not earn enough to buy food, and its ranks are being swelled enormously by small proprietoys, who, wanting in means to till their small farms, are obliged to sell them. DEPRESSION IN AGRICULTURE. That is the reason why public wealth isconcentrated in the hands of a few capitalists in each town, and also why so many uncultivated lands are seen, their owners, owing to their great extent, not being able to give them attention. The above evidence was taken before Dr. H. K. Carroll, commis- sioner; and evidence was placed before me proving that the sugar inter- ests had scarcely paid expenses for the ten years prior to 1899. AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. CHARACTER OF THE LABORING CLASSES. The laboring classes have surprisingly active minds, considering the conditions under which they have lived. They are mechanically inclined, kindly disposed, and respond readily to fair treatment. With opportunities and reasonable encouragement they will rapidly improve. Many American employers of Porto Rican laborers gave a very encouraging report of their industry and faithfulness. It was gratifying to note the desire to be self-supporting. The Women’s Aid Society of Porto Rico gives partial employment to about 400 poor women, many of whom are from the country. Garments are given to the very poor. Only 8 per cent of the women employed, however, accept alms, preferring to pay for their garments by installments at the rate of 5 cents per week. Their average earnings are 15 cents per week. 26 CAUSES OF DEPRESSION IN AGRICULTURE. It is pertinent to inquire the causes of the decline of agriculture under Spanish rule. They may be summarized as follows: (1) The system of credit or ‘advances ”—explained under head of (1) The system of credit or ‘advances ”—explained under head of ‘* coffee.” (2) The manufactures necessary to obtain full value of farm crops (1) The system of credit or ‘advances ”—explained under head of ‘* coffee.” (2) The manufactures necessary to obtain full value of farm crops (2) The manufactures necessary to obtain full value of farm crops discouraged. 3) Farms taxed for benefit of cities. (2) The manufactures necessary to obtain full value of farm crops discouraged. 3) Farms taxed for benefit of cities. 3) Farms taxed for benefit of cities. 4) Tendency to build up large farms. 4) Tendency to build up large farms. 5) The consumption tax on food. 5) The consumption tax on food. 6) Laws favoring the merchant cl 6) Laws favoring the merchant class. 7) No country roads; cost of transpor 7) No country roads; cost of transportation excessive. In som cases it costs $1.25 to transport 100 pounds 5 miles. (8) Economic conditions bad. Little agricultural machinery an (8) Economic conditions bad. Little agricultural machinery and few implements used on account of excessive import duties; labor depressed and unable to do good work. (9) Landlordism. Farms fell into the hands of town and city peo- (8) Economic conditions bad. Little agricultural machinery and few implements used on account of excessive import duties; labor depressed and unable to do good work. (9) Landlordism. Farms fell into the hands of town and city peo- (9) Landlordism. Farms fell into the hands of town and city peo- ple who knew little of farming. Many farmers were so involved that they could not control the management of their farms. (9) Landlordism. Farms fell into the hands of town and city peo- ple who knew little of farming. Many farmers were so involved that they could not control the management of their farms. 27 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. (10) Too narrow a range of agricultural production. The agricul- tural products imported amounted to about five-elevenths of the total imports. In 1895 the agricultural products imported amounted to $7,171,352 (gold), and the total nonagricultural to $9,644,101, or 42.60 per cent of the former to 57.40 per cent of the latter. EFFECTS OF SUCH CONDITIONS. The consumption tax was a tax levied by towns and cities upon the necessities of life, such as maize, rice, lard, sugar, flour, silk, char- coal (used for cooking), petroleum, etc. It was not placed on dry goods, jewelry, and similar articles. This tax was very heavy, as the following will illustrate: Consumption Consumption tax (silver). Muscovado sugar _.-----.-----.------ per cwt.. $2.00 OUI 2 css caine eee mamacccieclec S255 --do_.. 1.25 Rice _- Ree ne meee ceases meee cacsevuens a Okie “Ree Muscovado sugar _.-----.-----.------ per cwt.. $2.00 OUI 2 css caine eee mamacccieclec S255 --do_.. 1.25 Rice _- Ree ne meee ceases meee cacsevuens a Okie “Ree This tax was far-reaching in its effect. It not only nearly doubled the cost of living, but it prevented the establishment of small farms in the vicinity of cities, because the farmer must provide for the con- sumption tax before he could sell. This was practically prohibitory. The effect of landlordism is to obtain the largest present revenue The effect of landlordism is to obtain the largest present revenue from the land possible, regardless of the future. To this may be charged the complete destruction of all the timber within merchant- able distance of any good road or any market. Present fertility of soil is taxed to the utmost at the expense of future production. Improvements are cheap and rude. Under such conditions it was only a question of time when general Under such conditions it was only a question of time when general bankruptcy must be forced on the producers. It was certain to fol- low any general failure of crops, any great decline in prices or radical change in the character of the markets, or any monetary crisis which should destroy credit. Unfortunately for Porto Rico, all these things occurred at once. Change of flag necessitated new markets for a majority of her staples, the hurricane destroyed most of the crop in 1899, and credit to planters was totally withdrawn. The hurricane was a crowning calamity. It swept away not only crops, but the improvements, and devastated the island in all portions to an amount searcely to be estimated. While it visited the coffee plantations with special violence, it left its wreckage marks on every sugar plantation in the island. CAUSES OF DEPRESSION IN AGRICULTURE. The total exports of native products in 1895 were $15,190,856, of which amount $14,573,366 were agricultural. Tersely stated, they imported 50 cents’ worth of rice, flour, fish, etc., for subsistence to enable them to pro- duce one dollar’s worth of sugar, coffee, and tobacco for export. If there was any failure in the dollar crop, money must be borrowed to pay the 50 cents. (11) No agricultural schools nor journals to disseminate knowledge (11) No agricultural schools nor journals to disseminate knowledge upon agricultural subjects, without which no nation has taken front rank in agriculture. INCREASE OF SMALL FARMS. Naturally when a large number of small farms fell into the hands of the capitalist he consolidated them and placed them under one administration. If it was not convenient to work them, they were grazed, reducing the demand for labor. : The number of small farms should be greatly increased and their The number of small farms should be greatly increased and their products diversified to the limit of profit. The fruit and nut crop in ten years should exceed the combined annual export of all farm crops at this date. Winter vegetables, poultry, and dairy products should form large items in the export columns. SCIENTIFIC INQUIRY AND EXPERIMENTS. These improvements require scientific inquiry and experiments along practical lines. These experiments, to be of assured value, must be made upon Porto Rican soil. The tobacco industry comes nearer being able to sustain itself and provide for future improvement than the other industries. This, however, applies more to the large plant- ers and manufacturers than to the small producers, who at least need instruction. Few crops require more technical knowledge and skill in production and manufacture than tobacco. The production of the best varieties is a fine art and is complicated by fashion, which differs in different markets. Whatis a very high grade in one market is a low grade in another, and small producers can not investigate meth- ods as adapted to markets. How CAN DEPRESSION IN AGRICULTURE BE RELIEVED? J have stated the condition of agriculture and the cause of its decline somewhat fully in order to point out clearly the relief. It is evident that the only immediate relief that can be afforded the agricultural interests in Porto Rico must be provided through the established lines 28 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. of industry—sugar, tobacco, and coffee. While immediate steps should be taken greatly to increase the number of profitable agricul- tural industries, such changes can only be effected gradually and should be regarded rather as part of a wise economic policy for the future than as a measure to afford the immediate relief required. All thefarm labor of the island was formerly givenemployment. The insuf- ficiency of employment now arises chiefly from the prostration of the coffee farms. While the sugar and tobacco industries will provide about the normal amount of labor the present year, they can be greatly strengthened financially. Better drainage, the use of renovating crops, and a judicious system of crop rotation will enormously increase the product per acre, and more improved machinery will add largely to the sugar product. HOME PRODUCTION OF FOOD. A further means of improving agricultural conditions is for all the sugar, coffee, and tobacco plantations to produce a variety of food crops sufficient for their employees, and for each employee who is the head of a family to produce the food for his household. This should be a cardinal principle adopted by all planters. For an island as fertile as Porto Rico to import annually 50 cents’ worth of alimentary products in order to export one dollar’s worth of sugar, coffee, and tobacco is an unsafe policy and should be discontinued at once. INDUSTRIAL VILLAGES. The early establishment of a number of minor industries closely related to agriculture is of vital importance to future prosperity. The 29 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. object of such industries is to give profitable employment to the wives and children of farm laborers, so that the earning ability of the home may be doubled and in some cases quadrupled. Under such conditions, if the head of the family fails for any cause to earn his wage, the home goes on; the earning capacity is lessened, but the home is not destroyed. This has its moral as well as economic bearing. Many philanthropic Porto Ricans suggested that the farm laborers on the coffee and tobacco plantations scattered upon the mountains, without roads, society, or schools, children nude and semiwild, could never derive the full advantages of free education and be influenced by the elevation of society until they were gathered into small villages and became amenable to society. Ina republic this can not be done by force, but once establish small industrial villages in the country and the small, scattered mountain population will be attracted to the vil- lage by its superior earning capacity and its advantages for schools, society, and better living. This will be no detriment to the farms, because the village laborers will be within reach of every farm. The industrial village where all are workers, is fundamental in Japanese civilization. The manufacture of hats, straw goods, and matting, the production of raw silk, and the canning of tropical fruits are examples of the employments in question. BETTER HOMES. It is of vital importance to the future prosperity of Porto Rico that there should be a great improvement in the homes of the farm laborers, better houses, and more comforts. To this end a larger and more comfortable house must be devised that will be within the means of the laborer to build. This can be done with a slight addition of the labor expended upon it. In this connection the necessity of encour- aging the planting of trees for building purposes is apparent. PRESENT AGRICULTURAL RESOURCES AND POSSIBILITIES OF PORTO RIco. PRESENT AGRICULTURAL RESOURCES AND POSSIBILITIES OF PORTO RIco. PRESENT AGRICULTURAL RESOURCES AND POSSIBILITIES OF PORTO RIco. The exports of Porto Rico for the fiscal year ending June 30, 1901, may be estimated as follows: Estimated exports of Porto Rico for 1901. Estimated exports of Porto Rico for 1901. Articles. Pounds. Value. SUSOl's scccssatescacieecasscuseseeawauealsads miwedioaceed sume csc ewescu ewer ecteeus Molasses. -... porter wie gecia area a Tobacco and manufactures TONGS, cmt sige BN ws anc ot SRE A a cv i od vi cnn i ho liso 10, 605, 762 Nore: The total exports for 1897 were $11,011,524. POSSIBILITIES OF AGRICULTURE IN PoRTO RICO. As to the possibilities of agriculture in Porto Rico I submit the following statement: The United States imported in 1899, in addition to the imports of The United States imported in 1899, in addition to the imports of sugar, coffee, and tobacco, the following: Fibers (vegetable), manufactures of, including mattings, etc___..--.-- $45, 457, 687 Nits anid fads, 25 ok oe ae ates Oy pb tinin a Gr eenen bey clahiee sede 18, 317, 201 Silk; unmanufactured....2o tec. wecesdesveeemne vebecwectann 4a Sees 32, 479, 627 Total dono Gekbey asSssae scoaeamede. eee tecetes en deen aay ee 96, 254, 515 The industries of Porto Rico, properly developed, could produce a large portion of the foregoing without interfering with her present exports. In addition, it could produce camphor, india rubber, spices, and other articles of which we import large quantities. Thrifty speci- mens of india rubber plants and of camphor plants were seen. In case of such an expansion in the production of tropical products, Porto Rico would become a large consumer of the food products exported by the United States, which would be greatly to our trade advantage over the present plan of securing these articles. No por- tion of Porto Rico is over 20 miles from the coast, and with good roads all products could reach an ocean port in a few hours and by the conveyances of the farm. The transportation from the ports to New York is low and with increased freights will be much reduced. A few thousand dollars judiciously expended annually in the devel- opment of the agricultural resources of Porto Rico will result greatly to our benefit, because we shall then be buying our tropical imports with our surplus products. The appended list shows some articles, and their values, of import The appended list shows some articles, and their values, of import into the United States in 1899, all, or a portion of which, could be profitably produced in Porto Rico: ticles imported into the United States which might be produced in Porto Rico. Articles imported into the United States which might be produced in Porto Rico. Cocoa and the manufactures of ._.._-.-. --.-.--.------.------------ $5,360, 116 Coffee... ---- acicdorgeateeoelgeny ceecgey 000,070, 470 Fibers, vegetable, and manufactures of ........--..-------.---------- 45, 457, 687 Eruits.and nuts .<:22 <::cenc: cece ceeresceusels ces sek suere s sommes 18, 317, 201 Hats and bonnets, materials for ... _.....-. Articles. Nore: The total exports for 1897 were $11,011,524. The sugar crop of 1900 will be less than normal, but, by reason of the better price, will bring considerably more money. The tobacco crop is placed at normal, and the coffee crop at 50 per cent of normal. It will be seen that the estimated exports of the fiscal year ending June 1, 1901, are only $405,762 below those of 1897, the last year of Spanish possession, regardless of the destruction of the hurricane, which reduced the coffee export about $5,500,000. The farmers have given 30 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. more attention this year to the production of food crops. It may, therefore, be fairly claimed that, as a whole, the net income of Porto Rico from farm crops will be about the same as usual; but this will not relieve the distress, because it will not be as widely distributed as formerly, the deficiency occurring in the coffee districts. POSSIBILITIES OF AGRICULTURE IN PoRTO RICO. -------------- veces 25426, 726 India rubber and gutta- percha and manufactures of__.-.--.-.-.----- 82, 370, 098: Molasses _... wusmusuee es eewereckeees: eee s Sos 24: 3 ees oe8 789, 576 idle mrmnennd tNCLEROU cx vae coatene: aioceoconcasss asus tos ence ae 32, 479, 627 Spices IF a OE ER Re rete ea ee re eC 2, 782, 301 SUGAL ci. 2aebisceee adel aon shee tee eRe nes “ eeeese go e2 94, 964, 120 Tea. __ Keak dene DEMS Cai eel be ein cadeza 9, 675, 081 Tobacco and manufactures of _.._...----.----+-----.--00+--2e- nee -ee 11, 848, 357 Vanilla bean... 22s acsseceecueesed 22k sien Be se ee 1, 235, 412 Totals seasatec ease ae ee cca) ence serosa ace seas 812, 976, 771 AN AGRICULTURAL EXPERIMENT STATION. The most potent factor in accomplishing the improvement of agri- culture would bea properly equipped agricultural experiment station, 31 AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. with a wider range of investigation and application than is usually given to such institutions. First. Such a station should give immediate attention to the pro- with a wider range of investigation and application than is usually given to such institutions. First. Such a station should give immediate attention to the pro- ider range of investigation and application than is usually such institutions. Such a station should give immediate attention to the pro- First. Such a station should give immediate attention to the pro- duction of larger and better crops of coffee, sugar, and tobacco, at a less cost than at present. First. Such a station should give immediate attention to the pro- duction of larger and better crops of coffee, sugar, and tobacco, at a less cost than at present. Second. It should encourage the production of food products to Second. It should encourage the production of food products to the extent of home consumption. Third. It should promote the establishment of small farms for fruit Third. It should promote the establishment of small farms for frui and vegetables. Fourth. As soon as practicable it should prosecute investigation Third. It should promote the establishment of small farms for fruit and vegetables. Fourth. As soon as practicable it should prosecute investigations Fourth. As soon as practicable it should prosecute investigations that will lead to the improvement of farm stock with special reference to the requirements of the farms for work animals and of the markets for beef and dairy products. There is no good reason why Porto Rico should import 743,560 pounds of cheese annually at a cost of 16.4 cents per pound when there is an abundant supply of sweet grasses and rich milk on the island. Fifth. Thestation should diligently prosecute investigations in for- Fifth. Thestation should diligently prosecute investigations in for- estry to reestablish the woodlands. Sixth. It should be especially empowered and charged to introduce Sixth. It should be especially empowered and charged to introduce minor industries for the betterment of the families of farm laborers. Seventh. An important part of the station work should be the Seventh. AN AGRICULTURAL EXPERIMENT STATION. An important part of the station work should be the introduction of new varieties of seeds, plants, trees, and animals which are adapted to the climate and conditions found in Porto Rico; also to see that the best seeds and fertilizers are sold to farmers. For any country to fail to keep a watchful eye on the progress of other countries and take advantage of their natural resources and the improvements they have made in the products of the soil is to fall behind in the race of life and publicly acknowledge a lack of enter- prise. OBJECT LESSONS. It will be necessary to place the work of the station mainly in the form of object lessons and on a sufficient scale to show economic results. This will require more land than would be sufficient to establish theories or principles in agriculture. To carry out the plan of the station the earnest cooperation of the To carry out the plan of the station the earnest cooperation of the farmers should be secured by local associations and otherwise. The station should issue bulletins at regular periods, and the officers of the station should meet the local association for discussion of agricul- tural topics at least once a year. At the same time schools for women could be held, giving instruction in various home industries suited to their condition. AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. AGRICULTURAL RESOURCES, ETC., OF PORTO RICO. LOCATION OF THE STATION. There are a number of reasons why the experiment station should be located near San Juan. First. It is the opinion of all who have investigated the subject that First. It is the opinion of all who have investigated the subject that object lessons under farm conditions should form an essential feature of the station. It must, then, be at the place most accessible for the people of the whole island and for persons temporarily visiting the island. Second. The station must be located where it can have direct and Second. The station must be located where it can have direct and prompt communication with the Department of Agriculture at Wash- ington, because for some years it will not have equipment to do all the scientific work required, and reference must be constantly had to the several departments at Washington. Third. San Juan is the only city upon the island which has printing Third. San Juan is the only city upon the island which has printing establishments sufficiently equipped to issue bulletins in English and Spanish with reasonable facility. Fourth. Lands near San Juan can be purchased at reasonable prices. Fourth. Lands near San Juan can be purchased at reasonable prices INSTRUCTION IN AGRICULTURE IN THE COMMON SCHOOLS. A few simple oral lessons on the nature of seeds and plants, how to prepare the soil, and how to plant and care for the crop, with some small plats around the schoolhouse as object lessons, would be of in- estimable value to the youth and far-reaching in its results. Many of the lower classes in the country appear to have no knowledge of the principles of agriculture or of its successful practice. They half stir the soil, sow the garden seeds broadcast, and let Providence do the weeding. The commissioner of education for the island expressed a desire to do all in his power to promote agricultural education in the common schools. DESIRABILITY OF SECURING THE COOPERATION OF PORTO RICAN FARMERS IN EXPERIMENT WORK. It is highly important to secure the active cooperation of the farmers in investigations of the station and in the dissemination of information. The best plan is to organize local associations of farmers in every im- portant rural center in the island who will cooperate in testing seeds, plants, and methods of cultivation and aid in distributing bulletins, pamphlets, etc. In nearly every community can be found planters of liberal education and travel. Some were educated in the United States and can speak English. As far as consulted, all planters were enthusiastically in sympathy with the proposed work of the station. 32 FINANCIAL SUPPORT OF THE STATION. For the current expenses of an agricultural experiment station in Porto Rico at least as much money will annually be required as is now appropriated for such stations in the States and Territories ($15,000). The cost of the buildings needed by the station is estimated as follows: One building for director and assistants_..__. _.....---.-..-------.------- $5,000 One building for offices and laboratory...-_....----.--.----------------- 38, 000 One house for farm foreman......---.---.--------------------+--+ +--+ eee 1,000 Four cottages for farm laborers._._-....-...----------------------------- 800 BARNS See ueren st sje teased aes Me cor aae Gente tare mcia eae see Ame taae er donee. Ch OOO TOtal cede ict ines eaends Cohat aoeeoecscee ance deeds Ree eoskceeese 10, 800 Suitable land in the vicinity of San Juan will probably cost $25 per acre, and at least 200 acres should be obtained for the station there, for the purchase of which $5,000 will be required. For outlying experi- ments Government land on the island may be reserved. These reser- vations should include alluvial or level lands for sugar and rice, mountain lands for forestry, tobacco, and coffee, and sandy sea-coast lands for cocoanuts. O PLATE I. PLATE I. House Doc, No. 171. = eS, = WE —— pee GATHERING COCOANUTS. GATHERING COCOANUTS. House Doc. No. 171, PLATE Il. PLOWING FOR SUGAR CANE. PLATE Il. House Doc. No. 171, PLOWING FOR SUGAR CANE. HAULING CANE TO MILL. HAULING CANE TO MILL. PLATE Ill. House Doc. No. 171. House Doc. No. 171. PLATE Ill. CANE MILL. CANE MILL. SPREADING BAGASSE TO Dry. SPREADING BAGASSE TO Dry. House Doc. No. 171, PLaTe IV. EXPORTING SUGAR. PLaTe IV. House Doc. No. 171, EXPORTING SUGAR. a HAULING MOLASSES TO MARKET. a HAULING MOLASSES TO MARKET. HAULING MOLASSES TO MARKET. PLATE V. House Doc. No. 171, SuGAR PLANTER’S HOUSE, ARECIBO. SuGAR PLANTER’S HOUSE, ARECIBO. LaBoRER’S HUT, NEAR CARMEN. LaBoRER’S HUT, NEAR CARMEN. Pate VI. House Doc. No, 171. THREE-YEAR-OLD ORANGE TREES. THREE-YEAR-OLD ORANGE TREES. LaBorer’s HuT, COFFEE PLANTATION. LaBorer’s HuT, COFFEE PLANTATION. PLaTE VII. House Doc. No. 171. = eS Oe i as ee ee ee ES Barcelonera VEGA e e 0 S77. ARECIBO /SABELA QuesRani.tascwte’ MANAT ; £ 7 a VEGA-AL Tee a ve YSAYAMON =) Carotina AGUADILLA fi o. m RUSILLO-ALTO y) ve, MOCA % * COROZAL a "Or, owe OS ae se, NARANEITO CAGUAD TN ego AN SEBASTIAN : CraSece Morovis e oe RINCON etdate yt wAsvas-Bueng 3 @CIALITOS é ex. FINANCIAL SUPPORT OF THE STATION. LAS MARIAS Ne /7vA00 Comerro NASCO. 4 BARROS Pros i e@U4rura e GARRANQUITAS s £ @Maricao - ClORA "805, 0° * MAVAGUEZ ADJUNTAS 59 s SAN GERMAN ‘ ®c180-Bovo ® Yauco GUAYAN/. @LAUAS e UAYANILLA © S7) c-—==5 REFERENCES. Froaa FINISHED. eeeeeeeee= OAD BEING CONSTRUCTED. MAP OF ISLAND OF PoRTO RIGO, SHOWING STATE OF ROADS IN 1900. MAP OF ISLAND OF PoRTO RIGO, SHOWING STATE OF ROADS IN 1900. MAP OF ISLAND OF PoRTO RIGO, SHOWING STATE OF ROADS IN 1900.
https://openalex.org/W2009432223	https://www.hal.inserm.fr/inserm-01356877/document	English	null	Glycan Dependence of Galectin-3 Self-Association Properties	PloS one	2,014	cc-by	7,239	To cite this version: Hubert Halimi, Annafrancesca Rigato, Deborah Byrne, Géraldine Ferracci, Corinne Sebban-Kreuzer, et al.. Glycan Dependence of Galectin-3 Self-Association Properties. PLoS ONE, 2014, 9 (11), pp.e111836. 10.1371/journal.pone.0111836. inserm-01356877 Glycan Dependence of Galectin-3 Self-Association Properties Hubert Halimi, Annafrancesca Rigato, Deborah Byrne, Géraldine Ferracci, Corinne Sebban-Kreuzer, Latifa Elantak, Francoise Guerlesquin To cite this version: Hubert Halimi, Annafrancesca Rigato, Deborah Byrne, Géraldine Ferracci, Corinne Sebban-Kreuzer, et al.. Glycan Dependence of Galectin-3 Self-Association Properties. PLoS ONE, 2014, 9 (11), pp.e111836. 10.1371/journal.pone.0111836. inserm-01356877 Glycan Dependence of Galectin-3 Self-Association Properties Hubert Halimi, Annafrancesca Rigato, Deborah Byrne, Géraldine Ferracci, Corinne Sebban-Kreuzer, Latifa Elantak, Francoise Guerlesquin Abstract Human Galectin-3 is found in the nucleus, the cytoplasm and at the cell surface. This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD). There are still uncertainties about the relationship between the quaternary structure of Galectin-3 and its carbohydrate binding properties. Two types of self- association have been described for this lectin: a C-type self-association and a N-type self-association. Herein, we have analyzed Galectin-3 oligomerization by Dynamic Light Scattering using both the recombinant CRD and the full length lectin. Our results proved that LNnT induces N-type self-association of full length Galectin-3. Moreover, from Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance experiments, we observed no significant specificity or affinity variations for carbohydrates related to the presence of the N-terminal domain of Galectin-3. NMR mapping clearly established that the N-terminal domain interacts with the CRD. We propose that LNnT induces a release of the N-terminal domain resulting in the glycan-dependent self-association of Galectin-3 through N-terminal domain interactions. Citation: Halimi H, Rigato A, Byrne D, Ferracci G, Sebban-Kreuzer C, et al. (2014) Glycan Dependence of Galectin-3 Self-Association Properties. PLoS ONE 9(11): e111836. doi:10.1371/journal.pone.0111836 Editor: Lu-Gang Yu, University of Liverpool, United Kingdom Received July 11, 2014; Accepted October 1, 2014; Published November 4, 2014 Copyright: 2014 Halimi et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. imi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: The authors have no support or funding to report. authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. ave no support or funding to report Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * Email: guerlesquin@imm.cnrs.fr Competing Interests: The authors have declared that no competing interests exist. * Email: guerlesquin@imm cnrs fr . These authors contributed equally to this work. Galectin-3 displays a large range of cellular locations [3]. Abstract It is found in the nucleus, the cytoplasm and can be secreted via a non- classical pathway outside the cell [4]. In adults, Galectin-3 is ubiquitously distributed in tissues and it is thus involved in a large number of physiological and pathological processes such as cell proliferation, cell differentiation, cell survival and cell death [5–6]. Galectin-3 N-terminal domain is essential for its biological activities [2]. This unstructured domain is subject to metallopro- teinase proteolysis, which impacts on its biological functions. Cleavage at different positions has been shown to be a fine tuning of Galectin-3 activity and specificity. Serine and tyrosine phosphorylations have been described as post-translational mod- ifications [7]. These modifications are involved in Galectin-3 localization by regulating collagen domain cleavage [8]. Galectin- 3 has also an extracellular localization at the cell surfaces or in the extracellular matrix [4]. This lectin has been shown to mediate cell adhesion involving cell surface glycosylated components. Extra- cellular lattice formation resulting of Galectin-3/glycan interac- tions is an interesting problem, but a lot of contradictory structural information is found in the literature. The role of Galectin-3 N- terminal domain was suggested to be either in modulating the affinity of the lectin for carbohydrates or regulating the self- association of this chimera lectin. The CRD is responsible for the lectin activity of Galectin-3 [9], but the implication of the N- terminal domain in carbohydrate interaction is still an open Hubert Halimi1., Annafrancesca Rigato1,2., Deborah Byrne3, Ge´raldine Ferracci4, Corinne Sebban- Kreuzer1, Latifa ElAntak1, Francoise Guerlesquin1* Hubert Halimi1., Annafrancesca Rigato1,2., Deborah Byrne3, Ge´raldine Ferracci4, Corinne Sebban- Kreuzer1, Latifa ElAntak1, Francoise Guerlesquin1* 1 Laboratoire d’Inge´nierie des Syste`mes Mole´culaires, UMR 7255, CNRS, Aix-Marseille Universite´, Marseille, France, 2 U1006 INSERM, Aix-Marseille Universite´, Marseille, France, 3 Institut de la Microbiologie de la Me´diterrane´e, FR 3479, CNRS, Aix-Marseille Universite´, Marseille, France, 4 Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille, UMR 7286, Aix-Marseille Universite´, Marseille, France HAL Id: inserm-01356877 https://inserm.hal.science/inserm-01356877v1 Submitted on 26 Aug 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. November 2014 \| Volume 9 \| Issue 11 \| e111836 Introduction Galectins are small soluble lectins having carbohydrate-depen- dent extracellular and carbohydrate-independent intracellular activities [1]. Oligomerization is one of the unique features of secreted galectins forming ordered galectin-glycan lattices at the cell surface. Fourteen members of the galectin family have been identified in mammals and designated as galectin-1 to galectin-14. The common property of galectins is the presence of a carbohydrate-recognition domain (CRD) of about 130 amino acids with a highly conserved folding that confers affinity for b- galactoside-containing glycans [1]. Galectins are classified in three categories according to their structures [1]. The members of the prototype family which exist as monomers or homodimers and contain one CRD per subunit are Galectin-1, -2, -5, -7, -10, -11, -13 and -14. The tandem-repeat family accommodates two CRD domains in a single polypeptide chain connected by a non- conserved linker sequence of up to 70 amino acids. This family is composed of Galectin-4, -6, -8, -9 and -12. Galectin-3 is the unique member of the chimera family and its single polypeptide chain forms two distinct domains, a non-lectin N-terminal domain and a C-terminal domain constituting the CRD. Galectin-3 N- terminal domain encloses a short N-terminal segment necessary for secretion followed by collagen-like repeats connected to the C- terminal CRD domain [2]. November 2014 \| Volume 9 \| Issue 11 \| e111836 1 PLOS ONE \| www.plosone.org Galectin-3 Self-Association Figure 1. CRD and FL oligomerization states without glycan determined by DLS. Top, CRD (41 mM) size distribution by volume before and after filtration. Bottom, FL (37 mM) size distribution by volume before and after filtration. doi:10.1371/journal.pone.0111836.g001 Figure 1. CRD and FL oligomerization states without glycan determined by DLS. Top, CRD (41 mM) size distribution by volume before and after filtration. Bottom, FL (37 mM) size distribution by volume before and after filtration. doi:10.1371/journal.pone.0111836.g001 Figure 1. CRD and FL oligomerization states without glycan determined by DLS. Top, CRD (41 mM) size distribution by volume before and after filtration. Bottom, FL (37 mM) size distribution by volume before and after filtration. doi:10.1371/journal.pone.0111836.g001 question. Moreover, two models of Galectin-3 self-association have been reported: a C-type self-association involving the CRD [10] and a N-type self-association involving the N-terminal domain [11]. It has been reported that Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms disorganized heterogeneous cross-linked complexes [12]. added to the culture to induce overexpression of the proteins. NMR experiments NMR experiments were carried out at 303 K on a Bruker Avance III 600 MHz NMR spectrometer equipped with a TCI cryoprobe, and a Bruker Avance III 500 MHz NMR spectrom- eter. CRD chemical shift assignments were obtained from [13] and two sets of heteronuclear NMR experiments (HNCA and HNCOCA, and HNCO and HNCACO) were performed on the 15N and 13C-labeled CRD sample at 400 mM concentration in the presence and the absence of lactose. 1H-15N-HSQC titrations were performed on 15N-labeled proteins at 40 mM concentration for CRD and 30 mM for full length protein (FL), in the absence and the presence of various concentrations of ligands (lactose or Introduction After 4 hours at 37uC, cells were harvested. From a French press cell lysate, the His-Tag proteins were purified from the supernatant by affinity chromatography using a HiTrap pre-packed column on an AKTAPrime purifying system (GE Healthcare). Proteins were eluted using a linear imidazole gradient and dialyzed in a 5 mM potassium phosphate buffer at pH 7.4. EDTA was added to purify fractions of N-terminal domain and full length Galectin-3 to avoid cleavage by metalloproteinases. Protein purity was checked by coomassie blue staining of a SDS-PAGE and mass spectrometry analysis. For isotopic labeling, 15N-ammonium chloride was used as the sole source of nitrogen and 13C-C6glucose was used as the sole source of carbon. Using recombinant full length Galectin-3 (FL) and Galectin-3 CRD (CRD), we have investigated by Dynamic Light Scattering (DLS) the self-association properties of Galectin-3 in the presence or the absence of carbohydrates. A mechanism for the ligand- induced N-type association is proposed on the basis of our structural NMR data on CRD/N-terminal domain interactions in the presence or the absence of the lacto-N-neoTetraose (LNnT). Protein expression The cDNA sequences encoding N-terminal domain (13–113), CRD domain (114–250) and full length (1–250) human Galectin-3 were PCR amplified using appropriate primers (Table S1) in order to include a 6His-tag sequence at the N-terminus for the N- terminal domain and at the C-terminus for CRD and full length protein. The encoding sequences were then cloned into a pET22b(+) expression vector. After transformation of BL21(DE3) competent cells, the bacteria were grown on minimum cell medium M9 at 37uC until OD600 nm = 0.6. Then IPTG 1 mM was November 2014 \| Volume 9 \| Issue 11 \| e111836 PLOS ONE \| www.plosone.org 2 Galectin-3 Self-Association Figure 2. CRD and FL glycan induced oligomerization determined by DLS. A] CRD (41 mM) size distribution by volume before and after the addition of glycans. Top, monomeric CRD without glycan; middle, monomeric CRD with 2 mM lactose; bottom, monomeric CRD with 140 mM LNnT. B] FL (37 mM) size distribution by volume before and after the addition of glycans. Top, FL monomer without glycan; middle, FL monomer with 8 mM lactose; bottom, FL oligomers with 140 mM LNnT. doi:10.1371/journal.pone.0111836.g002 Figure 2. CRD and FL glycan induced oligomerization determined by DLS. A] CRD (41 mM) size distribution by volume before and after the addition of glycans. Top, monomeric CRD without glycan; middle, monomeric CRD with 2 mM lactose; bottom, monomeric CRD with 140 mM LNnT. B] FL (37 mM) size distribution by volume before and after the addition of glycans. Top, FL monomer without glycan; middle, FL monomer with 8 mM lactose; bottom, FL oligomers with 140 mM LNnT. doi:10 1371/journal pone 0111836 g002 LNnT). All NMR experiments were performed on samples in 5 mM potassium phosphate buffer at pH 7.4. Lactose was purchased from Sigma and LNnT from Elicityl Company. ZEN0040. The samples were measured in triplicate. Each measurement consisted of 11 runs, each run lasting for 10 seconds. We used a laser He-Ne at 633 nm with a scattering detection angle of 173u. All analyses were performed at 25uC. The solutions containing 41 mM CRD and 37 mM FL, in 5 mM potassium phosphate buffer at pH 7.4, were centrifuged at 14,000 rpm for 5 minutes and filtered through a 0.45 mm filter. Sodium phosphate buffer with a viscosity of 0.89 cp and a refractive index of 1.33 was used for all sample preparations. Surface Plasmon Resonance experiments SPR experiments were performed at 25uC on a NiHC sensor chip (Xantec) with a Biacore T200 instrument (GE Healthcare) using 10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 50 mM EDTA, 0.005% Tween-20 as the running buffer. Six his-tagged CRD and FL were immobilized (230 fmoles) by affinity on two independent experimental flow cells, and two flow cells without protein were used as control. A set of concentrations of lactose and LNnT were successively injected over all flow cells at a flow rate of 30 ml/min during one minute. Sensorgrams obtained from control flow cells were systematically subtracted from those obtained over CRD and FL. The KD values were calculated by plotting saturation binding curves using the equilibrium response at the plateau of all curves with BiaEvaluation software version 2.0 (GE Healthcare). Each value was obtained from at least two independent experiments performed in triplicate. Protein expression We used the standard refractive index 1.45 for a spherical protein to calculate the mass distribution of size. Protein analysis was performed using the instrument software based on the model determined from an L.curve. We used a standard operating procedure for protein analysis. All measurement conditions were optimized automatically by the instruments software. We added increments of 1 ml of 100 mM lactose to a 50 ml sample containing CRD and FL respectively. The concentrations of lactose used were from 2 mM to 8 mM. LNnT was added in 0.5 ml increments of a 7 mM stock to 50 ml sample containing CRD and FL respectively. The LNnT concentrations added to the FL sample were from 70 mM to 210 mM, in contrast to the higher amounts added to CRD, 70 mM to 1.2 mM. Oligomerization states of Galectin-3 investigated by DLS Oligomerization states of Galectin-3 investigated by DLS O go e at o states o a ect 3 est gated by S Taking into account the large number of contradictory results already published in the literature regarding the oligomeric states of Galectin-3, our first concern was to investigate the oligomer- ization states of CRD and full length Galectin-3 by DLS. After purification and freezing at 280uC, we could observe that both CRD and FL were present in two states: a small hydrodynamic size as already reported in the literature and a large hydrodynamic size, indicating an oligomerization of the Galectin-3 independent of the glycan presence and independent of the N-terminal domain, corresponding to the C-type association (Table 1 and Fig. 1). However, when comparing FL and CRD samples, it appeared that FL was more sensitive to oligomerization. We optimized a protocol including centrifugation (14,000 rpm during 5 mn) and filtering (0.45 mm) which allowed us to obtain a sample predominantly constituted of molecules with a hydrodynamic radius less than 5 nm (Table 1), consistent with the monomeric form of the lectin. We observed that the monomeric state of CRD or FL samples remained stable at room temperature for several hours following filtration. The samples also remained stable up to 47uC. With the aim to observe glycan induced oligomerization, we have performed all the DLS experiments using samples centri- fuged, filtered and analyzed at room temperature. Two carbohy- drates were tested, lactose, well established as a good ligand for Galectin-3 and LNnT [14]. Both x-ray structures of the CRD/ lactose complex (PDB 3ZSJ) and CRD/LNnT complex (PDB 4LBN) have been solved. In Table 1, the results of DLS experiments are summarized, showing the percentages of different hydrodynamic sizes (corresponding to different oligomerization states) for both CRD and FL in the absence and presence of the ligands. The comparison between the initial percentage of monomeric CRD and FL (size ,5 nm) and after lactose addition, clearly shows that lactose does not induce CRD nor FL oligomerization (Fig. 2). This data is in agreement with previous data found in the literature [15]. Interestingly, with the addition of 70 mM LNnT to the FL solution, two populations were present; the first with a hydrodynamic radius of 3.147 nm representing 66.7% of the total mass and the second with a radius of 4.643 nm, representing 33.3% (Table 1, sample 9). Dynamic Light Scattering We performed Dynamic Light Scattering (DLS) experiments using a Zetazizer Nano Series (Malvern Instruments, London, UK). The samples were analyzed in a disposable micro-cuvette November 2014 \| Volume 9 \| Issue 11 \| e111836 PLOS ONE \| www.plosone.org 3 Galectin-3 Self-Association NnT/70 mM# 3.147 66.7 4.643 33.3 0.822 n Domain, FL full length, BF before Filtration, AF after Filtration. AF* sample used for interaction studies. Negl Negligible. 1Size distribution from 3 measurements of 11runs. #The ents of 11 runs. Oligomerization states of Galectin-3 investigated by DLS The 4.643 nm population could be indicative of a pentameric state already described in the literature (Ahmad et al, 2004). Consequently, when we increased the concentration two fold of LNnT, the percentage of protein with low hydrodynamic size (less than 5 nm) dropped to zero, while an increase of two populations with larger hydrodynamic radii was observed. The first population with a hydrodynamic radius greater than 40 nm, represents 8.7% of the total mass and the second population with a radius greater than 100 nm, represents 91.3%. On the contrary, the presence of LNnT induced no effects on the CRD. The evidence clearly demon- strates that LNnT is able to induce oligomerization of the full- length protein, but not of the CRD alone. Such results strongly support the hypothesis of an essential role of the N-terminal domain in the mechanism of ligand-dependent oligomerization of Galectin-3 via an N-type self-association as recently reported in the literature [11]. Results and Discussion Table 1. Percentage distribution of size in hydrodynamic radius of CRD and FL in the presence and absence of glycans. Sample Galectin-3 Saccharide Size Distribution (% Mass)1 Polydispersity index ,5 nm ,100 nm .100 nm nm % nm % nm % nm % 1 CRD BF - 2.302 99.3 - - 48.84 Negl 324.9 0.7 0.743 2 CRD AF* - 2.163 99.9 - - - - 201.7 Negl 0.557 3 CRD AF Lactose/2 mM 2.663 99.6 - - - - 440.7 Negl 0.476 4 CRD AF LNnT/140 mM 2.313 99.6 - - - - 470.6 Negl 0.705 5 FL BF - 3.621 33 - - - - 291 67 0.785 6 FL AF* - 3.318 99.6 - - - - 309.2 Negl 0.471 7 FL AF Lactose/8 mM 3.155 99.6 - - - - 223.4 Negl 0.533 8 FL AF LNnT/140 mM - - - - 46.73 8.7 326.5 91.3 0.627 9 FL AF LNnT/70 mM# 3.147 66.7 4.643 33.3 - - - - 0.822 Galectin-3: CRD Carbohydrate Recognition Domain, FL full length, BF before Filtration, AF after Filtration. AF* sample used for interaction studies. Negl Negligible. 1Size distribution from 3 measurements of 11runs. #T oscillation of size throughout 9 measurements of 11 runs. Structural analysis of full length Galectin-3 To investigate the structural features of Galectin-3 ligand- induced oligomerization, we produced 15N-labelled N-terminal domain, CRD domain and full length Galectin-3 to perform an NMR structural study (Fig. 3). PLOS ONE \| www.plosone.org November 2014 \| Volume 9 \| Issue 11 \| e111836 4 Galectin-3 Self-Association N HSQC spectra of the free CRD domain of Galectin-3 (residues Ile132, Leu135, Val138, Lys139, Phe192, Glu193, . 1H-15N HSQC spectra carried out on a 500 MHz NMR spectrometer at 300 K. A] 15N-HSQC of full length Galectin-3 (in black) at ncentration and recombinant CRD (in red) at 40 mM concentration, B] 15N-HSQC of full length Galectin-3 (in black) at 30 mM concentration mbinant N-terminal domain (in red) at 30 mM concentration. 71/journal.pone.0111836.g003 Figure 3. 1H-15N HSQC spectra carried out on a 500 MHz NMR spectrometer at 300 K. A] 15N-HSQC of full length Galectin-3 (in black) at 30 mM concentration and recombinant CRD (in red) at 40 mM concentration, B] 15N-HSQC of full length Galectin-3 (in black) at 30 mM concentration and recombinant N-terminal domain (in red) at 30 mM concentration. doi:10.1371/journal.pone.0111836.g003 (residues Ile132, Leu135, Val138, Lys139, Phe192, Glu193, Phe198, Ile200, Gln201, Val202, Leu203, Glu205, Lys210, Ala212, Asp215, Ala216, Asp241, Thr243 and Ser244) (Fig. 5A). This result is in perfect agreement with the peptide analysis already reported by NMR spectroscopy indicating that the N- terminal domain of Galectin-3 interacts with residues of CRD located at the back of the molecule [17]. Moreover, residues located at the N- and C-terminal extremities of the CRD (residues Ile115 and Val116, and Tyr247, Thr248 and Met249) were also perturbed by the presence of the N-terminal domain as previously predicted by modeling of Galectin-3 involving the b-strands S1 and S12 [18]. The binding of the N-terminal domain of Galectin-3 on the N- and C-terminal segments of the CRD may explain the monomeric status of this galectin in solution. 1H-15N HSQC spectra of the free CRD domain of Galectin-3 (Fig. 3A) is in agreement with the already published assignment of this protein [13]. The 1H-15N HSQC of the free N-terminal domain (Fig. 3B) is characteristic of an unfolded domain with resonances between 8.5 and 7.5 ppm. The 1H-15N HSQC of the full length protein is the sum of the 1H-15N HSQC spectra of the N-terminal domain and of the CRD with slight chemical shift variations in perfect agreement with the already published assignment of the full length protein [16]. November 2014 \| Volume 9 \| Issue 11 \| e111836 Structural analysis of full length Galectin-3 In the three proteins (CRD, FL and N-terminal domain), the 6His-Tag was not assigned. 1H-15N HSQC spectra of the free CRD domain of Galectin-3 (Fig. 3A) is in agreement with the already published assignment of this protein [13]. The 1H-15N HSQC of the free N-terminal domain (Fig. 3B) is characteristic of an unfolded domain with resonances between 8.5 and 7.5 ppm. The 1H-15N HSQC of the full length protein is the sum of the 1H-15N HSQC spectra of the N-terminal domain and of the CRD with slight chemical shift variations in perfect agreement with the already published assignment of the full length protein [16]. In the three proteins (CRD, FL and N-terminal domain), the 6His-Tag was not assigned. To access the interactions between the CRD and N-terminal domains we compared 1H-15N HSQC spectra of the free CRD with that of CRD within the FL (Fig. 4C and Fig. 5A). Chemical shift variation analysis indicates that the markedly shifted resonances of CRD belong to some residues close to the sugar binding site and residues located at the backside of the lectin On the other hand, comparison of 1H-15N HSQC of the N- terminal domain alone and within the full length Galectin-3 was more difficult to interpret (Fig. 3B). Even if the assignment of the November 2014 \| Volume 9 \| Issue 11 \| e111836 November 2014 \| Volume 9 \| Issue 11 \| e111836 PLOS ONE \| www.plosone.org 5 Galectin-3 Self-Association Figure 4. Complex formation involving Galectin-3. A] 15N-HSQC spectra carried out on a 600 MHz NMR spectrometer equipped with a TCI cryoprobe at 300 K. Top left, free CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of lactose at 1.5 mM in red; top right, free full length Galectin-3 (FL) at 30 mM concentration in black and FL at 30 mM concentration in the presence of lactose at 1.5 mM in red; bottom left, CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of LNnT at 80 mM in red; bottom right, free full length Galectin-3 (FL) at 30 mM concentration in black and FL at 30 mM concentration in the presence of LNnT at 80 mM in red. Structural analysis of full length Galectin-3 Values shown were calculated using the equation Ddobs = [(DdHN 2 + DdN 2/25)]1/2. In green boxes are highlighted residues affected by lactose. In red boxes are highlighted residues affected by the N-terminal domain within the full length protein. doi:10 1371/journal pone 0111836 g004 Figure 4. Complex formation involving Galectin-3. A] 15N-HSQC spectra carried out on a 600 MHz NMR spectrometer equipped with a TCI cryoprobe at 300 K. Top left, free CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of lactose at 1.5 mM in red; top right, free full length Galectin-3 (FL) at 30 mM concentration in black and FL at 30 mM concentration in the presence of lactose at 1.5 mM in red; bottom left, CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of LNnT at 80 mM in red; bottom right, free full length Galectin-3 (FL) at 30 mM concentration in black and FL at 30 mM concentration in the presence of LNnT at 80 mM in red. B] Zoom in of HSQC: in left box, free CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of lactose at 0.1 mM in red, 0.8 mM in green and 1.5 mM in blue; in the right box, CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of LNnT at 28 mM in red and 90 mM in blue. C] Chemical shift variations observed on Galectin-3 CRD NH, top, induced by the N-terminal domain within FL (Table S2); center, induced by lactose (Table S3); bottom, induced by LNnT (Table S4). Values shown were calculated using the equation Ddobs = [(DdHN 2 + DdN 2/25)]1/2. In green boxes are highlighted residues affected by lactose. In red boxes are highlighted residues affected by the N-terminal domain within the full length protein. d i 10 1371/j l 0111836 004 doi:10.1371/journal.pone.0111836.g004 full length Galectin-3 has been recently reported [16], it was not possible to give a chemical shift mapping of the CRD domain interacting zone on the N-terminal unstructured domain in full length Galectin-3. Due to the various conformations of the flexible N-terminal domain, the presence of repeat segments and the poor resolution of the NMR spectra of the N-terminal domain, unambiguous sequence specific assignments within the complex were difficult to obtain. Structural analysis of full length Galectin-3 B] Zoom in of HSQC: in left box, free CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of lactose at 0.1 mM in red, 0.8 mM in green and 1.5 mM in blue; in the right box, CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of LNnT at 28 mM in red and 90 mM in blue. C] Chemical shift variations observed on Galectin-3 CRD NH, top, induced by the N-terminal domain within FL (Table S2); center, induced by lactose (Table S3); bottom, induced by LNnT (Table S4). Values shown were calculated using the equation Ddobs = [(DdHN 2 + DdN 2/25)]1/2. In green boxes are highlighted residues affected by lactose. In red boxes are highlighted residues affected by the N-terminal domain within the full length protein. doi:10.1371/journal.pone.0111836.g004 Figure 4. Complex formation involving Galectin-3. A] 15N-HSQC spectra carried out on a 600 MHz NMR sp Figure 4. Complex formation involving Galectin-3. A] 15N-HSQC spectra carried out on a 600 MHz NMR spectrometer equipped with a TCI cryoprobe at 300 K. Top left, free CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of lactose at 1.5 mM in red; top right, free full length Galectin-3 (FL) at 30 mM concentration in black and FL at 30 mM concentration in the presence of lactose at 1.5 mM in red; bottom left, CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of LNnT at 80 mM in red; bottom right, free full length Galectin-3 (FL) at 30 mM concentration in black and FL at 30 mM concentration in the presence of LNnT at 80 mM in red. B] Zoom in of HSQC: in left box, free CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of lactose at 0.1 mM in red, 0.8 mM in green and 1.5 mM in blue; in the right box, CRD at 40 mM concentration in black and CRD at 40 mM concentration in the presence of LNnT at 28 mM in red and 90 mM in blue. C] Chemical shift variations observed on Galectin-3 CRD NH, top, induced by the N-terminal domain within FL (Table S2); center, induced by lactose (Table S3); bottom, induced by LNnT (Table S4). Structural analysis of full length Galectin-3 Moreover, the affinities of CRD and FL were similar for LNnT indicating that the N-terminal domain does not mediate the lectin/LNnT interaction (0.1460.017 mM for CRD and 0.1260.016 mM for Structural analysis of Galectin-3 oligosaccharide complexes Structural analysis of full length Galectin-3 However, we could observe that numerous resonances of the N-terminal domain underwent perturbations when comparing the spectra of the N-terminal domain alone and within the full length protein, confirming that the N-terminal domain interacts with the CRD (Fig. 3A). x-ray structure to be involved in the formation of the CRD/lactose complex (PDB 3ZSJ). No significant difference was observed between the affected residues of the CRD alone and full length protein. This indicated that lactose does not affect N-terminal domain/CRD interactions (Fig. 4A and 4C). The KD obtained by SPR measurements with lactose (Fig. 6) were in agreement with our NMR data as similar affinities for CRD (1.2560.18 mM) and for FL (1.1260.2 mM) were observed. These results clearly established that the N-terminal domain did not mediate nor enhance lactose/CRD interactions. x-ray structure to be involved in the formation of the CRD/lactose complex (PDB 3ZSJ). No significant difference was observed between the affected residues of the CRD alone and full length protein. This indicated that lactose does not affect N-terminal domain/CRD interactions (Fig. 4A and 4C). The KD obtained by SPR measurements with lactose (Fig. 6) were in agreement with our NMR data as similar affinities for CRD (1.2560.18 mM) and for FL (1.1260.2 mM) were observed. These results clearly established that the N-terminal domain did not mediate nor enhance lactose/CRD interactions. 2. Complex formation of CRD and full length Galectin-3 with LNnT. Galectin-3 is known to interact with polyLacNAc oligosaccharides [14] and among them the structure of CRD/ LNnT complex was solved by x-ray (PDB 4LBN). SPR experiments show that both the CRD and the FL proteins have a higher affinity for LNnT than for lactose (Fig. 6). Moreover, the affinities of CRD and FL were similar for LNnT indicating that the N-terminal domain does not mediate the lectin/LNnT interaction (0.1460.017 mM for CRD and 0.1260.016 mM for 2. Complex formation of CRD and full length Galectin-3 with LNnT. Galectin-3 is known to interact with polyLacNAc oligosaccharides [14] and among them the structure of CRD/ LNnT complex was solved by x-ray (PDB 4LBN). SPR experiments show that both the CRD and the FL proteins have a higher affinity for LNnT than for lactose (Fig. 6). Structural analysis of Galectin-3 oligosaccharide complexes 1. Complex formation of CRD and full length Galectin-3 with lactose. Figures 4 and 5B show the NMR titrations conducted on the CRD with lactose. The residues for which we observed chemical shift variations correspond to ones shown in the November 2014 \| Volume 9 \| Issue 11 \| e111836 PLOS ONE \| www.plosone.org 6 Galectin-3 Self-Association Figure 5. Chemical shift mapping representation on Galectin-3 CRD structure. A] Effects of the N-terminal domain on the NH groups of amino acids of the CRD. The structure of CRD in the presence of LNnT (PDB 4LBN) is shown. In yellow is Trp181, in red is LNnT and in blue are the amino acids of the CRD with chemical shift variations in the presence of the N-terminal domain within the full length Galectin-3. B] Effects of lactose on the NH groups of amino acids of the CRD. The structure of CRD in the presence of lactose (PDB 3ZSJ) is shown. In yellow is Trp181, in red is the lactose and in green are amino acids of CRD with chemical shift variations induced by the presence of lactose. C] Effects of LNnT on the NH groups of amino acids of the CRD. The structure of CRD in the presence of LNnT (PDB 4LBN) is shown. In yellow is Trp181, in red is LNnT and in green are amino acids of the CRD with chemical shift variations induced by the presence of LNnT. doi:10.1371/journal.pone.0111836.g005 Figure 5. Chemical shift mapping representation on Galectin-3 CRD structure. A] Effects of the N-terminal domain on the NH groups of amino acids of the CRD. The structure of CRD in the presence of LNnT (PDB 4LBN) is shown. In yellow is Trp181, in red is LNnT and in blue are the amino acids of the CRD with chemical shift variations in the presence of the N-terminal domain within the full length Galectin-3. B] Effects of lactose on the NH groups of amino acids of the CRD. The structure of CRD in the presence of lactose (PDB 3ZSJ) is shown. In yellow is Trp181, in red is the lactose and in green are amino acids of CRD with chemical shift variations induced by the presence of lactose. C] Effects of LNnT on the NH groups of amino acids of the CRD. The structure of CRD in the presence of LNnT (PDB 4LBN) is shown. Structural analysis of Galectin-3 oligosaccharide complexes In yellow is Trp181, in red is LNnT and in green are amino acids of the CRD with chemical shift variations induced by the presence of LNnT. doi:10.1371/journal.pone.0111836.g005 Gly238 and Arg144 in agreement with the additional chemical shifts observed in the NMR spectra in the presence of LNnT (Fig. 4C). It is clear that these favorable interactions are consistent with a greater affinity for LNnT than for lactose, as calculated from SPR experiments (0.1460.017 mM for LNnT and 1.2560.18 mM for lactose with CRD) (Fig. 6). As mentioned in the literature, the conformation of LNnT and its contact at the surface of the protein limits the types of extensions suitable for Galectin-3 ligands. Any extension would, however, lie along the binding groove [14]. When comparing the effect of LNnT binding on the quality of the NMR spectra of CRD and full length Galectin-3, one can observe decreased peak intensity in the NMR spectra of the full length protein after LNnT addition (Fig. 4A). Such decrease for the bound protein is correlated to a glycan induced oligomerization observed in DLS experiments for the FL protein but not for the CRD alone, and results in a lower concentration of the soluble form. We thus concluded that LNnT induces a full length Galectin-3 N-type self-association. FL). The slow exchange of NMR chemical shift variations observed in the presence of LNnT and the fast exchange observed in the presence of lactose were also in agreement with a higher affinity for LNnT than for lactose (Fig. 4B). X-ray structural data on CRD/ligand interactions (PDB 3ZSJ and 4LBN) revealed that the binding of the core b-galactoside is highly conserved in all analyzed complexes. In particular, the interactions formed by Trp181 with O4 and O6 of the galactose moiety are always observed. 1H-15N HSQC spectra indicate that the ligand affects the CRD and FL spectra in a similar way (Fig. 4C and Fig. 5C). In the crystal structure of CRD/LNnT complex [19], there is a well- defined electron density for the three carbohydrate residues from the reducing end of LNnT (GlcNacb1-3Galb1-4Glc). These residues form identical hydrogen bonding with the protein as those in the lactose-bound structures. The GlcNac residue that is b1-3 linked to the lactose in the tetrasaccharide extends the Galectin-3 binding site by the formation of direct hydrogen bonds with the protein side chains (Arg144 and Asp148). Structural analysis of Galectin-3 oligosaccharide complexes Consistently, these two residues show additional chemical shift variations in the spectrum of CRD in the presence of LNnT (Fig. 4C). In addition, water- mediated interactions are observed between GlcNac carbonyl oxygen and the side chains of Arg144 and Asn160, and van der Waals contacts are found between GlcNac C6/O6 and Asp148 and His158. Additional chemical shifts are observed for these residues in the NMR spectra in the presence of LNnT (Fig. 4C). The terminal b1-4 galactose residue is relatively poorly defined in the electron density and it forms weak interactions with Structural implications of N-type self-oligomerization of Galectin-3 Acknowledgments The authors thank the NMR platform of the ‘‘Institut de Microbiologie de la Me´diterrane´e’’ for providing NMR facilities. Supporting Information overlap on the CRD, indicating that one interaction might alter the other (Fig. 5). These data bring us to conclude that LNnT removes the N-terminal domain from the CRD interface by competition, triggering the release of this N-terminal domain and resulting in the oligomerization of the full length Galectin-3 via a N-type glycan-dependent self-association. Supporting Information Table S1 (DOCX) Table S2 (DOCX) Table S3 (DOCX) Table S4 (DOCX) pp g Table S1 (DOCX) Table S2 (DOCX) Table S3 (DOCX) Table S4 (DOCX) Table S1 (DOCX) Table S2 (DOCX) Table S3 (DOCX) Table S4 (DOCX) yp g y p At the cell surface when Galectin-3 is overexpressed, two independent Galectin-3 self-association processes are the driving force of lattice formation. The first is a glycan-dependent N-type association where Galectin-3 forms heterogeneous oligomers through N-terminal domain interactions. The second is glycan- independent and also observed for the CRD alone, thus defined as C-type association. In the full length galectin-3, the C-type association is probably enhanced by the N-type association. In this work, we show how in the presence of LNnT, N-type oligomer- ization increases the exposure of the N- and C-terminal extremities of the CRD domain which are favorable to the C-type interactions. These two driving forces assisted by the specific positioning of glycans at the cell surface may induce a proper lattice formation. Such lattice is necessary to strengthen cell-cell interactions under dragging forces imposed by the fluid flow acting on cells or bacteria. The protein-protein interactions encountered at the N-terminal domain/CRD interface of Galectin-3 may be considered as new potential targets for drug design in cancer. Structural implications of N-type self-oligomerization of Galectin-3 On the basis of our NMR titrations, we analyzed the effects induced by the N-terminal domain, lactose and LNnT on the CRD chemical shifts (Fig. 4C). One can observe that the chemical shift variations on the CRD in the presence of LNnT are the sum of those induced by lactose and some due to the N-terminal domain. Thus, the N-terminal interface and the LNnT interface November 2014 \| Volume 9 \| Issue 11 \| e111836 PLOS ONE \| www.plosone.org 7 Galectin-3 Self-Association Figure 6. SPR measurement of lactose and LNnT binding to CRD and FL. Increasing concentrations of lactose (12.5–2000 mM, A, C) and LNnT (0.5–250 mM, B, D) were injected over immobilized CRD and FL. Typical sensorgrams obtained with CRD (A, B) are displayed. The measured plateau values were used to plot saturation binding curves (C, D). These curves were used to calculate the KD value for lactose and LNnT bindings to CRD and FL. One curve is representative of at least two independent experiments. doi:10.1371/journal.pone.0111836.g006 Figure 6. SPR measurement of lactose and LNnT binding to CRD and FL. Increasing concentrations of lactose (12.5–2000 mM, A, C) and LNnT (0.5–250 mM, B, D) were injected over immobilized CRD and FL. Typical sensorgrams obtained with CRD (A, B) are displayed. The measured plateau values were used to plot saturation binding curves (C, D). These curves were used to calculate the KD value for lactose and LNnT bindings to CRD and FL. One curve is representative of at least two independent experiments. doi:10.1371/journal.pone.0111836.g006 References 1. Leffler H, Carlsson S, Hedlund M, Qian Y, Poirier F (2004) Introduction to galectins. Glycoconj J. 19(7–9): 433–440. 12. Ahmad N, Gabius HJ, Andre´ S, Kaltner H, Sabesan S, et al. (2004) Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes. J. Biol. Chem. 279(12): 10841–10847. g y j 2. Gong HC, Honjo Y, Nangia-Makker P, Hogan V, Mazurak N, et al. (1999) The NH2 terminus of Galectin-3 governs cellular compartmentalization and functions in cancer cells. Cancer. Res. 59(24): 6239–6245. g p ( ) 13. Umemoto K, Leffler H (2001) Assignment of 1H, 15N and 13C resonances of the carbohydrate recognition domain of human Galectin-3. J Biomol NMR. 20(1): 91–92. , ( ) g , carbohydrate recognition domain of human Galectin-3. J Biomol NMR. 20(1): 91–92. ( ) 3. Newlaczyl AU, Yu LG (2011) Galectin-3-A jack-of-all-trades in cancer. Cancer Lett. 313, 123–128. 14. Stowell SR, Arthur CM, Mehta P, Slanina KA, Blixt O, et al. (2008) Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens. J. Biol. Chem. 283(15): 10109–10123. 4. Dumic J, Dabelic S, Flo¨gel M (2006) Galectin-3: an open-ended story. Biochim. Biophys. Acta 1760(4): 616–635. 5. Liu FT, Rabinovich GA (2005) Galectins as modulators of tumor progression. Cancer, 5, 29–41. 15. Morris S, Ahmad N, Andre´ S, Kaltner H, Gabius HJ, et al. (2004) Quaternary solution structures of galectins-1, -3, and -7. Glycobiology 14(3): 293–300. 6. Song L, Tang JW, Owusu L, Sun MZ, Wu J, et al. (2014) Galectin-3 in cancer. Clin. chim. Acta 431, 185–191. 16. Ippel H, Miller MC, Berbı´s MA, Suylen D, Andre´ S, et al. (2014) 1H, 13C, and 15N backbone and side-chain chemical shift assignments for the 36 proline- containing, full length 29 kDa human chimera-type Galectin-3. Biomol NMR Assign. DOI 10.1007/s12104-014-9545-3. 7. Mazurek N, Conklin J, Byrd JC, Raz A, Bresalier RS (2000) Phosphorylation of the beta-galactoside-binding protein Galectin-3 modulates binding to its ligands. J. Biol. Chem. 275(46): 36311–36315. 17. Berbı´s MA´ , Andre´ S, Can˜ada FJ, Pipkorn R, Ippel H, et al. (2014) Peptides derived from human Galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner. Biochem. Biophys. Res. Commun. 443(1): 126–131. J ( ) 8. Balan V, Nangia-Makker P, Kho DH, Wang Y, Raz A (2012) Tyrosine- phosphorylated Galectin-3 protein is resistant to prostate-specific antigen (PSA) cleavage. J. Biol. Chem. 287(8): 5192–5198. 9. Author Contributions Conceived and designed the experiments: HH AR DB GF CSK LE FG. Performed the experiments: HH AR DB GF CSK LE FG. Analyzed the data: HH AR DB GF CSK LE FG. Contributed reagents/materials/ analysis tools: HH AR DB GF CSK LE FG. Wrote the paper: HH AR DB GF CSK LE FG. November 2014 \| Volume 9 \| Issue 11 \| e111836 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8 Galectin-3 Self-Association References Seetharaman J, Kanigsberg A, Slaaby R, Leffler H, Barondes SH, et al. (1998) X-ray crystal structure of the human Galectin-3 carbohydrate recognition domain at 2.1-A˚ resolution. J. Biol. Chem. 273(21): 13047–52. 18. Barboni EA, Bawumia S, Henrick K, Hughes RC (2000) Molecular modeling and mutagenesis studies of the N-terminal domains of Galectin-3: evidence for participation with the C-terminal carbohydrate recognition domain in oligosaccharide binding. Glycobiology 10(11): 1201–1208. J ( ) 10. Yang RY, Hill PN, Hsu DK, Liu FT (1998) Role of the carboxyl-terminal lectin domain in self-association of Galectin-3. Biochemistry. 37(12): 4086–4092. domain in self-association of Galectin-3. Biochemistry. 37(12): 40 19. Collins PM, Bum-Erdene K, Yu X, Blanchard H (2013) Galectin-3 Interactions with Glycosphingolipids. J. Mol. Biol. 2836(13): 743–752. 11. Lepur A, Salomonsson E, Nilsson UJ, Leffler H (2012) Ligand induced galectin- 3 protein self-association. J Biol Chem. 287(26): 21751–21756. 3 protein self-association. J Biol Chem. 287(26): 21751–21756. November 2014 \| Volume 9 \| Issue 11 \| e111836 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 9
https://openalex.org/W2014737477	https://europepmc.org/articles/pmc4196091?pdf=render	English	null	Development of large-scale metabolite identification methods for metabolomics	BMC bioinformatics	2,014	cc-by	697	POSTER PRESENTATION Open Access * Correspondence: hunter.moseley@uky.edu 1Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40356, USA Full list of author information is available at the end of the article Materials and methods We have developed a novel algorithm to allow for the rapid detection of functional groups within existing meta- bolite databases such as KEGG Ligand and the Human Metabolome Database in order to create functional group resolved versions of both databases. These databases will allow for combined molecular formula and functional group (from CS tagging) queries to aid in metabolite identification based on accurate mass information with- out a priori knowledge. Published: 29 September 2014 doi:10.1186/1471-2105-15-S10-P36 Cite this article as: Mitchell et al.: Development of large-scale metabolite identification methods for metabolomics. BMC Bioinformatics 2014 15(Suppl 10):P36. doi:10.1186/1471-2105-15-S10-P36 Cite this article as: Mitchell et al.: Development of large-scale metabolite identification methods for metabolomics. BMC Bioinformatics 2014 15(Suppl 10):P36. Development of large-scale metabolite identification methods for metabolomics Joshua M Mitchell1, Teresa W-M Fan2,3,4, Andrew N Lane2,3,4, Hunter NB Moseley1,3,4* From UT–KBRIN Bioinformatics Summit 2014 Cadiz, KY, USA. 11-13 April 2014 distributions based upon both molecular formula and dis- tinct composition of functional groups. Furthermore, these two databases have only moderate overlap in molecular formulae. Thus, it is prudent to use multiple databases in metabolite assignment, since each of the major metabolite databases represents different portions of metabolism within the biosphere. In silico analysis of various CS- tagging strategies under different conditions for adduct formation demonstrate that the combination of FT-MS derived molecular formulas and CS-tagging can signifi- cantly increase the unique identification of isotopologues based on the entries in KEGG and HMDB databases. © 2014 Mitchell et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Authors’ details 1Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40356, USA. 2Graduate Department of Toxicology, University of Kentucky, Lexington, KY 40356, USA. 3Markey Cancer Center, University of Kentucky, Lexington, KY 40356, USA. 4Resource Center for Stable Isotope Resolved Metabolomics, University of Kentucky, Lexington, KY 40356, USA. Mitchell et al. BMC Bioinformatics 2014, 15(Suppl 10):P36 http://www.biomedcentral.com/1471-2105/15/S10/P36 Mitchell et al. BMC Bioinformatics 2014, 15(Suppl 10):P36 http://www.biomedcentral.com/1471-2105/15/S10/P36 Background Large-scale identification of metabolites is key to elucidat- ing and modeling metabolism at the systems level. Advances in metabolomics technologies, particularly ultra- high resolution mass spectrometry enable comprehensive and rapid analysis of metabolites, which is impractical to achieve by conventional methods. However, a significant barrier to meaningful data interpretation is the identifica- tion of a wide range of metabolites including unknowns and the determination of their role(s) in various metabolic networks. Our recent development of chemoselective (CS) probes to tag metabolite functional groups provides addi- tional structural constraints for metabolite identification, but remains limited by the lack of functional group- resolved metabolite databases. Authors’ details 1Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40356, USA. 2Graduate Department of Toxicology, University of Kentucky, Lexington, KY 40356, USA. 3Markey Cancer Center, University of Kentucky, Lexington, KY 40356, USA. 4Resource Center for Stable Isotope Resolved Metabolomics, University of Kentucky, Lexington, KY 40356, USA. * Correspondence: hunter.moseley@uky.edu 1Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40356, USA Full list of author information is available at the end of the article © 2014 Mitchell et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Results An isomeric analysis of both HMDB and KEGG demon- strates a high percentage of isomeric molecular formulas, indicating the necessity of techniques such as CS-tagging with detection via MS and NMR to help assign specific metabolites and their isotopologue and isotopomer
https://openalex.org/W4251734217	https://www.revistas.usp.br/rbefe/article/download/170541/161080	Portuguese	null	Sinais de vigorexia e uso de suplementos alimentares por praticantes de musculação de São Miguel do Oeste/SC	Revista Brasileira de Educação Física e Esporte	2,019	cc-by	6,283	Resumo Este trabalho teve como objetivo identificar a presença de indícios de vigorexia, o estado nutricional, percepções sobre a imagem corporal e o uso de suplementos em adultos jovens praticantes de musculação. Foram aplicados dois instrumentos para a coleta de dados, sendo um questionário para a verificação da percepção da imagem corporal com abordagem do grau de vigorexia e uso de suplementos e um conjunto de silhuetas. Avaliou-se ainda, o estado nutricional dos praticantes de musculação através do cálculo do Índice de Massa Corporal (IMC), associado ao percentual de gordura corporal. Participaram do estudo 52 indivíduos do sexo masculino, com idade média de 25,06 anos (±4,83) e com estado nutricional em que predominou um percentual de gordura corporal abaixo da média (82,7%). A utilização de suplementos nutricionais foi encontrada em 51,9% da amostra, sendo as fórmulas proteicas as mais utilizadas. Observou-se que 7,7% dos praticantes foram classificados possuindo severos indícios de vigorexia. No entanto, 28,8% apresentaram pelo menos 70% da pontuação necessária para serem classificados com severos indícios do transtorno, o que demonstrou comportamentos alterados em relação a exigência de um corpo ideal. Além disso, 63,5% dos praticantes de musculação relataram que não estavam satisfeitos com sua imagem corporal, e 94,2% gostariam de apresentar silhueta dois pontos acima da que achavam possuir, ou seja, desejavam ganhar massa muscular. Apesar do baixo índice de indivíduos que apresentaram severidade nos graus de vigorexia, é necessária uma maior atenção dos profissionais de educação física e nutricionistas que realizam o acompanhamento em academias de musculação na investigação e prevenção de possíveis danos à saúde de indivíduos que apresentam algum comportamento ligado ao transtorno, objetivando que estes sejam brevemente percebidos. Palvras-chave: Estado Nutricional; Suplementação; Musculação; Imagem Corporal. Palvras-chave: Estado Nutricional; Suplementação; Musculação; Imagem Corporal. Sinais de vigorexia e uso de suplementos alimentares por praticantes de musculação de São Miguel do Oeste/SC Caroline MOROSINI* Eloá Angélica KOENHLEIN* http://dx.doi.org/10.11606/1807-5509201900030443 http://dx.doi.org/10.11606/1807-5509201900030443 Universidade Federal da Fronteira Sul, Rea leza, PR, Brasil. Palvras-chave: Estado Nutricional; Suplementação; Musculação; Imagem Corporal. Método A densidade corporal foi calculada a partir dos resultados de três dobras cutâneas: peitoral, abdominal e coxa medial, coletadas em rodízio e por meio de três repetições para cálculo da média. Avaliou-se o estado nutricional dos praticantes, a partir da estimativa de porcentagem de gordura corporal, por meio da equação de densidade corporal generalizada de Jackson e Pollock16, juntamente com a equação de Siri17. Os dados foram comparados com o padrão de gordura corporal para o sexo masculino proposto por Lohman18. Além disso, calculou-se o índice de massa corporal (IMC) (peso/estatura²) e utilizou-se os critérios de classificação da Organização Mundial da Saúde (OMS)19. A presente pesquisa apresentou natureza quantitativa, transversal, de caráter descritivo utilizando, para tal, dados primários. Foram convidados a participar da pesquisa adultos jovens do sexo masculino, praticantes de musculação de quatro academias da região central da cidade de São Miguel do Oeste/SC, no período entre 13 de janeiro à 09 de fevereiro de 2015. Os critérios de inclusão adotados foram: homens com massa muscular desenvolvida e aparente, com idade entre 19 a 35 anos, que praticavam musculação por um período maior que seis meses e com frequência de duas ou mais vezes por semana na academia. Os participantes da pesquisa foram identificados com o auxílio do profissional de educação física responsável pela academia. Após a coleta dos dados antropométricos, foi aplicado um questionário para a verificação da percepção da imagem corporal com abordagem do grau de severidade de vigorexia e uso de suplementos, juntamente com questões sobre dados sócio demográficos com o objetivo de conhecer o perfil dos praticantes avaliados. A pesquisa foi aprovada pelo Comitê de Ética e Pesquisa da Universidade Federal da Fronteira Sul (CAAE: 39600914.2.0000.5564), sendo que os indivíduos que aceitaram participar do estudo assinaram o Termo de Consentimento Livre Esclarecido. Para a realização das medidas antropométricas, foi utilizada balança antropométrica digital com capacidade de peso para 200 quilos da marca Marte®, estadiômetro com capacidade de 2 metros, com escala de 0,5 centímetros, fita antropométrica e adipômetro científico da marca Sanny® com precisão de 0,1mm. Todos os procedimentos foram realizados em local apropriado de forma individualizada, sendo que o avaliado encontrava-se com roupas leves, sem calçado e sem adornos nos bolsos e na cabeça. Introdução Atualmente verifica-se que a sociedade impõe padrões sobre o que os indivíduos devem usar e como devem apresentar seus corpos¹. A maioria destas informações são comercializadas pela mídia, a qual pode ser considerada uma das principais causadoras de alterações da percepção corporal, iludindo e fixando a visão de que certa imposição sobre um corpo musculoso e forte está associada com sucesso, felicidade e saúde². a preocupação e a procura pela busca do corpo parecido com o padrão de beleza imposto se mostra crescente em indivíduos do sexo masculino, deixando para trás o tempo em que as preocupações com a imagem corporal predominavam nas mulheres. No entanto, esta preocupação com o corpo se manifesta de maneira diferente, sendo de maior atenção o transtorno conhecido como vigorexia ou dismorfia muscular5-7. O transtorno da vigorexia se apresenta como uma preocupação obsessiva na obtenção de um corpo forte e musculoso, onde o indivíduo dedica várias horas do dia a prática de exercícios físicos, sendo geralmente visível a associação de práticas Devido a estas informações estarem disponíveis a diferentes grupos, independente de sua faixa etária, sintomas de transtornos alimentares como a bulimia e anorexia vieram crescendo em crianças e adolescentes³. Porém, segundo Mota e Aguiar4, RRev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 443 Morosini C & Hoenhlein EA. alimentares específicas, como as dietas hiperproteicas e o uso e abuso de drogas ergogênicas5,8-10.l sem prescrição e devidas informações por parte dos profissionais habilitados como nutricionistas e médicos13. Diante dos fatores que influenciam o aparecimento da vigorexia, a crescente procura por academias é notória, como é possível observar nos dados do SEBRAE11, em que o Brasil ocupa a segunda colocação no mundo em número de academias de ginástica, nas quais a musculação trata-se da atividade mais procurada12. Assim, diante do exposto, o diagnóstico da vigorexia se torna um desafio, pois muitas vezes passa despercebido pelos profissionais da área da saúde, já que visualmente o indivíduo se apresenta fisicamente saudável14. Logo, quanto mais informações e conhecimentos sobre os sintomas deste transtorno, mais fácil se torna sua identificação precoce e a prevenção de possíveis complicações à saúde desses indivíduos15. Introdução Outro aspecto que cabe salientar refere- se ao uso de suplementos alimentares, pois concomitantemente ao aumento da procura por academias, essa prática vem se mostrando muito comum entre frequentadores e principalmente entre praticantes de musculação, que em sua maioria utilizam suplementos alimentares e farmacológicos Portanto, diante do exposto, o objetivo do estudo foi verificar a presença de indícios de vigorexia e uso de suplementos alimentares por homens jovens praticantes de musculação. Método No questionário proposto eram presentes, o conjunto de silhuetas proposto por Lima et al.20, questões formuladas por Rodrigues, Araújo e Alencar21 e para investigação mais detalhada do uso de suplementos, perguntas formuladas pelas autoras, abordando a frequência de uso, quantidade e origem da prescrição dos suplementos. As perguntas que possuíam como objetivo a identificação de indícios de vigorexia, traziam em 444 • Rev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 Sinais de vigorexia e uso de suplementos alimentares sua resposta assinalada um coeficiente específico de acordo com o seu envolvimento e implicância nos indícios do transtorno, permitindo, assim, a partir do escore (baixo= <16 pontos e elevado= >16 pontos), determinar a presença ou não de indícios de vigorexia nos praticantes de musculação investigados. silhueta que gostaria que o seu corpo parecesse. E a terceira, pedia ao praticante para assinalar qual a silhueta que melhor representava a forma com que as outras pessoas o enxergavam. Portanto, quando a variação entre a silhueta ideal e real se mostrou igual à zero, os indivíduos foram classificados como satisfeitos com sua imagem corporal. Já quando a diferença mostrou- se positiva (ideal > real), considerou-se um desejo de aumentar a massa muscular. Para a verificação da percepção da imagem corporal foi utilizado o conjunto de silhuetas proposto por Lima et al.20, o qual possui nove silhuetas para cada pergunta que lhe é acompanhada, variando de um indivíduo mais magro e com pouca massa muscular para o mais musculoso. Os dados foram analisados por meio de estatística descritiva, utilizando-se programa Microsoft Excel 2010, sendo os resultados expressos como média, desvio-padrão, frequência absoluta e relativa. A primeira pergunta se referia a qual silhueta que melhor representava a aparência corporal atual do indivíduo. A segunda questionava-se sobre, qual a RRev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 445 Resultados A amostra do estudo foi composta de 52 praticantes de musculação do sexo masculino, com idade média de 25,06 anos (±4,83), peso de 78,71kg (±8,97) e estatura de 1,77m (±0,07). Na TABELA 1, estão representados os dados quanto ao perfil dos praticantes de musculação avaliados. Notou-se que a maior parte da amostra apresentava grau de instrução com nível superior completo, com tempo de prática de musculação entre 6 meses a menos que 2 anos, frequência de realização de musculação de 2 a 4 vezes na semana com uma duração de 1 hora ou menos. RRev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 4 n % Grau de escolaridade Ensino médio incompleto 1 2% Ensino médio completo 16 32% Ensino superior incompleto 15 29% Ensino superior completo 20 38% Patologias prévias Sim 3 5,8% Não 49 94,2% Tempo de prática de muscu lação Entre 6 meses < 2 anos 32 61,5% Entre 2 anos a < 4 anos 14 26,9% Período maior que 4 anos 6 11,5% Frequência da prática de musculação 2 a 4 vezes na semana 37 71,2% 5 a 6 vezes na semana 15 28,8% Duração da sessão de muscu lação 1:00h ou menos 27 51,9% 1:00h a 2:00h 23 44,2% >2:00h 2 3,9% TABELA 1- Perfil dos praticantes de musculação de academias de São Miguel do Oeste/SC. Asma dos praticantes de musculação de academias de São Miguel do Oeste/SC. TABELA 1- Perfil dos praticantes de musculação de academias de São Miguel do Oeste/SC. Morosini C & Hoenhlein EA. Com relação a avaliação antropométrica, encontrou-se um valor médio para IMC de 25,12kg/m² (+2,67) e 9,19% de gordura corporal. Além disso, foi possível observar que 50% da amostra encontrava-se com sobrepeso e obesidade, e 82,7% com percentual de gordura corporal abaixo da média (TABELA 2). Esse resultado é característico em praticantes de musculação, pois o excesso de peso apontado pelo IMC pose ser explicado por meio da composição corporal, destacando a presença de massa muscular aumentada e baixo acúmulo de gordura. No que diz respeito aos suplementos alimentares, afirmaram fazer dos mesmos 51,9% (27) dos indivíduos, com frequência de uso de até 5 dias na semana e consumo de uma vez ao dia. Ainda, percebeu-se que a associação de fórmulas foi bastante comum na amostra estudada, sendo as fórmulas proteicas as mais citadas (TABELA 3). Resultados TABELA 2 - N % IMC <18,5kg/m² - Magreza 0 0% 18,6 - 24,9kg/m² - Eutrofia 26 50% 25 – 29,9kg/m² - Sobrepeso 23 44,2% >30kg/m² - Obesidade 3 5,7% % de Gordura Corporal Risco (<5%) 7 13,4% Abaixo da média (6 – 14%) 43 82,7% Média (15%) 0 0% Acima da média (16 – 24%) 2 3,9% Estado nutricional dos praticantes de musculação de academias de São Miguel do Oeste/SC de acordo com IMC e percentual de gordura corporal19,18. stado nutricional dos praticantes de musculação de academias de São Miguel do Oeste/SC de acordo om IMC e percentual de gordura corporal19,18. TABELA 2 -Estado nutricional dos praticantes de musculação de academias de São Miguel do Oeste/SC de acordo com IMC e percentual de gordura corporal19,18. TABELA 3 -Características do uso de suplementos alimentares por praticantes de musculação de academias de São Miguel do Oeste/SC. n % Associação de fórmulas 1 10 37% 2-3 11 40,8% 4-5 3 11,1% >5 3 11,1% Suplementos utilizados Proteicos 26 96,3% Ergogênicos** 13 48,1% Carboidratos¨ 6 22,2% Polivitamínicos 5 18,5% Outros° 3 11,1% Frequência do uso de suplementação diária Uma vez ao dia Antes 8 29,7% Depois 9 33,3% 2 vezes ao dia Antes e depois 4 14,9% 3 vezes 0 0% Não soube relatar 6 22,2% Whey Protein; Albumina; Caseína; Arginina; Glutamina; Aminoácidos de cadeia ramificada (BCAA). ¨Maltodextrina; Dextrose; Waxy Maize. *Creatina; cafeína. °Glucosamina; óxido nítrico (NO²). TABELA 3 -Características do uso de suplementos alimentares por praticantes de musculação de academias de São Miguel do Oeste/SC. TABELA 3 -Características do uso de suplementos alimentares por praticantes de musculação de academias de São Miguel do Oeste/SC. receberam indicação apenas de profissionais da educação física, 7% de nutricionista, e amigos e propagandas apresentaram percentuais abaixo de 4%, demonstrando baixa procura de indicação por profissionais habilitados e possíveis erros na melhor forma de utilização. Quanto a motivação para o uso de suplementos, 37% dos indivíduos relataram iniciar a suplementação por conta própria e 25,9% apontaram receber mais de uma indicação, seja ela por amigo, nutricionista, propagandas, profissional da educação física e vendedor de loja de suplementos. Observou-se ainda que 22,2% Por meio da análise da percepção corporal 446 • Rev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 Sinais de vigorexia e uso de suplementos alimentares tendência dos indivíduos acreditarem serem vistos com uma silhueta semelhante a descrita com a atual. Resultados No entanto, os mesmos idealizaram uma silhueta 2 pontos acima da descrita como atual, estabelecendo como a silhueta mais desejada a de número 7 (51,92%), seguindo a silhueta 6 (25%) e 5 (15,38%), como apresentado no FIGURA 1. utilizando-se as silhuetas, constatou-se que a maioria dos praticantes de musculação gostariam de apresentar uma silhueta maior do que a que eles achavam que possuíam, sendo a silhueta média apontada como atual de 4,26, para o corpo desejado de 6,36, e para o modo com que eles achavam ser vistos com a média de 4,57. Assim, notou-se uma Frequência de percepções da imagem corporal de praticantes de musculação de São Miguel do Oeste-SC. FIGURA 1- Frequência de percepções da imagem corporal de praticantes de musculação de São Miguel do Oeste-SC. Na investigação de indícios de vigorexia (TABELA 4), verificou-se que grande parte dos praticantes de musculação relataram se sentirem culpados quando não iam à academia (71,15%), que 42,2% dos indivíduos avaliados já haviam realizado sessões de musculação com presença de lesões, e que 63,46% declararam sentirem-se insatisfeitos com sua forma física. grande parte dos indivíduos de realização de uma dieta específica (69,23%), apontando que o consumo de suplementação possa estar à frente de uma dieta saudável e equilibrada em qualidade e quantidades suficientes de acordo com as necessidades nutricionais específicas. Notou-se ainda uma baixa frequência de relatos da utilização de ciclos de esteroides anabólicos (1,92%), o que pode estar associado ao número pequeno da amostra, como também a omissões de respostas. Enfatiza-se ainda, a relação entre a frequência de suplementação (51,9%) com a ausência em Frequência absoluta e relativa das respostas do questionário de indícios de vigorexia de praticantes de musculação de academias de São Miguel do Oeste/SC. TABELA 4 -Frequência absoluta e relativa das respostas do questionário de indícios de vigorexia de praticantes de musculação de academias de São Miguel do Oeste/SC. TABELA 4 -Frequência absoluta e relativa das respostas do questionário de indícios de vigorexia de praticantes de musculação de academias de São Miguel do Oeste/SC. Indícios de vigorexia SIM NÃO n % n % A musculação atrapalha na sua vida social ou familiar? 5 9,61% 47 90,38% Você faz uso de suplementos alimentares? 27 51,93% 25 48,07% Você faz uso de algum tipo de dieta especifica para seu treinamento? RRev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 447 Resultados 16 30,76% 36 69,23% continua RRev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 447 RRev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 447 RRev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 447 Morosini C & Hoenhlein EA. Indícios de vigorexia SIM NÃO n % n % Você já fez algum ciclo de esteroides anabolizantes? 1 1,92% 51 98,07% Você se sente culpado quando não vai à academia? 37 71,15% 15 28,84% Você esta satisfeito com sua forma física? 19 36,53% 33 63,46% Você já treinou lesionado? 22 42,30% 30 57,69% Mesmo sabendo das enfermidades ocasio nadas pelo excesso de treinamento você esta disposto a correr esse risco? 28 53,84% 24 46,15% Você passa muito tempo na frente do espe lho admirando seu corpo? 7 13,46% 45 86,53% continuação continuação entanto, quinze praticantes (28,84%), obtiveram uma pontuação de 70% da necessária para se classificarem como portadores de indícios de vigorexia, ou seja, uma pontuação de 11 a 15 pontos, demonstrando que alguns dos sintomas característicos do transtorno já são visualizados nos praticantes estudados. Na TABELA 5 são apresentados em frequência relativa e absoluta os resultados da somatória do questionário de indícios de vigorexia. Foi possível verificar que quatro indivíduos (7,69%) foram enquadrados com indícios de vigorexia, correspondendo a uma pontuação igual ou maior que 16 pontos. No TABELA 5 -Indícios de vigorexia em praticantes de musculação de academias de São Miguel do Oeste/SC. Indícios de vigorexia n % Indivíduos que apresentaram baixo indício de vigorexia (< que 11 pontos) 33 63,46% Indivíduos com pelos menos 70% da pontuação (11- 15 pontos) 15 28,84% Indivíduos com indícios de vigorexia (maior ou igual a 16 pontos) 4 7,69% TABELA 5 -Indícios de vigorexia em praticantes de musculação de academias de São Miguel do Oeste/SC. Indícios de vigorexia n % Indivíduos que apresentaram baixo indício de vigorexia (< que 11 pontos) 33 63,46% Indivíduos com pelos menos 70% da pontuação (11- 15 pontos) 15 28,84% Indivíduos com indícios de vigorexia (maior ou igual a 16 pontos) 4 7,69% vigorexia em praticantes de musculação de academias de São Miguel do Oeste/SC. 448 • Rev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 Discussão Portanto, os dados encontrados no estudo podem se tornar preocupantes quando visualizado o uso de forma errônea, em grandes quantidades e ainda sua real necessidade de uso, já que os mesmos são destinados para atender as necessidades nutricionais específicas de atletas e auxiliar no desempenho do exercício37. O uso de suplementos alimentares frequente por praticantes de musculação, associado à ausência de prescrição e devidas informações por parte dos profissionais habilitados como nutricionistas e médicos também foi observado na maioria dos participantes desse estudo. Resultados que também se mostraram elevados nos estudos de Fayh et al.13, no qual 39,1% dos homens praticantes de musculação faziam o uso de suplementos, e Rosa e Perina31 e Nogueira et al.32, nos quais mais da metade dos praticantes avaliados em seus estudos faziam o uso dos mesmos. Devido a isso, destaca-se a importância do profissional nutricionista na avaliação e na prescrição de uma dieta e suplemento que atenda a real necessidade de cada indivíduo, não gerando exageros e consequentemente o aparecimento de possíveis complicações ocasionadas pelo excesso de suplementação38. Outro ponto de extrema relevância, quando se trata de saúde e nutrição, é o conhecimento da fonte de indicação e prescrição, ou seja, se a utilização do suplemento foi baseada em uma indicação por profissional habilitado, já que muitos estudos vêm demonstrando a elevada prevalência da auto prescrição39,31. Dentre os suplementos mais consumidos, observou-se através de estudos como o de Gomes et al.33 que os mais utilizados por praticantes de musculação foram a base de proteína, especialmente o BCAA (aminoácidos de cadeia ramificada) e whey protein (proteína do soro do leite). Contribuindo com essa afirmação, Goston34 apresentou que os suplementos que possuíam maior consumo pelos praticantes de atividades físicas também foram os ricos em proteínas, sendo este resultado também relatado por Nogueira et al.32. Neste estudo, os resultados encontrados não se mostram distintos aos estudos observados, podendo haver o consumo em horários inapropriados, em quantidades e necessidades inadequadas prejudicando a saúde e o alcance dos objetivos40. A análise da percepção corporal por meio das silhuetas demonstrou que 94% dos praticantes estavam insatisfeitos com sua imagem corporal, pois desejavam aumentar ainda mais a musculatura, apontando como a silhueta mais desejada a de número 7, correspondendo às características de 26,1 kg/m² de IMC e 2,4% de gordura corporal20. Discussão Em relação ao estado nutricional, metade dos praticantes avaliados apresentaram excesso de peso de acordo com o IMC, e percentual de gordura corporal abaixo da média, ou seja, apresentavam massa muscular aumentada, semelhante aos estudos de Sehnem e Soares26, Theodoro, Ricalde e Amaro27 e Coelho et al.28, nos quais os participantes dos estudos possuíam as mesmas características físicas. A elevada escolaridade dos praticantes de musculação avaliados já foi descrita na literatura como característica importante nessa população. Dados que Ristow et al.22 demonstraram que 60,6% de sua amostra apresentavam mais de doze anos de estudo. Também verificou-se que indivíduos com maior escolaridade apresentam maior prevalência de atividade física23. De acordo com Okuma apud Ortiz, Isler e Darido24, indivíduos que apresentam maior nível de escolarização e maior renda familiar, apresentam melhores índices de adesão e a permanência em programas de exercício físico, do que aqueles que não apresentam estas características. Colaborando com esta afirmação Saba25, mostra que o padrão de vida e o nível de informação são fatores que interferem no inicio, na manutenção e no abandono de qualquer atividade física. Quanto ao tempo de prática, observou-se que a maioria dos praticantes estavam inseridos em um programa de musculação por um período entre 6 meses a menos que 2 anos, com regularidade de até 4 vezes na semana, caracterizando a amostra por frequentadores assíduos. Semelhante ao estudo de Ristow et al.22, onde 54,8% dos praticantes relataram praticar musculação mais de três vezes por semana, porém diferente ao encontrado por Santos 448 • Rev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 Sinais de vigorexia e uso de suplementos alimentares e Santos29, onde 56% dos avaliados realizavam cinco treinos semanalmente. associação da prática de musculação, colocando em segundo plano uma alimentação saudável e adequada, a qual além de benefícios a saúde colabora na obtenção dos resultados. A maior parte dos praticantes estudados relataram uma duração de treino curta, o que pode estar associado a ideia de que treinos mais curtos e intensos são mais efetivos. Porém, o uso de forma incorreta dessas estratégias podem ocasionar lesões e improdutividade quando a realização de treinos com cargas muito elevadas30. Discussão Dados semelhantes aos encontrados foram observados no estudo de Castanheira41, no qual utilizando a mesma escala de silhuetas, verificou que 89,7% dos indivíduos participantes desejavam um corpo mais musculoso, ou seja, encontravam-se insatisfeitos com sua imagem corporal, desejando uma silhueta dois pontos acima da atual. Além do número elevado dos praticantes estudados que referiram fazer o uso de suplementos, a maioria (55,5%) alegou utiliza-los até cinco vezes na semana e uma vez ao dia no horário após a sessão de musculação, semelhante ao que foi encontrado por Moreira, Navarro e Navarro35, onde 48,5% dos praticantes faziam o uso da suplementação diariamente. Sabe-se que a associação de uma dieta balanceada com a prática regular de atividade física pode ser um importante fator para a melhora da saúde, porém se torna benéfica quando tudo isso se apresenta em quantidades normais, melhorando a qualidade de vida e a saúde, maximizando o bem estar evitando a fadiga, as lesões e otimizando os depósitos de energia36,29. Contudo, apesar da maioria dos indivíduos fazerem o uso de suplementação, notou- se uma baixa frequência em relação ao uso de dieta específica para o treinamento, não evidenciando possíveis alterações de hábitos alimentares com a Em estudo de Zimmermann12, no qual foram investigados 61 adultos jovens praticantes de musculação do sexo masculino também foi encontrada a insatisfação com a imagem corporal em 95,08% dos indivíduos, apresentando ainda um desejo de alcançar uma silhueta em média dois pontos maiores do que a real. Assim, destaca-se a crescente preocupação entre os homens em relação RRev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 449 Morosini C & Hoenhlein EA. à procura de exercícios de fortalecimento muscular associada à melhora de sua aparência, demonstrando que a insatisfação corporal está bastante presente42. Porém, é aceitável que os indivíduos preocupem- se com sua aparência e queiram cuidar de si mesmos, demonstrando alguma insatisfação corporal. Contudo, quando esse objetivo torna-se uma obsessão, o meio pode se tornar favorável para o aparecimento da distorção de imagem, o qual sintoma é presente na vigorexia43. do baixo índice de indivíduos que apresentaram severidade nos graus de vigorexia, deve-se levar em consideração que aproximadamente 30% foram classificados como portadores de alguns sinais do transtorno, classificando a maioria da amostra como insatisfeitos com a própria imagem corporal. à procura de exercícios de fortalecimento muscular associada à melhora de sua aparência, demonstrando que a insatisfação corporal está bastante presente42. Discussão Porém, é aceitável que os indivíduos preocupem- se com sua aparência e queiram cuidar de si mesmos, demonstrando alguma insatisfação corporal. Contudo, quando esse objetivo torna-se uma obsessão, o meio pode se tornar favorável para o aparecimento da distorção de imagem, o qual sintoma é presente na vigorexia43. Além disso, observou-se o uso de suplementos alimentares sem prescrição por profissional habilitado, o que pode gerar possíveis complicações em função do excesso de nutrientes suplementados, sem avaliação de sua real necessidade. Ainda, cabe destacar, que a maioria dos praticantes não associavam a prática da atividade física com mudanças de hábitos alimentares específicos, o que pode demonstrar que a muitas vezes a suplementação está à frente de uma alimentação adequada de acordo com suas necessidades, colocando os suplementos como destaque na dieta. O questionário para identificação de possíveis indícios de vigorexia demonstrou que apenas 7,69% dos indivíduos apresentaram severos indícios do transtorno. Contudo, 15 praticantes, ou seja, 28,84% obtiveram pontuação correspondente a 70% da necessária para apresentar indícios elevados de vigorexia, enquadrando-os com comportamentos alterados em relação à exigência de um corpo ideal. Diferente ao encontrado por Zimmermann12 e Silva et al.44, onde respectivamente, 23% e 38% dos entrevistados foram classificados como portadores de severos indícios do transtorno. Contudo, cabe ressaltar que, apesar do número pequeno de indivíduos que apresentaram severos indícios de vigorexia na amostra, boa parte apresentou alguns comportamentos relacionados ao transtorno, como chama-se a atenção para o fato de que 71,15% dos pesquisados afirmaram se sentirem culpados quando não frequentavam a academia, 63,46% não se sentiam satisfeitos com a sua forma física e 42,30% afirmaram que já treinaram lesionados. Deste modo, acredita-se ser necessária uma maior atenção dos profissionais de educação física e nutricionistas que realizam o acompanhamento em academias de musculação na investigação e prevenção de possíveis danos à saúde de indivíduos que de alguma forma, leve ou expressiva apresentam algum comportamento ligado ao transtorno, objetivando que estes sejam brevemente percebidos. Sendo assim, como sugestão para futuros trabalhos desta natureza, destaca-se a avaliação dietética destes indivíduos, podendo deste modo verificar de forma mais aprofundada o consumo alimentar e observar possíveis falhas de excesso e deficiências nutricionais. Portanto, conclui-se através do estudo que apesar Abstract Signs of vigorexy and use of dietary supplementation among bodybuilders in the city of São Miguel do Oeste/SC Signs of vigorexy and use of dietary supplementation among bodybuilders in the city of São Miguel do Oeste/SC The aim of this study was to identify the presence of vigorexy evidences, the nutritional status, body image perceptions and the use of supplements in young adults’ bodybuilders. Two instruments for data collection were applied, one questionnaire to verify the body image perception with the degree of vigorexy approach and use of supplements and a silhouettes set. It was also evaluated the nutritional status of bodybuilders by calculating the Body Mass Index (BMI), together with the percentage of body fat. The study included 52 males with a mean age of 25.06 years (± 4.83) and nutritional status that prevailed a percentage of body fat below average (82.7%). The use of nutritional supplements was found in 51.9% of the sample, the protein formulas were the most commonly used. It was observed that 7.7% of practitioners were classified having severe evidences of vigorexy. However, 28.8% had at least 70% of points required to be classified with severe evidence of trouble, which demonstrated altered behaviors from the requirement of an ideal body. In addition, 63.5% of body builders reported that they were not satisfied with their body image, and 94.2% would like to present silhouette two points above that thought has, or wanted to gain muscle 450 • Rev Bras Educ Fís Esporte, (São Paulo) 2019; Jul-Set 33(3):443-452 Sinais de vigorexia e uso de suplementos alimentares mass. Despite of the low rate of individuals that presenting severe degrees of vigorexy, it is necessary a greater attention by physical educators and nutritionists who do the monitoring in fitness centers in the investigation and prevention of possible damage to the individual health who have some behavior related to the disorder, aiming that they are perceived soon. mass. Despite of the low rate of individuals that presenting severe degrees of vigorexy, it is necessary a greater attention by physical educators and nutritionists who do the monitoring in fitness centers in the investigation and prevention of possible damage to the individual health who have some behavior related to the disorder, aiming that they are perceived soon. Keywords: Nutritional State; Supplementation; Bodybuilding; Body Image. Referências 1. Camargo TPP, Costa SPV, Uzunian LG, Viebig RF. Vigorexia: revisão dos aspectos atuais deste distúrbio de imagem corporal. Rev bras psicol esporte. 2008;2(1):1-15. 2. Conti MA, Frutuoso MFP, Gambardella AMD. Excesso de peso e a insatisfação corporal em adolescentes. Rev Nutr. 2005;18:491-497. 3. Campagna VN, Souza ASL. Corpo e imagem corporal no inicio da adolescência feminina. Boletim de Psicologia. 2006;56:9-35. 4. Mota CG, Aguiar EF. Dismorfia Muscular: uma nova síndrome em praticantes de musculação. Rev Bras Ciênc Saúde. 2011;27:49-56. 5. 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https://openalex.org/W3049746024	https://hal.science/hal-03034344/document	English	null	Steady-state heat transfer in microcracked media	Mechanics & industry	2,020	cc-by	6,713	Steady-state heat transfer in microcracked media Sharan Raj Rangasamy Mahendren, Hélène Welemane, Olivier Dalverny, Amèvi Tongne Steady-state heat transfer in microcracked media Sharan Raj Rangasamy Mahendren, Hélène Welemane, Olivier Dalverny, Amèvi Tongne To cite this version: Sharan Raj Rangasamy Mahendren, Hélène Welemane, Olivier Dalverny, Amèvi Tongne. Steady- state heat transfer in microcracked media. Mechanics & Industry, 2020, 21 (5), pp.2-11. 10.1051/meca/2020034. hal-03034344 HAL Id: hal-03034344 https://hal.science/hal-03034344v1 Submitted on 1 Dec 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible This is an author’s version published in: http://oatao.univ-toulouse.fr/26761 Official URL: https://doi.org/10.1051/meca/2020034 1 Introduction [9–11]. For steady-state heat conduction, Sevostianov [12] and others [13,14] apply the direct methodology based on temperature jump across insulating crack lips. For Hoenig [15], Hatta and Taya [16], Benveniste and Miloh [17] and more recently Shafiro and Kachanov [18], the equivalent inclusion method appears again as a key issue. While several studies account for the arbitrary value of matrix/inclusion conductivity and arbitrary crack’s ori- entation or shape, most of the existing papers generally provide thermal conductivity of microcracked media in the non-interacting case. Nguyen et al. [19] give closed- form expression for different schemes but consider only one orientation of the crack. Nevertheless, that is not the only challenge. Opening or closing of microcrack (also known as unilateral effect) can have a different influence on the material, in turn on the overall properties. Conse- quences of both induced anisotropy and unilateral effect on the elastic problem have been studied by few authors [1,20], but the same cannot be said for the heat conduction problem. Defects have an influence on the macroscopic behaviour of a material, each on a different scale. The overall behaviour of the material can be characterized by its microstruc- ture. This transition from micro-to-macro can be modelled using averaging techniques (homogenization) in order to derive the effective properties of a material. Homogenization studies often concentrate on the elas- tic behaviour of a microcracked material. In so-called direct methodology, cracks are represented as material dis- continuities with parallel faces. The displacement jumps induced by the cracks allow deriving their contribu- tion to the overall response. For instance, Kachanov [1] and Nemat-Nasser and Hori [2] have provided effective stiffness expressions for arbitrarily oriented microcracks. Eshelby’s equivalent inclusion method [3] also offers rele- vant solutions when considering cracks as flat ellipses (in 2D) or ellipsoids (in 3D). For instance, Mura [4] has stud- ied various ellipsoidal limit cases and Mori and Tanaka [5] have enhanced the representation to the case of mul- tiple interacting inhomogeneities. Note that energy-based bounds developed by Ponte Casta˜neda and Willis [6] allow accounting for different spatial cracks distribution. The modelling of the steady-state behaviour within microcracked media can also be achieved through numer- ical simulation. Carson et al. [21] apply Finite Element Method (FEM) to find the conductivity of non-insulated porous of various shapes and sizes, while Tang et al. [22] propose a similar modelling for concrete with con- ductive heterogeneities. Shen et al. Received: 23 October 2019 / Accepted: 17 April 2020 Abstract. Material behaviour is often affected by the heterogeneities existing at the microscopic level. Especially the presence of cracks, voids, etc collectively known as defects, can play a major role in their overall response. Homogenization can be used to study the influence of these heterogeneities and also to estimate the effective properties of a given material. Several research works have been dedicated to determining the elastic behaviour of microcracked media. Yet, thermal properties are not investigated as much. Moreover, the question of unilateral effect (opening/closing of cracks) still remains an important issue. So, this paper aims to provide the effective thermal conductivity of 2D microcracked media with arbitrarily orientated cracks, either open or closed. With the help of Eshelby-like approach, homogenization schemes (dilute and Mori-Tanaka) and bounds (Ponte Casta˜neda-Willis) are developed to provide the closed-form expressions. In addition, these results are compared to numerical simulations performed based on finite element modelling. Keywords: Homogenization / heat conduction / microcracking / unilateral effect / finite element method Keywords: Homogenization / heat conduction / microcracking / unilateral effect / finite element method To cite this version: Rangasamy Mahendren, Sharan Raj and Welemane, Hélène and Dalverny, Olivier and Tongne, Amèvi Steady-state heat transfer in microcracked media. (2020) Mechanics & Industry, 21 (5). 2-11. Any correspondence concerning this service should be sent to the repository administrator: tech-oatao@listes-diff.inp-toulouse.fr Scientific challenges and industrial applications in mechanical engineering Philippe Le Grognec (Guest Editor) Scientific challenges and industrial applications in mechanical engineering Philippe Le Grognec (Guest Editor) ∗e-mail: helene.welemane@enit.fr This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Steady-state heat transfer in microcracked media Sharan Raj Rangasamy Mahendren, H´el`ene Welemane∗, Olivier Dalverny, and Am`evi Tongne Universit´e de Toulouse; INP/ENIT; LGP; 47 Avenue d’Azereix, 65016, Tarbes, France Received: 23 October 2019 / Accepted: 17 April 2020 Mechanics &Industry Mechanics & Industry 21, 519 (2020) Mechanics & Industry 21, 519 (2020) © S.R. Rangasamy Mahendren et al., Published by EDP Sciences 2020 https://doi.org/10.1051/meca/2020034 Mechanics & Industry 21, 519 (2020) © S.R. Rangasamy Mahendren et al., Published by EDP Sciences 2020 https://doi.org/10.1051/meca/2020034 Available online at: www.mechanics-industry.org Available online at: www.mechanics-industry.org 2 Theoretical framework where λ is the local symmetric second-order thermal conductivity tensor. Current works on the effective thermal properties are influenced by the similarities between elasticity and steady-state heat conduction variables [8]: The matrix is considered to be isotropic, continuous and has the thermal conductivity tensor λm = λm I (λm is the scalar thermal conductivity and I is the second-order identity tensor). This matrix is weakened by randomly distributed single-family of parallel microcracks (Fig. 1a). These cracks are modelled as a flat oblate ellipse (mean semi-axes a and c with c ≪a; Fig. 1b) with unit normal n and 2D volume fraction fc = π d ω. Here d = Na2 is the scalar crack density (N is the number of cracks per unit area) as defined by Budiansky and O’Connell [24] and ω = c/a ≪1 is their mean aspect ratio. Elasticity Heat conduction stress σ heat flux q strain ε temperature gradient g stiffness C thermal conductivity λ Hooke’s law Fourier’s law Elasticity Heat conduction stress σ heat flux q strain ε temperature gradient g stiffness C thermal conductivity λ Hooke’s law Fourier’s law Let denote Ωthe area of the 2D Representative Volume Element (RVE) of the microcracked media, ∂Ωits outer boundary and u the outward unit normal to ∂Ω(Fig. 1a). The macroscopic temperature gradient G (respectively heat flux Q) can be defined as the mean temperature (resp. external heat flux) on the boundary ∂Ω. Under sta- tionary thermal conditions, the macroscopic temperature gradient G (resp. heat flux Q) corresponds to the average of the corresponding microscopic quantity g (resp. q): A uniform macroscopic temperature gradient G is imposed at the outer boundary δΩof the RVE. Assuming an initial natural state, the microscopic and macroscopic quantities can be linked linearly as [25]: g(x) = A(x) · G, ∀x ∈Ω (4) (4) where A is the second-order gradient localization tensor. Similar to (3), the overall behaviour of the RVE can be given by: G = 1 \|Ω\| Z ∂Ω T(x) u(x) dL = 1 \|Ω\| Z Ω g(x) dΩ= ⟨g⟩ (1) G = 1 \|Ω\| Z ∂Ω T(x) u(x) dL = 1 \|Ω\| Z Ω g(x) dΩ= ⟨g⟩ (1) Q = 1 \|Ω\| Z ∂Ω q(x) · u(x) x dL = 1 \|Ω\| Z Ω q(x) dΩ= ⟨q⟩(2) Q = −λhom · G (5) (5) where λhom is the overall thermal conductivity of the microcracked media. 1 Introduction [23] use a plastic damage model to create cracks under tensile load and then consider steady-state conduction to find the conduc- tivity of the microcracked concrete with high aggregate Based on the physical analogy with elasticity (as in pio- neering works of Bristow [7], see also [8]), some authors have extended these modelling approaches to thermal, electrical and permeability properties of cracked media S.R. Rangasamy Mahendren et al.: Mechanics & Industry 21, 519 (2020) 2 (a) (b) Fig. 1. (a) RVE with single family of parallel microcracks, (b) crack geometry. (a) volume. One can cite also works of Tran et al. [14] based on an adaptive scheme Boundary Element Method (BEM) to find conductivity of a domain containing sev- eral cracks. Once again, we note that the crack orientation and unilateral effect are not given enough attention. The present work intends to propose an Eshelby-like modelling approach for the steady-state heat transfer in a 2D microcracked medium. The effective thermal con- ductivity is derived based on the geometry of cracks considered as thin aspect ratio inclusions, and on the rel- evant choice of cracks properties according to their status (open or closed). The theoretical basis of the 2D linear thermal problem is stated in Section 2. As a demonstra- tion, for different estimations (dilute and Mori-Tanaka schemes and Ponte Casta˜neda-Willis bound), closed-form expressions for a single-family of parallel cracks are pre- sented in Section 3. In addition to the analytical solution, we also propose a numerical analysis of the thermal prob- lem by means of finite element simulations. Modelling and description of the simulated area are given in Section 4. The results obtained from micromechanics and numer- ical simulation are finally compared and discussed in Section 5. (a) (a) (b) (b) Fig. 1. (a) RVE with single family of parallel microcracks, (b) crack geometry. locally follow the Fourier’s linear thermal law: q(x) = −λ(x) · g(x), ∀x ∈Ω (3) (3) 2 Theoretical framework Assuming the condition ⟨A⟩= I and (4), the effective thermal conductivity of the microcracked media comes to: Q = 1 \|Ω\| Z ∂Ω q(x) · u(x) x dL = 1 \|Ω\| Z Ω q(x) dΩ= ⟨q⟩(2) where T(x), g(x) and q(x) respectively represent the local temperature, local temperature gradient and local heat flux at any point x of Ω. λhom = λm + fc (λc −λm) · ⟨A⟩c (6) (6) where ⟨·⟩r= 1 Ωr R Ωr · dΩdenotes the mean value over the area Ωr of the phase r for r = {m, c}. The RVE studied here includes two phases (matrix and microcracks), where each phase is homogeneous and S.R. Rangasamy Mahendren et al.: Mechanics & Industry 21, 519 (2020) 3 where tensor R is defined as: where tensor R is defined as: The single-inhomogeneity problem studied by Eshelby [3] considers a single ellipsoidal inclusion embedded inside an infinite matrix subjected to macroscopic stress or strain tensors at infinity. This elasticity problem can be extended to thermoelasticity [8,26]. Here, matrix and cracks exhibit matrix-inclusion topology. In such case, the local tem- perature gradient in the crack can be approximated by the uniform local field obtained for an ellipsoid embed- ded in an infinite matrix subjected to uniform boundary conditions G∞. R (ω, ξ) = ω ! 1 −ξ h I −SE ! 1 −ξ i−1 , ∀ω ≪1, ∀ξ (10) (10) ( ) The above equation is valid for all the mean aspect ratio ω ≪1 and all the ratio ξ of scalar conductivity between defects and matrix. The present study focuses on the case of flat ellipse-shaped microcracks (c ≪a) for which aspect ratio tends to zero. Besides, we intend to account for different crack status: Taking all this into account, the estimated solutions for localization tensor over the crack’s phase ⟨A⟩c can be determined. And they depend on the depolarization tensor SE (similar to the Eshelby tensor of the elastic problem) whose factors are given in [8]. In our case, the said tensor for a flat oblate ellipse can be given as: – open cracks: one has λc = 0, so ξ = 0 and R (ω →0, ξ = 0) = n ⊗n, – closed crack: one has λc = λ∗̸= 0, so ξ ̸= 0 and R (ω →0, ξ ̸= 0) = 0. 2 Theoretical framework SE (ω) = 1 1 + ω n⊗n + ω 1 + ω (I−n⊗n), ∀ω ≪1 (7) Accordingly (9) can be simplified into: Accordingly (9) can be simplified into: (7) ( ) Note that the configuration of flat cracks corresponds to the limit case where ω →0, which has to be introduced only at the very end of the mathematical developments. As mentioned before, the unilateral effect is one of the main focus of this work. So, two different results can be obtained at the end, based on the state of the crack (open or closed). In either case, cracks are assumed to be isotropic λc = λc I (λc is the crack’s scalar thermal con- ductivity) with a different value of λc depending on the state of the crack: λdil hom = ( λm · h I −π d n ⊗n i , if cracks are open λm , if cracks are closed (11 (11) ( ) When we are to consider some interactions between cracks, the Mori-Tanaka scheme may provide an inter- esting solution [5]. The boundary condition here is given by G∞= ⟨g⟩m and the localization tensor reads: – when the cracks are open, λc = 0, which supports the adiabatic conditions on the crack lips, – when the cracks are open, λc = 0, which supports the adiabatic conditions on the crack lips, ⟨A⟩MT c = ⟨A⟩dil c · h (1 −fc) I + fc ⟨A⟩dil c i−1 (12) (12) – when the cracks are closed, λc = λ∗, which accounts for some level of heat transfer continuity; this assumption is inspired by the works of Deud´e et al. [20], where closed cracks are represented by a fictitious isotropic material with scalar conductivity λ∗̸= 0. – when the cracks are closed, λc = λ∗, which accounts for some level of heat transfer continuity; this assumption is inspired by the works of Deud´e et al. [20], where closed cracks are represented by a fictitious isotropic material with scalar conductivity λ∗̸= 0. This leads to: λMT hom = λm · h I + π d R i−1 (13) (13) 3 Calculation of the effective thermal conductivity Since we want to study the influence of the crack’s orientation on con- ductivity, further simulations are done by rotating the whole group of cracks which maintains a constant dis- tance between them for all orientations. To be precise, the so-called Reference Configuration (RC) corresponds to the distribution of cracks rather grouped near the cen- tre of A to reduce edge effects (Fig. 2). While keeping the circular spatial distribution, other configurations are also studied in the following to show the influence of cracks’ location. λP CW hom = λm · " I −π d R · I + π d 2 R −1# (16) (16) Based on the state of the flat defects, one gets: λP CW hom =      λm · I −π d 1 1 + π d 2 n ⊗n , if cracks are open λm , if cracks are closed (17) The generalized scalar conductivity of a material λ(v) related to the direction of unit vector v is defined by: , if cracks are closed (17) l ( ) Note that PCW bound will provide the same result as dilute and MT schemes when no spatial distribution is considered for the former and elliptical distribution for the latter [27]. λ(v) = v · Q v · G (18) (18) when the material is subjected to uniform temperature gradient G = Gv v. For the numerical model, zero flux condition is imposed on the left and right edges (with outer normals ± t) of area A. At the same time, tem- peratures T1 and T2 (∆Tv = T1 −T2 > 0) are applied respectively on the top and bottom edges (with outer normals ± v) of the cell; the temperature on each side is uniform. Such latter boundary condition, namely tem- ∆T Some main comments can be made regarding these theoretical developments. First, the three modelling approaches show crack induced anisotropy for open cracks. Yet, equations (11), (14) and (17) provide dif- ferent expressions of the effective conductivity tensor through the tensorial term n ⊗n. We also observe that as d →0, all estimations lead to the same result which corresponds to the matrix conductivity λdil hom, λMT hom, λP CW hom →λm . 3 Calculation of the effective thermal conductivity As before, the specific behaviour of flat cracks according to their status gives the following: We impose a uniform macroscopic thermal gradient G at the outer boundary δΩof the RVE. This is similar to the classical strain-based formulation in elasticity. As a first, we will estimate the effective conductivity through different schemes and bounds. λMT hom =    λm · I −π d 1 1 + π d n ⊗n , if cracks are open λm , if cracks are closed (14) When there is a dilute concentration of cracks (small d), it is considered that there is no interaction between them. The remote condition in this case can be given by G∞= G. Hence, the localization tensor can be given by: (14) Ponte Casta˜neda-Willis developed an energy-based upper bound to find effective stiffness [6]. This bound takes into account the shape of the inclusion (through SE) and also the spatial distribution of cracks through an additional tensor Sd. The simplified localization tensor can be given by: Ponte Casta˜neda-Willis developed an energy-based upper bound to find effective stiffness [6]. This bound takes into account the shape of the inclusion (through SE) and also the spatial distribution of cracks through an additional tensor Sd. The simplified localization tensor can be given by: ⟨A⟩dil c = h I −SE ! 1 −ξ i−1 with ξ = λc λm (8) (8) Substituting (8) in (6), we get the general expression: Substituting (8) in (6), we get the general expression: ⟨A⟩P CW c = ⟨A⟩dil c · I + fc ! 1 −ξ Sd · ⟨A⟩dil c −1 (15) λdil hom = λm · h I −π d R i (9) ⟨A⟩P CW c = ⟨A⟩dil c · I + fc ! 1 −ξ Sd · ⟨A⟩dil c −1 (15) λdil hom = λm · h I −π d R i (9) (15) (9) 4 S.R. Rangasamy Mahendren et al.: Mechanics & Industry 21, 519 (2020) For simplicity, a circular spatial distribution is adopted, for which Sd = 1 2 I. Now (6) can be written as: The cracks are positioned inside the simulated area using circular spatial distribution (in agreement with spa- tial distribution assumed for PCW bound). 3 Calculation of the effective thermal conductivity On the other hand, we observe a complete deactivation of microcracking when the defects are closed λdil hom = λMT hom = λP CW hom = λm . Note that detailed developments and extension to 3D case can be found in [27]. perature gradient G = Gv v = ∆Tv L v, creates an overall heat flux Q (= Qt t + Qv v) inside the simulated area. On a global point of view, the two edges with zero flux act as adiabatic walls, allowing the heat flux Q to be mainly oriented along the v direction. From definition (18), the numerical effective conductivity in direction v is then provided by: λnum(v) = Qv Gv (19) 4 Numerical simulations λnum(v) = Qv Gv (19) (19) In the following, numerical simulations are performed using finite element software Abaqus®. (t, v) denotes an orthonormal coordinate system. The simulated area A is a square (size L = 1 m) that follows steady-state heat conduction. The matrix is designed as an unit 2D shell with its own scalar conductivity λm. Assuming there are N = 10 cracks in the area, the radius of the crack can be given by a = 2 r d N . In what follows, the range of consid- ered density is less than 0.1, related crack’s radius has, therefore, a maximum value of 0.1 m. where Qv is the average heat flux along the v direction. L It can be calculated as Qv = 1 L Z L 0 HFL2 dt with HFL2 It can be calculated as Qv = 1 L Z L 0 HFL2 dt with HFL2 being the heat flux density in v direction along the path on the top/bottom edge. Alternatively, Qv can be found using Reaction flux RFLi calculated on each node i on the top/bottom edge when considering unit dimension in n the transverse direction, i.e. Qv = 1 L n X i=1 RFLi. the transverse direction, i.e. Qv = 1 L X i=1 RFLi. Influence of matrix element size on the average heat flux Qv (RC; (n, v) = 0◦). corresponds to the closed case (λc = 50% λm) and shows continuity in heat transfer. corresponds to the closed case (λc = 50% λm) and shows continuity in heat transfer. distribution has no major influence on the resulting con- ductivity. Accordingly, the RC will be considered for all further simulations done in the study. As first illustration, Figure 5 shows the heat flux vec- tor at integration points for density d = 0.1 (RC; (n, v) = 45◦). Figure 5a corresponds to the open case (λc = 0) and shows that the cracks acts as a thermal barrier accord- ing to the adiabatic behaviour on their lips. Figure 5b the transverse direction, i.e. Qv = 1 L X i=1 RFLi. The finite element type used for both the matrix and crack is quadratic triangular DC2D6 (see Fig. 3 for RC; (n, v) = 45◦). Fixing 100 elements inside each crack, the influence of the size of the matrix elements on the heat flux has been studied (Tab. 1). We have used a very fine mesh to prevent improper scattering of the flux around the crack tips. In that case, the model has approximately 73500 elements and 148000 nodes including 201 nodes on the outer edges. Note that the computation time remains acceptable (less than a minute). Moreover, estimations of λnum(v) obtained for different cracks distributions have been compared for the most critical case, i.e. (n, v) = 0◦ (see Fig. 4). From Table 2, it is observed that cracks Cracks are usually represented as seams for the open state (duplicated nodes). Yet, this cannot account for the heat transfer during crack closure. So, the crack is mod- elled here as an elliptical inclusion (created as a partition on the 2D shell) with normal n and scalar conductivity λc. Since creating a crack with zero aspect ratio is not possible (Ωc = 0), the cracks are designed with an aspect ratio 0 ̸= ω ≪1 (so fc ≪1). For a given fc, the value of the scalar conductivity λc determines if the cracks are open (λc = 0) or closed (λc = λ∗̸= 0). Such a description of the crack geometry and the unilateral effect is in line with the theoretical framework used in Section 2. S.R. Rangasamy Mahendren et al.: Mechanics & Industry 21, 519 (2020) 5 Fig. 2. Simulated area showing spatial distribution of cracks for the Reference Configuration (RC). Fig. 2. Simulated area showing spatial distribution of cracks for the Reference Configuration (RC). Fig. 3. Simulated area mesh (RC; (n, v) = 45◦). trix element size on the average heat flux Qv (RC; (n, v) = 0◦). Size of the matrix elements 0.1 0.05 0.025 0.01 Total no of elements 13804 42950 52976 73466 Heat flux Qv, W.m−2 10484.4 10468.1 10468.0 10468.4 Table 1. Influence of matrix element size on the average heat flux Qv (RC; (n, v) = 0◦). Size of the matrix elements 0.1 0.05 0.025 0.01 Total no of elements 13804 42950 52976 73466 Heat flux Qv, W.m−2 10484.4 10468.1 10468.0 10468.4 Table 1. 5 Results and discussion This section intends to compare theoretical developments and FE numerical simulations. From (18) and (5), the S.R. Rangasamy Mahendren et al.: Mechanics & Industry 21, 519 (2020) 6 Fig. 4. Influence of cracks position for (n, v) = 0◦. Fig. 4. Influence of cracks position for (n, v) = 0◦. Fig. 4. Influence of cracks position for (n, v) = 0◦. Table 2. Influence of cracks distributions ((n, v) = 0◦). Configuration λnum/λm Deviation w.r. RC Reference configuration (RC) 0.727 - Configuration 1 0.730 0.47% Configuration 2 0.732 0.66% Table 2. Influence of cracks distributions ((n, v) = 0◦). acks distributions ((n, v) = 0◦). Configuration λnum/λm Deviation w.r. RC Reference configuration (RC) 0.727 - Configuration 1 0.730 0.47% Configuration 2 0.732 0.66% theoretical scalar conductivity λ(v) comes to: theoretical scalar conductivity λ(v) comes to: of the scheme or crack density. As pointed out ear- lier, for the theoretical models, we see that as d → 0, λdil hom ≈λMT hom ≈λP CW hom (d = 0.1 in Fig. 6a, d = 0.05 in Fig. 6b). This can be attributed to the fact that as d decreases, the size of the crack decreases (respectively from a = 0.1 m to a = 0.07 m), making the interaction between the cracks less influential and at one point there is no interaction between the cracks essen- tially leading to a dilute configuration. We also see that as the crack becomes smaller, so does its influence on the conductivity (λ(n) ≈0.73 λm for a = 0.1 m whereas λ(n) ≈0.86 λm for a = 0.07 m). Figure 6 also illustrates λth(v) = v · λhom · v (20) This can be estimated for different schemes (th = {dil, MT, PCW}) and compared to λnum(v). ( { }) ( ) Recalling previous results from Section 3, open cracks contribute to the degradation of the thermal conduc- tivity, mainly along the direction n normal to the crack surface. This case is true for the simulations as well (see Fig. 6). Both the theoretical and simulated results show us damage-induced anisotropy irrespective S.R. Rangasamy Mahendren et al.: Mechanics & Industry 21, 519 (2020) 7 Fig. 5. Heat flux vectors at integration points inside the simulated area (RC; (n, v) = 45◦; d = 0.1; a = 0.1 m; ω = 0.001). Fig. 5. 5 Results and discussion Heat flux vectors at integration points inside the simulated area (RC; (n, v) = 45◦; d = 0.1; a = 0.1 m; ω = 0.001). x vectors at integration points inside the simulated area (RC; (n, v) = 45◦; d = 0.1; a = 0.1 m; ω = 0.001). ig. 5. Heat flux vectors at integration points inside the simulated area (RC; (n, v) = 45◦; d = 0.1; a = 0.1 m Fig. 6. Generalized thermal conductivity λ(v) normalized by its initial value for a material weakened by a single array of parallel open microcracks of unit normal n. Fig. 6. Generalized thermal conductivity λ(v) normalized by its initial value for a material weakened by a single array of parallel open microcracks of unit normal n. the consistency between the theoretical and simulated results. It is interesting to observe that for lesser angles (n, v) < 45◦, simulated results tend towards PCW and for higher angles, they approach the dilute case. Indeed, inter- actions are greater when the cracks are mostly orthogonal to the heat flux. But, if cracks tend to be aligned with the direction of the temperature gradient then the influence of cracks decreases and heat flux is less disturbed, tending to the dilute case (see Fig. 7). to the degradation of conductivity (see (11), (14) and (17)), i.e. the effective conductivity in any direction is recovered to its initial value at the cracks’ closure. So the generalized scalar conductivity in unit direction v for closed cracks can be given as: λ(v) = λm, ∀v. Just like the open crack, simulated and theoretical results are consistent for the closed crack (see Fig. 8). We also see that the former has only a negligible amount of degra- dation of thermal conductivity (less than 0.035% for d = 0.1 and less than 0.02% for d = 0.05 when considering λ∗= 50% λm). ( ) On the other hand, dilute, Mori-Tanaka and PCW approaches show that closed cracks do not contribute S.R. Rangasamy Mahendren et al.: Mechanics & Industry 21, 519 (2020) 8 S.R. Rangasamy Mahendren et al.: Mechanics & Industry 21, 519 (2020) Fig. 7. Heat flux vectors at integration points for various orientations of open cracks (RC; d = 0.1; a = 0.1 m; ω = Fig. 7. 5 Results and discussion Normal thermal conductivity λ(n) normalized by its initial value for various aspect ratios (d = 0.1; a = 0.1 m); points represent simulation results (RC); log10 scale is used for abscissa. 5 Results and discussion Heat flux vectors at integration points for various orientations of open cracks (RC; d = 0.1; a = 0.1 m; ω = 0.001). x vectors at integration points for various orientations of open cracks (RC; d = 0.1; a = 0.1 m; ω = 0.001). Fig. 7. Heat flux vectors at integration points for various orientations of open cracks (RC; d = 0.1; a = From (11), (14) and (17) we know that the theoretical results are not a function of the aspect ratio ω since they all correspond to the limit case ω →0 ! λdil hom, λMT hom and λP CW hom only depend on λm, d and n . But as discussed earlier, it is not possible to simulate an ellipse with zero aspect ratio. So it seems natural to study the influence of the aspect ratio on the simulated results. Since the maximum degradation is along the direction n normal to the crack, we intend to focus only on λ(n). Figure 9a corresponds to open crack and Figure 9b corresponds to closed crack with fixed values of dilute, MT and PCW denoted as reference. In both cases, the simulated results are really sensitive to the aspect ratio ω and get closer to the PCW bound when ω →0. Especially in the closed case, the simulations tend to the full recovery of λ(n), same as the theoretical models. Note that all the sim- ulations linked to varying aspect ratio are performed by varying the crack thickness c and keeping the crack density d and radius a as constants. S.R. Rangasamy Mahendren et al.: Mechanics & Industry 21, 519 (2020) 9 Fig. 8. Generalized thermal conductivity λ(v) normalized by its initial value for a material weakened by a single array of parallel closed microcracks of unit normal n (RC; λ∗= 50% λm). Fig. 8. Generalized thermal conductivity λ(v) normalized by its initial value for a material weakened by a single array of parallel closed microcracks of unit normal n (RC; λ∗= 50% λm). Fig. 9. Normal thermal conductivity λ(n) normalized by its initial value for various aspect ratios (d = 0.1; a = 0.1 m); points represent simulation results (RC); log10 scale is used for abscissa. Fig. 9. References [1] M. Kachanov, Advances in Applied Mechanics, vol. 30, Academic Press, New York, 1993 [2] S. Nemat-Nasser, M. Hori, Micromechanics: Overall Prop- erties of Heterogeneous Materials, in: Applied Mathematics and Mechanics, vol. 37, Elsevier Science, Amsterdam, 1993 Further studies could now be conducted to extend such analytic procedure (homogenization and numerical sim- ulations) for flux-based boundary condition from which effective thermal resistivity can be derived. Considering earlier work of the authors [27], it would also be relevant to compare theoretical and numerical results in the 3D case. [3] J.D. Eshelby, The determination of the elastic field of an ellipsoidal inclusion, and related problems, Proc. R. Soc. A 421, 379–396 (1957) [4] T. Mura, Micromechanics of Defects in Solids, Martinus Nijhoff, Boston, 1987 [5] T. Mori, K. Tanaka, Average stress in matrix and average elastic energy of materials with misfitting inclusions, Acta Metallica 21, 571–574 (1973) ( ) [6] P. Ponte Casta˜neda, J. Willis, The effect of spatial distri- bution on the effective behavior of composite materials and cracked media, J. Mech. Phys. Solids 43, 1919–1951 (1995) 6 Conclusion and perspectives (t, v) Orthonormal coordinate system of the simulated area a, c Crack’s mean semi-axes, m d Scalar crack density fc Crack’s volume fraction in 2D g, G Microscopic and macroscopic tempera- ture gradient respectively q, Q Microscopic and macroscopic heat flux respectively ∆Tv = T1 −T2 Difference between temperatures T1 and T2 along the direction v, K u Outward unit normal to ∂Ω ξ Ratio of scalar conductivities between crack and matrix Gt, Gv Temperature gradient along the direc- tion t and v respectively, K.m−1 Qt, Qv Average heat flux along the direction t and v respectively, W.m−2 T(x), g(x), q(x) Temperature, temperature gradient and heat flux respectively at point x HFL Heat flux in a structure RC Reference Configuration RFL Reaction flux RVE Representative Volume Element Fig. 10. Influence of scalar conductivity λ∗on the nor- mal thermal conductivity λ(n) normalized by its initial value (RC; d = 0.1; a = 0.1 m); log10 scale is used for abscissa. and properties of cracks were taken into account in the same way as for theory. The consistency of theoretical and numerical results have been demonstrated through following points. For open cracks, we observe that the microcracked medium exhibits an induced anisotropy with main degradation of conductivity in the direction normal to the cracks. Also, as crack density tends to zero, all models recover the matrix property. On the other hand, cracks closure leads to a complete deactivation of dam- aging effects. Finally the sensitivity of numerical results based on the aspect ratio of defects has been shown. 6 Conclusion and perspectives Also, the theoretical results for the closed case do not depend on the fictitious scalar conductivity λ∗. This may not be true for the simulations. So, a series of simula- tions were performed with varying λ∗and for different aspect ratios (d and a are still constants). The values for λ∗are given as a proportion of λm such that λ∗= α λm with α = {1, 5, 10, 25, 50, 80, 100}[%]. Figure 10 shows that there is a clear influence of the scalar conductiv- ity λ∗on the numerical thermal conductivity. For α ≤ 10%, we see a drastic decrease in the conductivity, this is due to the fact that we are slowly approaching the open case (α = 0). We also observe that as ω →0 the influ- ence of λ∗diminishes and representation of closed cracks by means of an ellipse with fictitious scalar conductiv- ity λ∗becomes independent of the λ∗value, just like the theoretical results. In this work, we have presented the closed-form expres- sions for the effective thermal conductivity of 2D microc- racked media under the steady-state heat condition. The theoretical background is based on the equivalent inclu- sion method where cracks are represented as the limit case of thin elliptic inclusions. Special attention has been paid to the unilateral effect by considering specific properties of the cracks according to their state. Different estimations of the overall thermal behaviour that take into account (Mori-Tanaka scheme and PCW bound) or not (dilute) cracks interactions have been provided, both for open and closed cracks. The determination of effective ther- mal conductivity was also performed by means of finite element simulations. For this numerical part, geometry S.R. Rangasamy Mahendren et al.: Mechanics & Industry 21, 519 (2020) 10 Fig. 10. Influence of scalar conductivity λ∗on the nor- mal thermal conductivity λ(n) normalized by its initial value (RC; d = 0.1; a = 0.1 m); log10 scale is used for abscissa. Fig. 10. 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https://openalex.org/W2804102328	https://europepmc.org/articles/pmc5957326?pdf=render	English	null	Mosquito saliva alone has profound effects on the human immune system	PLoS neglected tropical diseases	2,018	cc-by	15,077	RESEARCH ARTICLE Megan B. Vogt1, Anismrita Lahon2, Ravi P. Arya2, Alexander R. Kneubehl2, Jennifer L. Spencer Clinton2, Silke Paust3, Rebecca Rico-Hesse2* Megan B. Vogt1, Anismrita Lahon2, Ravi P. Arya2, Alexander R. Kneubehl2, Jennifer L. Spencer Clinton2, Silke Paust3, Rebecca Rico-Hesse2* 1 Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, Texas, United States of America, 2 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America, 3 Department of Pediatrics, Baylor College of Medicine, Houston, Texas, United States of America * rebecca.rico-hesse@bcm.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Rhoel Ramos Dinglasan, University of Florida, UNITED STATES Received: December 28, 2017 Accepted: April 10, 2018 Published: May 17, 2018 Copyright: © 2018 Vogt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data are available in the Flow Repository (https://flowrepository.org) from the following links: Human PBMCs: https:// flowrepository.org/id/FR-FCM-ZYWP. NSG Mice Prelim studies: https://flowrepository.org/id/FR- FCM-ZYWR. NSG Mice Later studies: https:// flowrepository.org/id/FR-FCM-ZYWQ. OPEN ACCESS Citation: Vogt MB, Lahon A, Arya RP, Kneubehl AR, Spencer Clinton JL, Paust S, et al. (2018) Mosquito saliva alone has profound effects on the human immune system. PLoS Negl Trop Dis 12 (5): e0006439. https://doi.org/10.1371/journal. pntd.0006439 Editor: Rhoel Ramos Dinglasan, University of Florida, UNITED STATES Abstract Mosquito saliva is a very complex concoction of >100 proteins, many of which have unknown functions. The effects of mosquito saliva proteins injected into our skin during blood feeding have been studied mainly in mouse models of injection or biting, with many of these systems producing results that may not be relevant to human disease. Here, we describe the numerous effects that mosquito bites have on human immune cells in mice engrafted with human hematopoietic stem cells. We used flow cytometry and multiplex cyto- kine bead array assays, with detailed statistical analyses, to detect small but significant vari- ations in immune cell functions after 4 mosquitoes fed on humanized mice footpads. After preliminary analyses, at different early times after biting, we focused on assessing innate immune and subsequent cellular responses at 6 hours, 24 hours and 7 days after mosquito bites. We detected both Th1 and Th2 human immune responses, and delayed effects on cytokine levels in the blood, and immune cell compositions in the skin and bone marrow, up to 7 days post-bites. These are the first measurements of this kind, with human immune responses in whole animals, bitten by living mosquitoes, versus previous studies using incomplete mouse models and salivary gland extracts or needle injected saliva. The results have major implications for the study of hematophagous insect saliva, its effects on the human immune system, with or without pathogen transmission, and the possibility of deter- mining which of these proteins to target for vaccination, in attempts to block transmission of numerous tropical diseases. * rebecca.rico-hesse@bcm.edu Introduction Approximately 750,000 people die of mosquito transmitted diseases each year, including malaria, dengue, West Nile, Zika, and chikungunya fevers [1, 2]. The incidence of these dis- eases is expected to rise significantly in the next few decades as the host ranges of multiple spe- cies of mosquitoes increase due to climate change [3, 4]. Not only do mosquitoes transmit diseases, but they also may increase the severity of the diseases they transmit. In experimental infections of mice, delivery of arboviruses via mosquito bite or via needle injection in conjunc- tion with uninfected mosquito bite results in more severe disease than delivery of virus alone via needle injection [5–9]. In mouse models of infections by malaria and leishmania parasites, mosquito and sandfly saliva have also been shown to enhance infectivity and disease progres- sion [10, 11]. Competing interests: The authors have declared that no competing interests exist. Mosquito saliva is a complex mixture of proteins that allows the mosquito to acquire a blood meal from its host (necessary for egg maturation), by circumventing vasoconstriction, platelet aggregation, coagulation, and inflammation or hemostasis (reviewed in [12]). It is well known that mosquito saliva contains proteins that are immunogenic to humans, and some allergic responses can be very severe [13, 14]. Because mosquito saliva can be immunogenic, it is speculated that mosquito saliva may enhance pathogenicity of arboviruses by manipulating the host’s immune response. The human immune system can be divided into innate (natural killer (NK) cells, neutro- phils, monocytes, macrophages, mast cells, and dendritic cells) and adaptive (T and B cells) arms. Upon activation, innate immune cells release cytokines and chemokines, kill cells via cytotoxic molecules (NK cells only), and phagocytose pathogens, which aids in pathogen destruction and allows specialized cells to process antigens for presentation to T and B cells. Successful T and B cell activation by their cognate antigen induces their differentiation into clonal effector or long-lived antigen specific memory cells both of which are crucial to protect us from recurrent, severe disease (reviewed in [15]). T cells are often classified based on expression of the CD4 or CD8 co-receptors. Upon acti- vation, CD4 T cells can differentiate into a variety of T helper cells (Th1, Th2, etc.), each with unique functions, whereas CD8 T cells become cytotoxic T lymphocytes (CTLs) [16, 17]. A small proportion of T cells outside of the thymus express both CD4 and CD8. Author summary Mosquito saliva proteins have numerous effects on the immune system, and we describe here the use of mice with a humanized immune system to study the effects of mosquito bites on human cells. Our results show that the number of immune cell types affected is much larger than previously described, and some immune responses to mosquito bites can be detected up until 7 days post-bite. The biological significance of these changes remains to be determined, but it might explain how some pathogens, such as viruses, can Funding: This work was supported by the National Institutes of Health grants to RRH (grant numbers R01 AI098715, R01 AI099483). This project was supported by the Cytometry and Cell Sorting Core 1 / 27 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 Mosquito saliva effects at Baylor College of Medicine with funding from the NIH (P30 AI036211, P30 CA125123, and S10 RR024574) and the expert assistance of Joel M. Sederstrom. Additionally, this project was supported by the Antibody Based Proteomics Core at Baylor College of Medicine with funding from the NIH (P30 NC1-CA125123) and CPRIT (RP120092) with the expert assistance of Shixia Huang. Stereophotography was done with the support of the Optical Imaging & Vital Microscopy Core, Mary E. Dickinson, PhD, Academic Director,at Baylor College of Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. at Baylor College of Medicine with funding from the NIH (P30 AI036211, P30 CA125123, and S10 RR024574) and the expert assistance of Joel M. Sederstrom. Additionally, this project was supported by the Antibody Based Proteomics Core at Baylor College of Medicine with funding from the NIH (P30 NC1-CA125123) and CPRIT (RP120092) with the expert assistance of Shixia Huang. Stereophotography was done with the support of the Optical Imaging & Vital Microscopy Core, Mary E. Dickinson, PhD, Academic Director,at Baylor College of Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. spread through the body in these cells, replicate to higher extents, and even remain in some tissues for far longer than detected in blood. spread through the body in these cells, replicate to higher extents, and even remain in some tissues for far longer than detected in blood. Mosquito saliva effects cytokines stimulate naïve CD4 T cells to differentiate into Th1 T cells, which produce the cyto- kines IFNγ, IL-2, and TNFβ and stimulate the activation of CTLs. Th1 cells also stimulate B cells to produce specific IgG isotypes that mediate effector functions against intracellular path- ogens [29]. The Th2 response typically involves a strong humoral response to eradicate extra- cellular pathogens and parasites. When overactive, a Th2 response can cause allergies or asthma. During a Th2 response, activated mast cells or T cells produce the cytokine IL-4, which stimulates naïve CD4 T cells to become Th2 cells. These Th2 cells produce more IL-4 as well as the cytokines IL-5, IL-6, IL-9, IL-10, and IL-13, which together, inhibit phagocytic inflammation and promote the proliferation and survival of mast cells and other cells needed for the clearance of extracellular pathogens. Th2 cells also stimulate B cells to produce antibod- ies of the IgE isotype. Th1 and Th2 responses are not necessarily mutually exclusive, as mixed Th1/Th2 immune response can occur; however, cytokines produced in each response do actively inhibit production of cytokines and processes involved in the other response (reviewed in [17, 30, 31]). In this study, we sought to investigate the effects of mosquito saliva on the human immune system. Previous studies have addressed this phenomenon but used mouse models genetically predisposed to a Th1 (C57BL6 mice) or Th2 (BALB/c mice) response, and/or used mosquito salivary gland extracts instead of secreted mosquito saliva or mosquito bites [32, 33]. These studies have produced conflicting results as to the anti-inflammatory and viral enhancing properties of saliva proteins [13]. Furthermore, these studies did not use detailed analyses of cellular responses to mosquito bites or use mouse models that mimic human infection [34, 35]. It is clear that mouse models of human disease do not replicate many innate and adaptive immune responses [36], so we sought to create a model in which we could measure the human cytokine and cellular responses, in vivo. Thus, we have developed a humanized mouse model of mosquito biting, to study the specific effects of mosquito saliva on the pathogenesis of mos- quito-borne viruses. Materials and methods Mosquito rearing Aedes aegypti (Rockefeller) mosquitoes were obtained from BEI resources as eggs (MRA-734). Mosquitoes were maintained under standard insectary conditions (~28˚C, 80% relative humidity) with a 12-hour light/dark cycle maintained by the Philips Hue Smart Lighting sys- tem. Larvae were raised in water pans and fed on a mixture of ground rabbit chow (Purina)- liver powder (Bio-Serv)-yeast (Bio Serv) in a 4:1:1 ratio, ad libitum. Emerged mosquitoes were moved to mesh cages and fed on 10% sucrose (Sigma) solution ad libitum. Colony mainte- nance was performed by feeding mosquitoes on anesthetized C57/BL6 mice (IACUC AN- 6151). In the subsequent days following blood feeding, eggs were collected, desiccated, and stored for a maximum of 6 months. Introduction These double positive (DP) T cells arise from CD4 or CD8 T cells that upregulate expression of the other co- receptor in the periphery through an unknown mechanism [18]. DP T cells can have similar, albeit more exaggerated, functions to CTLs [18–23]; moreover, they can regulate or exacerbate inflammation through the production of cytokines, including IL-10, IL-4, and IFNγ [18, 24, 25]. Another type of T cell that performs similar functions to DP T cells is the natural killer T (NKT) cell. These cells, which develop in the thymus and express a rearranged T cell receptor and NK cell markers, can be cytotoxic. Activated NKT cells are also potent and rapid cytokine producers and can polarize immune responses through the production of large amounts of IFNγ, IL-4, and/or IL-17A [26–28]. Immune responses can be classified according to the type of helper T cell involved; the two canonical immune responses are the Th1 response and the Th2 response. The Th1 response typically involves a cell-mediated response to eradicate intracellular pathogens. Antigen pre- senting cells activated by intracellular pathogens produce the cytokines IL-12 and IL-18. These PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 2 / 27 Our results are the only to measure the effects of mosquito bite on specific components of the human immune system, especially early after saliva injection, and they indicate long-lasting effects of these components, up to 7 days in human skin and bone mar- row cells. Mosquito saliva effects and be alive as evidenced by saliva droplets in the oil, and response to physical stimulus, respectively. Saliva was extracted immediately from the oil by pooling the salivations and add- ing one volume of cold phosphate buffered saline (PBS). The salivation/PBS mixture was pulse vortexed and centrifuged at 10,000g for 5min. The aqueous layer was removed and the protein concentration was quantified using the BCA assay (Pierce). Isolation and stimulation of human PBMCs Source leukocytes were obtained from the Gulf Coast Regional Blood Center (Houston, TX). PBMCs were isolated via density centrifugation using Leucosep centrifuge tubes (Gre- nier Bio-one) and lymphocyte separation media (Corning) according to manufacturers’ protocols. To remove any residual red blood cell contamination, PBMC pellets were then treated with red blood cell lysis solution (eBiosciences) according to manufacturer’s instruc- tions. PBMCs were resuspended in RPMI 1640 media with 10% fetal bovine serum (FBS) and were plated at 1x106 cells/well in a 24 well plate. PBMCs were stimulated with mosquito saliva (2μg salivary proteins per well), or the equivalent of 4 mosquito bites [37], to corre- spond with the number of infected mosquito bites required to produce dengue fever in humanized mice [7], and lipopolysaccharide (1mg/L media; Sigma), pokeweed mitogen (5mg/L media; Sigma), or untreated media. Supernatant samples were collected daily for five days post stimulation to assess cytokine production. On day five post stimulation, all PBMCs were assessed via flow cytometry. Production of humanized mice Humanized mice were engrafted as previously described [7]. Briefly, male and female NSG breeders were obtained from The Jackson Laboratory, and mice were bred in the Transgenic Mouse Facility at Baylor College of Medicine. One day post birth, each pup from these breedings was sublethally irradiated with 100 centigrays and intrahepatically injected with 3x105 CD34+ stem cells. These stem cells were isolated from human umbilical vein cord blood from either the University of Texas MD Anderson Cord Blood Bank (Houston, TX) or the Texas Cord Blood Bank (San Antonio, TX) using the Dynabeads CD34 positive selec- tion kit (Invitrogen) following the manufacturer’s instructions. Levels of engraftment of human hematopoietic cells were tested 6 to 8 weeks later using flow cytometry to target human and mouse CD45+ cells (S1 Table). Mosquito salivation Mosquito saliva was collected from female Aedes aegypti Rockefeller mosquitoes 4–5 days post-eclosion. Adult female mosquitoes were immobilized by removal of wings and legs. The proboscis was inserted into a 10μL microcapillary tube (Drummond) containing microscopy- grade immersion oil (Zeiss). The mosquitoes were allowed to salivate into the oil at insectary conditions for 45 minutes. After salivation, the mosquitoes were confirmed to have salivated 3 / 27 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 Mosquito saliva effects Multiplex cytokine bead array assay Cytokine and chemokine levels in PBMC supernatant and mouse serum samples were determined using the Milliplex 41-plex human cytokine/chemokine magnetic bead panel (Millipore) according to the manufacturer’s instructions. Samples were collected on either the Bio-Plex (Bio-Rad) using Bio-Plex Manager software or the Magpix (Millipore) using xPonent software (Luminex). Tissue collection and processing Six hours, 24 hours, or 7 days post mosquito bite, mice were humanely euthanized via isoflur- ane overdose. Upon cessation of breathing, mice were exsanguinated via intracardiac bleed. Blood was stored in heparin treated and untreated microcentrifuge tubes for further process- ing. Skin from rear footpads was removed using surgical scissors and stored separately in PBS with 2% FBS (PBS/FBS) and 5μg/mL collagenase. Spleens and femurs were also removed from each mouse and stored separately in PBS/FBS. Blood stored in untreated tubes was allowed to clot at room temperature for at least thirty min- utes. The clotted blood was then centrifuged at 1500g for ten minutes at 4˚C. Following this cen- trifugation, serum was transferred to new microfuge tubes and stored at -70˚C until used in multiplex cytokine bead array assay. Blood stored in heparinized tubes were transferred to 50mL conical tubes. Red blood cells were lysed using RBC lysis solution (eBioscience) according to the manufacturer’s protocol. The remaining white blood cell pellet was resuspended at 1x104 to 1x106 cells/mL in PBS/FBS. These cells were stored at 4˚C until stained for flow cytometry analysis. Skin footpads were cut into small pieces and incubated in PBS/FBS and 5mg/mL collage- nase at 37˚C for 1 hour. Following digestion, skin pieces were ground over a 40μm cell strainer into a 50mL conical tube. Skin cells were washed twice in PBS/FBS and resuspended at 1x104 to 1x106 cells/mL in PBS/FBS. Spleens were burst by grinding between two frosted microscope slides. Spleen contents were then ground over a 40μm strainer into a 50mL conical tube. Red blood cells were lysed using RBC lysis solution (eBioscience) according to the manufacturer’s protocol. Remaining cells were resuspended at 1x106 – 1x107 cells/mL in PBS/FBS. Bone marrow was flushed out of femurs using a 25G needle filled with PBS/FBS. Marrow was ground over a 40μm strainer into a 50mL conical tube. Red blood cells were lysed and remaining cells were resuspended as with the spleen cells. Flow cytometry Blood, bone marrow, skin, and spleen cells from hu-NSG mice and stimulated human PBMCs were transferred to 96 well plates and incubated with antibodies against extracellular targets (Table 1) on ice for 30 minutes. Cells were fixed and permeabilized using the FoxP3 Transcrip- tion Factor Staining Buffer Kit (eBioscience) following the manufacturer’s protocol. Following permeabilization, cells from hu-NSG mice were incubated with antibodies against intracellular targets (Table 1) on ice for 30 minutes. Cells were washed, resuspended in PBS/FBS, and stored at 4˚C until analysis. Samples were analyzed on the LSRII Fortessa (BD) using the HTS mod- ule. Data were collected using the FACSDiva software (BD). Data were analyzed using FlowJo (v10.2; FlowJo, LLC) (S2 Fig). Mosquito biting of Hu-NSG mice Mosquito biting of reconstituted humanized mice was carried out as previously reported [7], although with uninfected mosquitoes. In short, 4 to 7 days post-emergence, female mosquitoes were starved for 24 hours in dram vials (4–6 mosquitoes per vial) capped in a fine, white poly- ester mesh (Bio-Serv). Dram vials were kept at insectary conditions (28˚C, 80% humidity) for the duration of the 24-hour starving. Mosquitoes were then transferred to a BSL3 facility, and the dram vials were held against a footpad of anesthetized, humanized mice, allowing the mos- quitoes to feed (IACUC AN-6151). A “bite” was defined visually by mosquito engorgement and did not include probing; approximately 4 bites total occurred for each mouse, on both footpads. This number was chosen based on our previous studies demonstrating that 4 infected mosquitoes are required to bite each humanized mouse to consistently produce den- gue fever [7]. 4 / 27 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 b Some panels contain multiple antibodies to the same target but conjugated to different fluors. These antibodies were not used in conjunction with each other but were substituted out due to manufacturer availability. Statistical analysis Statistical analysis was performed using Prism (v6.0; GraphPad) software. Outliers were removed using ROUT analysis (Q = 1%). Data were analyzed via two-way ANOVA and t-test using Holm-Sidak correction for multiple comparisons. 5 / 27 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 a Panels P1, P2, and P3 all refer to panels used in the preliminary flow studies reported in Figs 3 and 4. Panels 1 and 2 refer to panels used in the remainder of the flow cytometry experiments reported in Figs 5 and 6. Mosquito saliva increases the frequency of natural killer T cells in human peripheral blood mononuclear cells Our previous studies demonstrated that mosquito saliva enhances dengue infection in human- ized mice [7]. Therefore, we hypothesized that mosquito saliva modulates the human immune response to enable virus replication. To determine which type of effects we might see in human immune cells, we studied the ex vivo responses of primary human peripheral blood mononuclear cells (PBMCs) to mosquito saliva. Human PBMCs were isolated from two separate blood donors and then treated with mos- quito saliva (equal to 4 mosquito bites: 2.0 μg [37]) or untreated media. We examined the immune cell populations present 5 days post treatment via flow cytometry (Fig 1). Natural killer T cells (NKT; CD3+CD56+) were found at a higher frequency following mosquito saliva treatment; however, we saw no statistical differences in the number of activated cells (CD69+). When activated, NKT cells can rapidly produce large amounts of IFNγ, IL-4, and/or IL-17A. Fig 1. Mosquito saliva increased frequency of CD4+CD8+ double positive T cells and natural killer T cells. Human PBMCs from two donors were stimulated with either mosquito saliva or untreated media. Flow cytometry was performed on these cells 5 days post stimulation to assess frequencies of different immune cell populations. These frequencies are expressed as the mean percentage of CD45+ cells with error bars indicating 1 standard error of the mean (SEM). The subset of the T cell plot represents the percentage of NKT cells that were activated. T-tests were performed using GraphPad Prism. Multiple comparisons were corrected for using the Holm-Sidak method, which increased the significance threshold to p < 0.01. #, p<0.1; ##, p<0.05; , p<0.01; p<0.005. Abbreviations: NK = Natural Killer; MF = Macrophage; DC = Dendritic Cell; DP = Double Positive. Cell markers used to define cell populations: T cells, CD45 +CD3+; NK Cells, CD45+CD3-CD56+; B Cells, CD45+CD3-CD19+; Monocytes/MFs, CD45+CD3-CD14+; Neutrophils, CD45+CD3-CD66b+; Myeloid Cells, CD45+CD3-CD11c+; DP T Cells, CD45+CD3+CD4+CD8+; CD4 T Cells, CD45+CD3+CD4+CD8-; CD8 T Cells, CD45+CD3+CD4-CD8+; NKT Cells, CD45 +CD3+CD56+; Activated NKT Cells, CD45+CD3+CD56+CD69+. https://doi.org/10.1371/journal.pntd.0006439.g001 Fig 1. Mosquito saliva increased frequency of CD4+CD8+ double positive T cells and natural killer T cells. Human PBMCs from two donors were stimulated with either mosquito saliva or untreated media. Flow cytometry was performed on these cells 5 days post stimulation to assess frequencies of different immune cell populations. These frequencies are expressed as the mean percentage of CD45+ cells with error bars indicating 1 standard error of the mean (SEM). Mosquito saliva effects Ethics statement All experiments involving mice were done in accordance with guidelines of the Institutional Animal Care and Use Committee at Baylor College of Medicine (IACUC Protocol AN-6151), and the recommendations in the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council, National Academy of Sciences, 2011). The Institutional Review Board at Baylor College of Medicine determined that our use of human blood products in this study did not constitute human subjects research. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 Mosquito saliva effects Table 1. Antibodies used for flow cytometry experiments. Target Extracellular (EC), Intracellular (IC), or Nuclear (N) Panel(s)a Clone Fluorb Manufacturer CD1c EC 2 L161 Pacific Blue Biolegend CD3 EC P1, P2, P3 UCHT1 PE/CF594 BD Biosciences 1, 2 UCHT1 BUV661 BD Biosciences CD4 EC P2, P3, 1 OKT4 BV650 Biolegend CD8a EC P2 RPA-T8 BV570 Biolegend 1 RPA-T8 BV605 Biolegend CD11b EC P1, P3, 2 ICRF44 BV605 Biolegend CD11c EC P1, P3 Bu15 Pacific Blue Biolegend 2 3.9 BV650 Biolegend CD14 EC P1, P3, 2 HCD14 AF700 Biolegend CD16 EC P1, P3 3G8 BV570 Biolegend 1 3G8 AF700 Biolegend CD19 EC P1, P3, 2 HIB19 PE/Cy7 Biolegend CD20 EC P1 2H7 BV650 Biolegend CD25 EC P2, 1 BC96 APC/Cy7 Biolegend CD45 EC P1, P2, P3, 1, 2 2D1 Amcyan BD Biosciences CD56 EC P1 MEM-188 PerCP/Cy5.5 BD Biosciences P2, 1 HCD-56 BV421 Biolegend CD62L EC P2 DREG-56 AF700 Biolegend CD66b EC P1, 2 1A4 PE BD Biosciences CD69 EC 1, P2 FN50 PE/Cy5 Biolegend CD80 EC 2 2D10 APC BD Biosciences CD86 EC 2 IT2.2 PE/Cy5 BD Biosciences CD123 EC 2 7G3 PE/CF594 BD Biosciences CD177 EC 2 MEM-166 FITC Biolegend FoxP3 N P2, 1 150D AF647 Biolegend HLA-DR EC P1, 2 L243(G46-6) APC/Cy7 BD Biosciences IFNα2b IC P3 7N4-1 AF647 BD Biosciences IFNγ IC P3, 1 4S.B4 AF488 Biolegend IL-2 IC P3 MQ1-17H12 APC/Cy7 Biolegend IL-4 IC P2 MP4-25D2 PE/Cy7 Biolegend P2 MP4-25D2 BV711 Biolegend 1 MP4-25D2 PE/Dazzle 594 Biolegend IL-8 IC P3 G265-8 BV510 Biolegend IL-10 IC P2, 1 JES3-19F1 PE Biolegend IL-12 p40 IC P3 eBioHP40 PerCP/eFluor 710 eBioscience IL-17 IC 1 N49-653 PerCP/Cy5.5 Biolegend Ki67 N P2 B56 AF488 BD Biosciences P2 B56 BV711 BD Biosciences 2 B56 BV786 BD Biosciences TGFβ IC 1 TWF-2F8 PE/Cy7 Biolegend TNFα IC P3 MAb11 BV711 Biolegend 1 Mab11 BV785 Biolegend TNFβ IC P3 359-81-11 PE Biolegend a Panels P1, P2, and P3 all refer to panels used in the preliminary flow studies reported in Figs 3 and 4. Panels 1 and 2 refer to panels used in the remainder of the flow Table 1. Antibodies used for flow cytometry experiments. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 6 / 27 Mosquito saliva effects These cytokines can polarize the immune system towards a Th1, Th2, or Th17 response, respectively. [26–28]. These cytokines can polarize the immune system towards a Th1, Th2, or Th17 response, respectively. [26–28]. Mosquito saliva suppresses cytokine production in human PBMC cultures Cytokines are secreted signaling proteins of the innate immune system and are crucial in establishing an effective immune response; thus, we investigated whether mosquito saliva has an effect on cytokines produced by human PBMCs ex vivo. Supernatants were collected daily from human PBMCs treated with either mosquito saliva or untreated media; cytokine levels were assessed using multiplex cytokine bead array assays. As positive controls, PBMCs were stimulated with lipopolysaccharide or pokeweed mitogen to verify that these PBMCs were able to produce cytokines (S1 Fig). Mosquito saliva-treated PBMCs had lower supernatant concentrations at all time points of most cytokines examined compared to those of the untreated control, with the most significant decreases at days 1 and 2 post treatment (Fig 2). The most significantly lowered cytokines were PDGF-AA, PDGF-BB, and RANTES, all of which are secreted in response to endothelium wall breach and promote clotting and immune cell recruitment [38, 39]. These functions would antagonize mosquito feeding; thus, suppression of these cytokines by mosquito saliva is not unexpected. However, RANTES does regulate CD8+ T cell functions during West Nile virus and HIV infections, so suppression of this cytokine by mosquito saliva could impact the anti-viral immune response [40]. We also observed a decrease in levels of IP-10 in saliva-treated PBMCs; IP-10 is a chemokine that can attract a wide variety of immune cells, including monocytes, macrophages, and dendritic cells [41]. In the context of dengue virus infection, IP-10 has been shown to have a protective effect by competitively inhibiting the access of dengue virus to heparin sulfate, a putative host cell receptor [42, 43]; thus, mosquito saliva could potentially enhance infection by decreasing IP-10 levels. Currently, we are unable to determine whether mosquito saliva actively inhibits the process of cytokine production or merely kills the cells responsible for producing the cytokines. Fig 2. Mosquito saliva decreases cytokine production in human PBMCs. Human PBMCs from two donors were stimulated with either mosquito saliva or untreated media. Supernatant cytokine concentrations were assessed daily via multiplex cytokine bead array assay. Data presented are log10- fold change of saliva stimulated PBMCs compared to media stimulated PBMCs of the same donor; the darker the color, the higher the concentration of the cytokine in the supernatant of the saliva-stimulated PBMCs. Slashed squares represent missing data. https://doi.org/10.1371/journal.pntd.0006439.g002 Fig 2. Mosquito saliva decreases cytokine production in human PBMCs. Mosquito saliva increases the frequency of natural killer T cells in human peripheral blood mononuclear cells The subset of the T cell plot represents the percentage of NKT cells that were activated. T-tests were performed using GraphPad Prism. Multiple comparisons were corrected for using the Holm-Sidak method, which increased the significance threshold to p < 0.01. #, p<0.1; ##, p<0.05; , p<0.01; p<0.005. Abbreviations: NK = Natural Killer; MF = Macrophage; DC = Dendritic Cell; DP = Double Positive. Cell markers used to define cell populations: T cells, CD45 +CD3+; NK Cells, CD45+CD3-CD56+; B Cells, CD45+CD3-CD19+; Monocytes/MFs, CD45+CD3-CD14+; Neutrophils, CD45+CD3-CD66b+; Myeloid Cells, CD45+CD3-CD11c+; DP T Cells, CD45+CD3+CD4+CD8+; CD4 T Cells, CD45+CD3+CD4+CD8-; CD8 T Cells, CD45+CD3+CD4-CD8+; NKT Cells, CD45 +CD3+CD56+; Activated NKT Cells, CD45+CD3+CD56+CD69+. https://doi.org/10.1371/journal.pntd.0006439.g001 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 7 / 27 Preliminary flow cytometry studies After observing alterations in cytokine production and NKT cell frequency in PBMCs treated with mosquito saliva, we expanded our study to investigate the effects of mosquito saliva on the immune system of an entire organism. To this end, we used the hu-NSG mouse model (humanized NOD/SCID/IL2-gamma chain null mice (NSG), generated by infusion of human hematopoietic stem cells into NSG mice), which reproduces human myeloid and human lym- phoid cell responses and is susceptible to infection by dengue virus [7, 44–47]. To determine the impact of mosquito bite on the population of various immune cells, donor matched hu- NSG mice were each bitten by an average of 4 mosquitoes on their rear footpads while under ketamine/xylazine anesthesia. Mice were euthanized at 12 hours, 24 hours, or 48 hours post- bite. Skin (rear footpads), blood, spleen, and bone marrow were examined for immune cell population changes to capture both the local and the systemic changes in response to mosquito bite. Unbitten mice served as negative controls. Figs 3 and 4 describe the changes in T cell and other leukocyte populations, respectively. To summarize our results, at 12 hours post-bite, IL-2 producing CD4 T cells (CD45+, CD3+, CD4+, IL-2+) were decreased in the blood in the exposed compared to control mice. Also at 12 hours post-bite, B cells (CD45+, CD3-, CD19+) cells were decreased in the skin. At 24 hours post-bite, NK cells (CD45+, CD3-, CD56+) were increased in the blood. Also, at 24 hours post-bite, CD8 T cells (CD45+, CD3+, CD8+) were increased in the bone mar- row while CD14+ myeloid cells (CD45+, CD3-. CD14+, CD11c+) were decreased in the bone marrow. In the skin at 24 hours post-bite, IL-12 producing monocytes and macro- phages (CD45+, CD3-, CD14+, IL-12+) were increased. Lastly, at 48 hours, CD4 T cells (CD45+, CD3+, CD4+) were decreased in the blood, and NK cells (CD45+, CD3-, CD56+) were decreased in the bone marrow. Collectively, these results indicate that mosquito saliva modulates the human immune system represented in our humanized mice, and many sig- nificant changes occur in the early hours after mosquito bite. These responses include migration of leukocytes and T cells into the blood, skin and bone marrow; this indicated that further effects could probably be measured downstream, later in time after bite. Mosquito saliva effects Mosquito saliva suppresses cytokine production in human PBMC cultures Human PBMCs from two donors were stimulated with either mosquito saliva or untreated media. Supernatant cytokine concentrations were assessed daily via multiplex cytokine bead array assay. Data presented are log10- fold change of saliva stimulated PBMCs compared to media stimulated PBMCs of the same donor; the darker the color, the higher the concentration of the cytokine in the supernatant of the saliva-stimulated PBMCs. Slashed squares represent missing data. https://doi.org/10.1371/journal.pntd.0006439.g002 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 8 / 27 Mosquito bite impacts lymphocyte and myeloid cells across multiple tissues Because our preliminary flow cytometry study revealed that mosquito bite impacts the immune system of the hu-NSG mice, we performed a similar experiment using different time points (6 hours, 24 hours, and 7 days post-bite) to deepen our knowledge of the effects of mos- quito saliva on the innate (6 hours, 24 hours) and adaptive (7 days) immune responses. For experiments involving these new time points, mice were bitten and euthanized and their tis- sues collected as in the preliminary flow cytometry study. Figs 5 and 6, along with Tables 2 and 3, describe the changes in T cell and other leukocyte populations, respectively. At 6 hours post-bite in the blood, we observed an increase in NKT cells (CD45+, CD3+, CD56+), CD8 T cells (CD45+, CD3+, CD4-, CD8+), and DP T cells (CD45+, CD3+, CD4+, CD8+), which all have cytotoxic capabilities, and a decrease in regulatory T cells (Tregs; CD45+, CD3+, CD4+, FoxP3+), which can dampen inflammatory responses through secre- tion of IL-10. Also, B cells (CD45+, CD3-, CD19+), NK cells (CD45+, CD3-, CD56+) and myeloid cells (CD45+, CD3-, CD11c+; dendritic cells, monocytes, macrophages, neutro- phils) were increased at 6 hours in the blood. The increase of B cells we observed is likely a result of increased production of B cells in the bone marrow. Most myeloid cells are anti- gen-presenting cells; their increase in the blood may indicate that they are travelling either to or from the bite site. In the spleen, there was an increase in NK cells, but a decrease in the PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 9 / 27 Mosquito saliva effects Fig 3. Preliminary assessment of mosquito saliva on human T cell populations in hu-NSG mice. Hu-NSG mice were each bitten on their rear footpads by approximately four mosquitoes. Unbitten mice were used as controls. At 12, 24, or 48 hours post-bite, mice were euthanized and the indicated tissues were collected. Human T cell populations were assessed via flow cytometry. Graphs show the mean frequency of immune cell populations (with outliers removed) as a percentage of Mosquito saliva effects Fig 3. Preliminary assessment of mosquito saliva on human T cell populations in hu-NSG mice. Hu-NSG mice were each bitten on their rear footpads by approximately four mosquitoes. Unbitten mice were used as controls. At 12, 24, or 48 hours post-bite, mice were euthanized and the indicated tissues were collected. Mosquito saliva effects human CD45+ cells in bitten (n = 6, 12 or 48 hours post-bite; n = 5, 24 hours post-bite) or control mice (n = 4, 12 or 48 hours post-bite; n = 5, 24 hours post-bite). Error bars represent 1 SEM. T-tests were performed using Graphpad Prism. Multiple comparisons were corrected for using the Holm-Sidak method, which increased the significance threshold to p < 0.01. #, p<0.1; ##, p<0.05; , p<0.01; p<0.005. Cell markers used to define cell populations: T Cells, CD45+CD3+; Proliferating T Cells, CD45+CD3+Ki67+; CD4 T Cells, CD45+CD3+CD4+; Proliferating CD4 T Cells, CD45+CD3+CD4+Ki67+; CD8 T cells, CD45+CD3+CD8+; Activated CD8 T Cells, CD45+CD3+CD8+CD69-; Proliferating CD8 T Cells, CD45+CD3+CD8+Ki67+. https://doi.org/10.1371/journal.pntd.0006439.g003 https://doi.org/10.1371/journal.pntd.0006439.g003 total number of T cells (CD45+, CD3+), and in the bone marrow there were decreases in both total T cells and Tregs. Considering that we observed an increase in multiple T cell subsets in the blood, the reduction of T cells in these two organs could be a result of migra- tion to the blood. Taken together, these results indicate that the immune system is not at steady-state and may be shifting toward a Th1 response; future studies will verify that con- clusion by determining which cytokines, if any, are being produced by the DP T, NKT, NK, and CD11c+ cells. At 24 hours post-bite, Tregs were increased in the blood and decreased in the spleen. The decrease of Tregs in the spleen is coincident with an increase of Tregs in the blood and could indicate that the Tregs migrated from the spleen to the blood. If the increase of Tregs in the blood were associated with an increase in serum IL-10 levels, then this could indicate an anti- inflammatory response. In the skin, DP T cells were decreased at 24 hours post-bite. Because DP T cells are often associated with inflammatory responses, this could indicate a decrease in inflammation in the skin. We also saw a decrease in myeloid cells in the spleen and NK cells in the bone marrow. Taken together, these results indicate a shift towards an anti-inflammatory response. At 7 days post-bite, DP T cells were increased in the blood, skin, and bone marrow. These DP T cells are typically found at sites of inflammation, indicating that there may be skin At 7 days post-bite, DP T cells were increased in the blood, skin, and bone marrow. These DP T cells are typically found at sites of inflammation, indicating that there may be skin inflammation occurring at this time point. There was also a decrease in Tregs and an increase in NK cells in the skin, providing a further indication of inflammation in the skin. We also saw a decrease in neutrophils and myeloid cells in the blood. Because inflammation and DP T cells are associated with both Th1 and Th2 responses, further experiments will need to be done to determine the role of DP T cells following mosquito bite. We saw a decrease in neutrophils and myeloid cells in the blood and a decrease in myeloid cells and monocytes and macro- phages cells in the bone marrow. Lastly, there was an increase in CD8 T cells but a decrease in myeloid cells in the spleen. CD8 T cells are typically associated with a Th1 response and can produce large amounts of pro-inflammatory cytokines. Further experiments will determine whether mosquito saliva activates these CD8 T cells and stimulates them to produce cytokines. While T cells and NK cells indicate an inflammatory response, the overall data suggest a mixed Th1/Th2 response. Mosquito bite impacts lymphocyte and myeloid cells across multiple tissues Human T cell populations were assessed via flow cytometry. Graphs show the mean frequency of immune cell populations (with outliers removed) as a percentage of Fig 3. Preliminary assessment of mosquito saliva on human T cell populations in hu-NSG mice. Hu-NSG mice were each bitten on their rear footpads by approximately four mosquitoes. Unbitten mice were used as controls. At 12, 24, or 48 hours post-bite, mice were euthanized and the indicated tissues were collected. Human T cell populations were assessed via flow cytometry. Graphs show the mean frequency of immune cell populations (with outliers removed) as a percentage of PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 10 / 27 https://doi.org/10.1371/journal.pntd.0006439.g003 Mosquito saliva effects Human leukocyte populations were assessed via flow cytometry. Graphs show the mean frequency of immune cell populations (with outliers removed) as a percentage of human CD45+ cells in bitten (n = 6, 12 or 48 hours post-bite; n = 5, 24 hours post-bite) or control mice (n = 4, 12 or 48 hours post-bite; n = 5, 24 hours post-bite). Error bars represent 1 SEM. T-tests were performed using Graphpad Prism. Multiple comparisons were corrected for using the Holm-Sidak method, which increased the significance threshold to p < 0.01. #, p<0.1; ##, p<0.05; , p<0.01; p<0.005. Abbreviations: DC = Dendritic Cell; MF = Macrophage; NK = Natural Killer. Cell Markers used to define cell populations: Myeloid Cells, CD45+CD3-CD11c+; Activated Myeloid Cells, CD45+CD3-CD11c+HLADR+; Monocytes, CD45+,CD3-,CD11c+,CD11b+; B Cells, CD45+CD3-CD19+; NK Cells, CD45+CD3-CD56+; Activated NK Cells, CD45+CD3-CD56+CD69+; Proliferating NK Cells, CD45+CD3-CD56+Ki67+; Monocytes/MFs, CD45+CD3-CD14+. https://doi.org/10.1371/journal.pntd.0006439.g004 Despite a lack of significant changes across broad classes of cytokines, we did see significant changes in concentrations of specific cytokines. At 6 hours post-bite, we saw an increase in the pro-inflammatory cytokine IL-1α, but a decrease in the pro-inflammatory cytokines IL-12p40 and TNFα. In addition to promoting inflammation, IL-12p40 stimulates the production of IFNγ (the major Th1 cytokine) by NK cells and T cells [48]. We also saw a decrease in the Th2 cytokines IL-5 and IL-9 at 6 hours post- bite. Both of these cytokines are produced by T cells [49, 50]; from our examination of T cell populations in the hu-NSG mice following mosquito bite (Fig 5), we saw a significant decrease in total T cells in the spleen and bone marrow and a trend toward a decrease in total T cells in the skin and blood at 6 hours post-bite. This decrease in T cells may account for the decreased serum concentrations of IL-5 and IL-9. We also saw a decreased serum concentration of the chemokines IL-8, MCP-1, and MDC at this time point. Lastly, we saw an increase in the growth factor VEGF at this time point; VEGF is often released in response to endothelium wall breach to stimulate the growth of endothelial cells [51, 52]. Since the mosquito bites likely caused limited vascular injury in the mice, it is not unexpected that we observed an increase in serum VEGF in the bitten mice compared to the control mice. Mosquito bite alters cytokine levels in hu-NSG mice To further our examination of mosquito bites on the immune system of humanized mice, we investigated whether serum levels of human cytokines changed in mosquito-bitten mice. Serum was collected at 6 hours, 24 hours, and 7 days post-bite from the same mice used to examine immune cell populations, and cytokine levels were measured via multiplex cytokine bead array assays. Fig 7A shows the fold-change of each bitten mouse compared to the mean of the control group. Cytokines were grouped by the following functions to assist in analysis: chemokines, growth factors pro-inflammatory cytokines, anti-inflammatory cytokines, Th1 cytokines, and Th2 cytokines. A majority of the bitten mice did not appear to differ from the control in cytokine levels; however, the fold change differences in Th2 cytokines and anti- inflammatory cytokines increased over time. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 11 / 27 Mosquito saliva effects Fig 4. Preliminary assessment of mosquito saliva on human leukocyte populations in hu-NSG mice. Hu-NSG mice were each bitten on their rear footpads by approximately four mosquitoes. Unbitten mice were used as controls. At 12, 24, or 48 hours post-bite, mice were euthanized and the indicated tissues were collected. Mosquito saliva effects Fig 4. Preliminary assessment of mosquito saliva on human leukocyte populations in hu-NSG mice. Hu-NSG mice were each bitten on their rear footpads by approximately four mosquitoes. Unbitten mice were used as controls. At 12, 24, or 48 hours post-bite, mice were euthanized and the indicated tissues were collected. Fig 4. Preliminary assessment of mosquito saliva on human leukocyte populations in hu-NSG mice. Hu-NSG mice were each bitten on their rear footpads by approximately four mosquitoes. Unbitten mice were used as controls. At 12, 24, or 48 hours post-bite, mice were euthanized and the indicated tissues were collected. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 12 / 27 To summarize, the serum cytokine concentrations at 6 hours post- bite indicate a dysregula- tion of inflammatory signaling, a decrease in Th1 and Th2 signaling, an alteration in chemo- tactic signaling, and an increase in endothelium repair signaling. g g p g g At 24 hours post-bite, we saw an increase in the pro-inflammatory and anti-viral cytokine IFNα [53]. This cytokine is usually produced in response to viral infection, so its presence after uninfected mosquito bite is unexpected. To our knowledge, this is the first report of IFNα pro- duction following mosquito bite. We saw a decrease in the anti-inflammatory cytokine IL- 1RA, a cytokine that competitively inhibits IL-1α and IL-1β from binding to the IL-1 receptor [54, 55]. The increase in this cytokine may be an attempt to regulate the increased IL-1α observed at 6 hours post-bite. We saw an increase in the Th2 cytokine IL-9; this increase con- trasts with our observation at 6 hours post-bite where a decrease in IL-9 was coincident with a decrease in the number of total T cells. At 24 hours post-bite, numbers of total T cells in the bitten mice is equivalent to that of the control mice, which may account for the increase in serum concentrations of IL-9. We saw an increase in the chemokines fractalkine and MDC. An increase in these chemokines in the serum could induce an influx monocytes and other cells that express the appropriate chemokine receptors into the blood [56–58]. We did not see any shifts in monocyte populations in the mice at 24 hours post-bite, but perhaps this shift occurred at a time point that was not analyzed. Lastly, we saw an increase in the growth factors VEGF and IL-3. As previously mentioned, VEGF is involved in endothelium wall repair; IL-3 is also involved in endothelium wall repair and promotes the production of platelets, which are consumed during an endothelium wall breach [59]. To summarize, the serum cytokines present at 24 hours post-bite indicate an interferon response, an anti-inflammatory response, a Th2 response, an influx of monocytes, and a continued endothelium repair response. At 7 days post-bite, we saw a decrease in the pro-inflammatory cytokine sCD40L in the bit- ten mice compared to the control mice. In addition to stimulating an inflammatory response, this cytokine is also important for immunoglobulin class switching by B cells [60–62]. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 In the PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 13 / 27 Mosquito saliva effects Fig 5. Mosquito saliva changes human T cell populations in hu-NSG mice. Hu-NSG mice were each bitten by approximately four mosquitoes on a rear footpad. Unbitten mice were used as controls. At 6 hours post-bite, 24 hours post-bite, or 7 days post-bite, mice were euthanized and indicated tissues were collected. Immune cell populations were assessed via flow cytometry. Graphs show mean cell counts of bitten (n = 12) and control groups (n = 12) (with outliers removed) as the number of cells per tissue type. Error bars represent 1 SEM. T-tests were performed using GraphPad Prism. Multiple comparisons were corrected for using the Holm-Sidak method, which increased the significance threshold to p < 0.01. #, p<0.1; ##, p<0.05; , p<0.01; p<0.005. Abbreviations: DP = Double Positive; NK = Natural Killer. Cell markers used to define cell populations: DP T Cells, CD45+CD3+CD4+CD8+; CD4 T Cells, CD45+CD3+CD4 +CD8-; Regulatory T Cells, CD45+CD3+CD4+CD8-FoxP3+; CD8 T Cells, CD45+CD3+CD4-CD8+; NKT Cells, CD45+CD3+CD56+. https://doi.org/10.1371/journal.pntd.0006439.g005 Fig 5. Mosquito saliva changes human T cell populations in hu-NSG mice. Hu-NSG mice were each bitten by approximately four mosquitoes on a rear footpad. Unbitten mice were used as controls. At 6 hours post-bite, 24 hours post-bite, or 7 days post-bite, mice were euthanized and indicated tissues were collected. Immune cell populations were assessed via flow cytometry. Graphs show mean cell counts of bitten (n = 12) and control groups (n = 12) (with outliers removed) as the number of cells per tissue type. Error bars represent 1 SEM. T-tests were performed using GraphPad Prism. Multiple comparisons were corrected for using the Holm-Sidak method, which increased the significance threshold to p < 0.01. #, p<0.1; ##, p<0.05; , p<0.01; p<0.005. Abbreviations: DP = Double Positive; NK = Natural Killer. Cell markers used to define cell populations: DP T Cells, CD45+CD3+CD4+CD8+; CD4 T Cells, CD45+CD3+CD4 +CD8-; Regulatory T Cells, CD45+CD3+CD4+CD8-FoxP3+; CD8 T Cells, CD45+CD3+CD4-CD8+; NKT Cells, CD45+CD3+CD56+. https://doi.org/10.1371/journal.pntd.0006439.g005 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 14 / 27 Mosquito saliva effects Fig 6. Mosquito saliva alters human leukocyte populations in hu-NSG mice. Hu-NSG mice were each bitten by four mosquitoes on a rear footpad. Unbitten mice were used as controls. At 6 hours post-bite, 24 hours post-bite, or 7 days post-bite, mice were euthanized and indicated tissues were collected. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 Mosquito saliva effects Table 2. Changes in T cell populations in bitten hu-NSG mice compared to control mice. 6 hours post-bite 24 hours post-bite 7 days post-bite Blood # Regulatory T Cells " DP T Cells " CD8+ T Cells " NKT Cells " Regulatory T Cells " DP T Cells Skin No Significant Changes # DP T Cells # Regulatory T Cells " DP T Cells Spleen # Total T Cells # Regulatory T Cells " CD8+ T Cells Bone marrow # Total T Cells # Regulatory T Cells No Significant Changes " DP T Cells https://doi.org/10.1371/journal.pntd.0006439.t002 Table 2. Changes in T cell populations in bitten hu-NSG mice compared to control mice. context of a viral infection, decreasing or preventing immunoglobulin class switching could be detrimental for the host. We saw an increase in serum concentrations of the chemokine frac- talkine and a decrease in the chemokines IL-8 and MCP-1. Since both fractalkine and MCP-1 attract similar cell types (namely monocytes), it is difficult to predict whether changes in con- centrations of these two chemokines affect efflux or influx of cells into the blood [56, 57, 63]. While we did not see a significant change in monocyte numbers in the blood, we did see a decrease of monocytes in the bone marrow at this time point. Also, the decrease in IL-8 in the serum did correspond with a decrease in neutrophils (the target of IL-8) in the blood and a trend toward an increase in neutrophils in the skin [64]. We also observed an increase in the growth factors IL-3 and IL-7. IL-7 enhances the growth of early B and T cells and stimulates the proliferation of mature T cells [65, 66]. This increase in IL-7 correlated with an increase in CD8 T cells in the spleen and DP T cells in the blood, skin, and bone marrow. To summarize, at 7 days post-bite, we saw a decrease in pro-inflammatory cytokines, an increase in some but a decrease in other chemokines, and an increase in growth factors. g We were unable to detect the following cytokines in the serum of either the bitten or the control mice at any of the time points: Flt-3 ligand, GRO, IL-1β, IL-2, IL-4, IL-6, IL-13, IL-15, MCP3, TGFα and TNFβ. Of these, GRO, IL-2, IL-4, IL-6, IL-13 and TGFα can be detected in the serum of healthy humans [67–70]. Immune cell populations were assessed via flow cytometry. Graphs show mean cell counts of bitten (n = 12) and control groups (n = 12) (with outliers removed) as the number of cells per tissue type. Error bars represent 1 SEM. T-tests were performed using GraphPad Prism. Multiple comparisons were corrected for using the Holm-Sidak method, which increased the significance threshold to p < 0.01. #, p<0.1; ##, p<0.05; , p<0.01; p<0.005. Abbreviations: NK = Natural Killer, Mϕ = Macrophage; DC = Dendritic Cell. Cell markers used to define cell populations: Hematopoietic Cells, CD45+; T Cells, CD45+CD3+; NK Cells, CD45 +CD3-CD56+; B Cells, CD45+CD3-CD19+; Monocytes/MFs, CD45+CD3-CD14+; Neutrophils, CD45+CD3-CD66b+; Myeloid Cells, CD45+CD3-CD11c+; Other, CD45+ but negative for all other markers examined. https://doi.org/10.1371/journal.pntd.0006439.g006 Fig 6. Mosquito saliva alters human leukocyte populations in hu-NSG mice. Hu-NSG mice were each bitten by four mosquitoes on a rear footpad. Unbitten mice were used as controls. At 6 hours post-bite, 24 hours post-bite, or 7 days post-bite, mice were euthanized and indicated tissues were collected. Immune cell populations were assessed via flow cytometry. Graphs show mean cell counts of bitten (n = 12) and control groups (n = 12) (with outliers removed) as the number of cells per tissue type. Error bars represent 1 SEM. T-tests were performed using GraphPad Prism. Multiple comparisons were corrected for using the Holm-Sidak method, which increased the significance threshold to p < 0.01. #, p<0.1; ##, p<0.05; , p<0.01; p<0.005. Abbreviations: NK = Natural Killer, Mϕ = Macrophage; DC = Dendritic Cell. Cell markers used to define cell populations: Hematopoietic Cells, CD45+; T Cells, CD45+CD3+; NK Cells, CD45 +CD3-CD56+; B Cells, CD45+CD3-CD19+; Monocytes/MFs, CD45+CD3-CD14+; Neutrophils, CD45+CD3-CD66b+; Myeloid Cells, CD45+CD3-CD11c+; Other, CD45+ but negative for all other markers examined. https://doi.org/10.1371/journal.pntd.0006439.g006 ed Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 15 / 27 T cells are the primary producers of IL-2, IL-4, IL-6, and IL-13 [71–78]. Because hu-NSG mice do not have a human thymus, the T cells present in these mice are not HLA restricted to human cells and may not respond appropriately to signals from other human immune cells in the mice. This could account for the lack of these cytokines in the serum. Both GRO and TGFα are produced by keratinocytes and monocytes in the skin [79, 80]. Since hu-NSG mice do not have human keratinocytes, these mice would produce low to no levels of GRO and TGFα. Lastly, since we only investigated cytokines present in the serum of these mice, our data do not exclude the possibility that these cytokines were produced in the tissues. Future studies will investigate what cytokines are produced at the site of mos- quito bite and at other tissues throughout the body. Table 3. Changes in B cells, natural killer cells, and myeloid cells in bitten hu-NSG mice compared to control mice. 6 hours post-bite 24 hours post-bite 7 days post-bite Blood " B cells " NK Cells " Myeloid Cells No significant changes # Neutrophils # Myeloid Cells Skin No Significant Changes No significant Changes " NK Cells Spleen " Natural Killer Cells # Myeloid Cells # Myeloid Cells Bone Marrow No Significant Changes # NK Cells # Monocytes/Macrophages # Myeloid Cells h //d i /10 13 1/j l d 0006439 003 e 3. Changes in B cells, natural killer cells, and myeloid cells in bitten hu-NSG mice compared to control mice. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 16 / 27 Discussion Mosquitoes deposit numerous salivary proteins into their host’s skin while acquiring a blood meal. Past work has shown that mosquito saliva enhances the pathogenicity of dengue, West Nile, and other arbovirus infections. From these results, we hypothesized that mosquito saliva modulates the host immune system in a way that promotes arbovirus replication and transmis- sion. In this work, we used human PBMCs treated with mosquito saliva and humanized mice bitten by mosquitoes to determine what effects mosquito saliva alone has on the human immune system. Our results suggest that the human immune response to mosquito saliva is significant and complex: mosquito saliva alters the frequencies of several immune cell popula- tions, in multiple tissues, at several times after blood feeding. Mosquito saliva also affects serum cytokine levels, with the most notable trend being an increase in anti-inflammatory and Th2 cytokines compared to unbitten, control mice, at 7 days post- bite. The fact that these effects last up to 7 days post-bite is especially interesting, and is of concern in the context of allergic reactions. The long-lasting effects in the humanized bone marrow and skin cells could explain how some of the viruses transmitted by mosquitoes could possibly still be viable in these tissues, or how they could serve as replication reservoirs. Relatively few studies have investigated the effects of mosquito saliva on human cells ex vivo; fewer have investigated cytokine production from human cells following stimulation with mosquito saliva. Salivary gland extracts from the Armigeres subalbatus mosquito (a vector of Japanese encephalitis and West Nile viruses) induce apoptosis in human PBMCs and THP- 1 cells (human monocytic cell line) [81]. In another ex vivo study, monocyte-derived dendritic cells differentiated from human PBMCs produced higher amounts of the cytokine IL-12p70 when stimulated with mosquito saliva collected from Ae. aegypti mosquitoes [82]. In a third study, mosquito saliva from Anopheles stephensi mosquitoes (a vector of malaria) induced the degranulation of the human mast cell line LAD-2 [83]. We did not detect a significant decrease in cell populations or an increase in IL-12p70 production in our ex vivo studies; however, this is most likely due to differences in study design. Mosquito saliva effects Fig 7. Mosquito saliva alters concentrations of select serum cytokines in hu-NSG mice. (A) Serum cytokine production in hu-NSG mice following mosquito bite. Hu-NSG mice were each bitten by approximately four mosquitoes on a rear footpad. Unbitten mice were used as controls. At the indicated times, mice were euthanized and serum cytokine levels were determined via multiplex cytokine bead array assay. Data presented are the log10 fold change of specific cytokine concentrations in each bitten mouse compared to the mean concentration of the control mice. Darker colors represent a higher concentration of a given cytokine in the serum of the bitten mice. Pure black represents a positive 1 log10 fold change (highest possible), while pure white represents a negative 1 log10 fold change (lowest possible). Slashed squares represent missing data or outlier data. Data for the following cytokines were not included in the analysis because all mice had serum concentrations below the limit of detection: Flt3 ligand, GRO, IL-1β, IL-2, IL-4, IL-6, IL-13, IL-15, MCP3, TGFα, and TNFβ. (B) Significant differences in serum cytokine levels between bitten mice (black) and control mice (gray). Graphs show mean concentrations (pg/mL) of cytokines; error bars are 1 SEM. T-tests were performed on serum cytokine concentration data (with outliers removed) from (A) using GraphPad Prism. Multiple comparisons were corrected for using the Holm-Sidak method, which increased the significance threshold to p < 0.01. #, p<0.1; ##, p<0.05; , p<0.01; p<0.005. https://doi.org/10.1371/journal.pntd.0006439.g007 Mosquito saliva effects PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 17 / 27 Mosquito saliva effects cells after mosquito bite; these decreases may point toward a Th2 response. When analyzing the serum cytokine response, we did observe a significant trend toward Th2 cytokine produc- tion at 7 days post-bite. Th2 immune responses occur most often in response to parasitic infec- tions or allergen exposure, and they tend to dampen inflammatory and cytotoxic responses, both of which are needed to clear viral infections. Thus, if mosquito saliva were to trigger a Th2 response in the context of an arbovirus infection, the virus would be able to replicate to higher levels than it would in the presence of a Th1 response. In most arbovirus infections, higher viral load corresponds to more severe disease. Thus, a saliva-induced Th2 immune response could explain observations in experimental models of arbovirus infections that mos- quito saliva enhances pathogenesis. At various times post mosquito bite, we also observed increases in cell populations typically associated with a Th1 immune response: natural killer T cells, natural killer cells, and CD8+ T cells. While these cells can kill virus-infected cells and are generally important for clearing viral infections, they have been implicated in increased disease severity of dengue infection in humans [85]. In cases of dengue hemorrhagic fever in human children, increased numbers of CD69+ natural killer cells, CD4+ T cells, and CD8+ T cells have been observed [86–88]. Some speculate that the increase in these cell populations is a response to severe infection, not a mechanism of pathogenesis; however CD8+ T cells isolated from these severely infected indi- viduals were primed towards producing large amounts of pro-inflammatory cytokines and not towards killing viral infected cells [88, 89]. Further investigations will need to be done to deter- mine whether the increased CD8+ T cell populations we observed were pro-inflammatory CD8+ T cells. Nevertheless, the possibility remains that even though mosquito saliva increases some subsets of immune cells typically associated with a Th1 immune response, these cells could be responsible for increased disease severity in humans. Our study is not the only one to show that mosquito saliva produces a mixed Th1/Th2 response. Pingen et al discovered that while a mosquito bite does induce some elements of a Th2 response, it did not prevent elements of a Th1 response in C57BL/6 mice [90]. Discussion We used mosquito saliva as opposed to sali- vary gland extract (a heterogeneous mixture of saliva and the cells that make it) and mono- cyte-derived dendritic cells and mast cells are rarely found in the blood and therefore would not be a component of our human PBMCs. Other mouse studies of the effects of mosquito saliva on the immune system concluded that mosquito saliva primes the immune response toward a Th2 response (reviewed in [84]); how- ever, our results indicate a mixed Th1/Th2 response. We observed increases in both regulatory T cells and CD4/CD8 double positive T cells. As these cells are rare, and more potent cytokine producers and killers than conventional single positive T cells [18–22], it is interesting to spec- ulate that mosquito saliva may be uniquely capable to induce the differentiation of single posi- tive T cells into this cytotoxic double positive T cell subset. Further studies are needed to examine whether these unique double positive T cells play an important role in host protection from flavivirus infection. Furthermore, we noticed decreases in total T cells and natural killer 18 / 27 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 Supporting information pp g S1 Fig. Stimulation with pokeweed mitogen or LPS alters immune cell population frequen- cies and cytokine production in human PBMCs. Human PBMCs were isolated from two different human donors and stimulated with mosquito saliva, pokeweed mitogen, LPS, or untreated media. Pokeweed mitogen and LPS were used to confirm that the human PBMCs used in these experiments were capable of responding to immunomodulatory compounds. (A) Immune cell population changes following stimulation with pokeweed mitogen and LPS. Stimulated human PBMCs were analyzed via flow cytometry five days post stimulation. Data presented are the mean frequencies of individual cell populations as a percentage of CD45+ cells. The subset of the T cell plot represents the percentage of NKT cells that were activated. Error bars represent 1 standard error of the mean. Abbreviations: NK = Natural Killer; MF = Macrophage; DC = Dendritic Cell; DP = Double Positive. Cell markers used to define cell pop- ulations: T cells, CD45+CD3+; NK Cells, CD45+CD3-CD56+; B Cells, CD45+CD3-CD19+; Monocytes/MFs, CD45+CD3-CD14+; Neutrophils, CD45+CD3-CD66b+; DCs/Monocytes/ MFs, CD45+CD3-CD11c+; DP T Cells, CD45+CD3+CD4+CD8+; CD4 T Cells, CD45+CD3+ CD4+CD8-; CD8 T Cells, CD45+CD3+CD4-CD8+; NKT Cells, CD45+CD3+CD56+; Acti- vated NKT Cells, CD45+CD3+CD56+CD69+. (B) Cytokine production by human PBMCs fol- lowing stimulation with pokeweed mitogen and LPS. Supernatants from stimulated human PBMCs were collected daily up to five days post stimulation and analyzed for cytokine concen- trations. Data shown are the log10 fold change of each of the stimulated conditions compared to the media stimulated control for the each donor and time point; the darker the color, the higher production of cytokines in treated PBMCs (saliva, pokeweed mitogen, or LPS). Areas with slashes represent samples where no data were collected for that cytokine. (TIF) S2 Fig. Gating strategy for flow cytometry experiments. These flow charts describe the gat- ing strategies used to analyze flow cytometry data in this study. (A) This was the gating strategy used for the data presented in Figs 3 and 4. Grey boxes represent gates that were made during the analysis of all three panels. Blue, red, and yellow boxes represent gates that were made dur- ing the analysis of Panels P1, P2, and P3, respectively. Green boxes represent gates that were PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 20 / 27 S1 Fig. Stimulation with pokeweed mitogen or LPS alters immune cell population frequen- cies and cytokine production in human PBMCs. Mosquito saliva effects mosquito bite [44, 47, 96]. In addition, we expect to test for the biological significance of these immune cell changes, which might lead to stimulation of infected cells to migrate to important sanctuary tissues (e.g., bone marrow or brain), where viral reservoirs could be established, away from the full forces of the immune system. In the case of arboviruses, many establish infections in brain or bone marrow cells of human patients, leading to specific pathologies such as encephalitis or bone loss and leukopenia and thrombocytopenia [97–99]. It is currently unclear if mosquito saliva contributes to these tissue infections and more severe pathologies. Mosquitoes and the diseases they transmit are of growing public health concern. Often, there are no prophylaxes for these diseases other than mosquito control and no treatments other than palliative care. Understanding how mosquito saliva interacts with the human immune system not only helps us understand mechanisms of disease pathogenesis but also could provide possibilities for treatments. If we know which mosquito saliva components enhance pathogenesis of diseases, we could create a human vaccine to counteract these effects for multiple arbovirus infections. A similar approach has been used to vaccinate and protect mice against a sandfly saliva protein (maxadilan) that enhances the infection and progression of Leishmania major [100]. These approaches have been commercialized and used to interrupt tick transmission of cattle diseases [101], and we expect that the definition of these factors would help provide the same approaches in humans. They con- cluded that mosquito saliva enhances disease by recruiting arbovirus-susceptible cells to the bite site, in their model of Semliki Forest virus infection. Zeidner et al. reached the same con- clusion, in "flavivirus-susceptible" (C3H) mice bitten by Aedes mosquitoes, with some T cells and cytokines detectable in the serum up to 7 days post-bite; however, this study did not include flow cytometry, for identification of cell subsets, and these mice are not good models of human infection [34]. In our study, we observed an increase in monocytes and macrophages to the bite site, but only after 7 days post-bite. In the context of arbovirus infection, the migra- tion we observed would be too late to impact the initial replication or dissemination of the virus but could allow for infected cells to return to the skin where they could transmit the virus to new mosquitoes. This is a significant observation that could possibly explain epidemiologic events, where arbovirus transmission is often limited to distinct, small areas of hypertransmis- sion, and where non-viremic hosts could be sources of infection [91–93]. All of the mouse studies of mosquito saliva effects reported previously have used mice that differ from human immune systems: AG129 (which lack type I and type II interferon recep- tors), BALB/c mice (which are predisposed to Th2 immune responses), and C57BL/6 mice (which are predisposed to Th1 immune responses) [32, 33, 90, 94]. The mouse model we used in this study does not have the same limitations because these mice have been reconstituted with a human immune system, including innate immunity [95]. However, this model does not have either a full human complement system or a fully functional T cell compartment [95]. In future studies, we seek to address these limitations by using other types of reconstituted humanized mice (e.g., BLT, DRAG), which would reproduce different parts of the human immune system (such as active T helper cells, immunoglobulin class-switching, etc.) following PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 19 / 27 Author Contributions Conceptualization: Megan B. Vogt, Silke Paust, Rebecca Rico-Hesse. Data curation: Megan B. Vogt. Formal analysis: Megan B. Vogt, Silke Paust, Rebecca Rico-Hesse. Funding acquisition: Rebecca Rico-Hesse. Funding acquisition: Rebecca Rico-Hesse. Investigation: Megan B. Vogt, Anismrita Lahon, Ravi P. Arya, Alexander R. Kneubehl, Jenni- fer L. Spencer Clinton, Rebecca Rico-Hesse. Methodology: Megan B. Vogt, Anismrita Lahon, Ravi P. Arya, Alexander R. Kneubehl, Jenni- fer L. Spencer Clinton, Rebecca Rico-Hesse. Project administration: Rebecca Rico-Hesse. Project administration: Rebecca Rico-Hesse. Supervision: Silke Paust, Rebecca Rico-Hesse. Validation: Silke Paust, Rebecca Rico-Hesse. Visualization: Megan B. Vogt, Alexander R. Kneubehl, Silke Paust, Rebecca Rico-Hesse. Writing – original draft: Megan B. Vogt, Silke Paust, Rebecca Rico-Hesse. Writing – review & editing: Megan B. Vogt, Alexander R. Kneubehl, Jennifer L. Spencer Clin- ton, Silke Paust, Rebecca Rico-Hesse. S1 Table. List of humanized mice used in these experiments. Mice are listed according to experimental group, with mouse ID, sex, and human CD45+ engraftment levels given. (DOCX) S1 Table. List of humanized mice used in these experiments. Mice are listed according to experimental group, with mouse ID, sex, and human CD45+ engraftment levels given. (DOCX) Acknowledgments The authors would like to thank Mayra Sanabria-Hernandez, Enid Mondragon, Donovan Berens, and Kassandra Peterson for their help in humanized mice production, flow cytometry, and mosquito rearing. We thank Chih-Wei Hsu for stereomicroscope photography. The authors would like to thank Mayra Sanabria-Hernandez, Enid Mondragon, Donovan Berens, and Kassandra Peterson for their help in humanized mice production, flow cytometry, and mosquito rearing. We thank Chih-Wei Hsu for stereomicroscope photography. Mosquito saliva effects made during the analysis of both Panels P1 and P3, and orange boxes represent gates that were made during the analysis of both Panels P2 and P3. (B) This was the gating strategy used for the data presented in Figs 1, 5 and 6. Grey boxes represent gates that were made during the analysis of both Panel 1 and Panel 2 data. Red boxes represent gates that were made only dur- ing the analysis of Panel 1 data. Blue boxes represent gates that were made only during the analysis of Panel 2 data. Boxes containing italicized text represent gates that were only used in the analysis of humanized mice samples and not in the analysis of human PBMC samples. (TIF) Supporting information Human PBMCs were isolated from two different human donors and stimulated with mosquito saliva, pokeweed mitogen, LPS, or untreated media. Pokeweed mitogen and LPS were used to confirm that the human PBMCs used in these experiments were capable of responding to immunomodulatory compounds. (A) Immune cell population changes following stimulation with pokeweed mitogen and LPS. Stimulated human PBMCs were analyzed via flow cytometry five days post stimulation. Data presented are the mean frequencies of individual cell populations as a percentage of CD45+ cells. The subset of the T cell plot represents the percentage of NKT cells that were activated. Error bars represent 1 standard error of the mean. Abbreviations: NK = Natural Killer; MF = Macrophage; DC = Dendritic Cell; DP = Double Positive. Cell markers used to define cell pop- ulations: T cells, CD45+CD3+; NK Cells, CD45+CD3-CD56+; B Cells, CD45+CD3-CD19+; Monocytes/MFs, CD45+CD3-CD14+; Neutrophils, CD45+CD3-CD66b+; DCs/Monocytes/ MFs, CD45+CD3-CD11c+; DP T Cells, CD45+CD3+CD4+CD8+; CD4 T Cells, CD45+CD3+ CD4+CD8-; CD8 T Cells, CD45+CD3+CD4-CD8+; NKT Cells, CD45+CD3+CD56+; Acti- vated NKT Cells, CD45+CD3+CD56+CD69+. (B) Cytokine production by human PBMCs fol- lowing stimulation with pokeweed mitogen and LPS. Supernatants from stimulated human PBMCs were collected daily up to five days post stimulation and analyzed for cytokine concen- trations. Data shown are the log10 fold change of each of the stimulated conditions compared to the media stimulated control for the each donor and time point; the darker the color, the higher production of cytokines in treated PBMCs (saliva, pokeweed mitogen, or LPS). Areas with slashes represent samples where no data were collected for that cytokine. (TIF) S2 Fig. Gating strategy for flow cytometry experiments. These flow charts describe the gat- ing strategies used to analyze flow cytometry data in this study. (A) This was the gating strategy used for the data presented in Figs 3 and 4. Grey boxes represent gates that were made during the analysis of all three panels. Blue, red, and yellow boxes represent gates that were made dur- ing the analysis of Panels P1, P2, and P3, respectively. Green boxes represent gates that were 20 / 27 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006439 May 17, 2018 Mosquito saliva effects 2. WHO Factsheet Vector-borne diseases Factsheet #387: World Health Organization; 2014. Available from: http://www.who.int/kobe_centre/mediacentre/vbdfactsheet.pdf. 3. Campbell LP, Luther C, Moo-Llanes D, Ramsey JM, Danis-Lozano R, Peterson AT. Climate change influences on global distributions of dengue and chikungunya virus vectors. 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https://openalex.org/W4288459169	https://pure.uva.nl/ws/files/109282519/Recycling_gradient_elution_liquid_chromatography.pdf	English	null	Recycling gradient-elution liquid chromatography for the analysis of chemical-composition distributions of polymers	Journal of chromatography A/Journal of chromatography	2,022	cc-by	13,386	Recycling gradient-elution liquid chromatography for the analysis of chemical- composition distributions of polymers Niezen, L.E.; Staal, B.B.P.; Lang, C.; Philipsen, H.J.A.; Pirok, B.W.J.; Somsen, G.W.; Citation for published version (APA): Niezen, L. E., Staal, B. B. P., Lang, C., Philipsen, H. J. A., Pirok, B. W. J., Somsen, G. W., & Schoenmakers, P. J. (2022). Recycling gradient-elution liquid chromatography for the analysis of chemical-composition distributions of polymers. Journal of Chromatography A, 1679, Article 463386. https://doi.org/10.1016/j.chroma.2022.463386 Citation for published version (APA): Niezen, L. E., Staal, B. B. P., Lang, C., Philipsen, H. J. A., Pirok, B. W. J., Somsen, G. W., & Schoenmakers, P. J. (2022). Recycling gradient-elution liquid chromatography for the analysis of chemical-composition distributions of polymers. Journal of Chromatography A, 1679, Article 463386. https://doi.org/10.1016/j.chroma.2022.463386 UvA-DARE (Digital Academic Repository) General rights General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare uva nl) Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Citation for published version (APA): Niezen, L. E., Staal, B. B. P., Lang, C., Philipsen, H. J. A., Pirok, B. W. J., Somsen, G. W., & Schoenmakers, P. J. (2022). Recycling gradient-elution liquid chromatography for the analysis of chemical-composition distributions of polymers. Journal of Chromatography A, 1679, Article 463386. https://doi.org/10.1016/j.chroma.2022.463386 a r t i c l e i n f o Article history: Received 31 May 2022 Revised 13 July 2022 Accepted 27 July 2022 Available online 28 July 2022 Keywords: Gradient Recycling Liquid Chromatography Gradient elution Polymer analysis Article history: Received 31 May 2022 Revised 13 July 2022 Accepted 27 July 2022 Available online 28 July 2022 Keywords: Gradient Recycling Liquid Chromatography Gradient elution Polymer analysis Synthetic polymers typically show dispersity in molecular weight and potentially in chemical compo- sition. For the analysis of the chemical-composition distribution (CCD) gradient liquid chromatography may be used. The CCD obtained using this method is often convoluted with an underlying molecular- weight distribution (MWD). In this paper we demonstrate that the inﬂuence of the MWD can be reduced using very steep gradients and that such gradients are best realized utilizing recycling gradient liquid chromatography (LC ⟳LC). This method allows for a more-accurate determination of the CCD and the assessment of (approximate) critical conditions (if these exist), even when high-molecular-weight stan- dards of narrow dispersity are not readily available. The performance and usefulness of the approach is demonstrated for several polystyrene standards, and for the separation of statistical copolymers consist- ing of styrene/methyl methacrylate and methyl methacrylate/butyl methacrylate. For the latter case, ap- proximate critical compositions of the copolymers were calculated from the critical compositions of two homopolymers and one copolymer of known chemical composition, allowing for a determination of the CCD of unknown samples. Using this approach it is shown that the copolymers elute signiﬁcantly closer to the predicted critical compositions after recycling of the gradient. This is most clear for the lowest- molecular-weight copolymer ( M w = 4.2 kDa), for which the difference between measured and predicted elution composition decreases from 7.9% without recycling to 1.4% after recycling. p y g y g © 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) © 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) chemical composition (CCD) and sequence or block length (BLD) exist and speciﬁc distributions, such as on degree-of-substitution and/or tacticity are important characteristics of speciﬁc types of polymers. To analyze and understand the relationship between these distributions and the resulting material properties, typically some form of liquid chromatography (LC) is utilized [1–5] . Recycling gradient-elution liquid chromatography for the analysis of chemical-composition distributions of polymers Leon E. Niezen a , b , ∗, Bastiaan B.P. Staal c , Christiane Lang c , Harry J.A. Philipsen d , Bob W.J. Pirok a , b , Govert W. Somsen b , e , Peter J. Schoenmakers a , b a Analytical Chemistry Group, van ’t Hoff Institute for Molecular Sciences, Faculty of Science, University of Amsterdam, Science Park 904, Amsterdam, XH 1098, The Netherlands Hoff Institute for Molecular Sciences, Faculty of Science, University of Amsterdam, Science Park 904, Amsterdam, XH al Chemistry Group, van ’t Hoff Institute for Molecular Sciences, Faculty of Science, University of Amsterdam, Science Park 904, Amsterdam Netherlands a Analytical Chemistry Group, van ’t Hoff Institute for Molecular Sciences, Faculty of Science, Un 1098, The Netherlands b Centre for Analytical Sciences Amsterdam (CASA), The Netherlands c BASF SE, Carl-Bosch-Strasse 38, Ludwigshafen am Rhein 67056, Germany Centre for Analytical Sciences Amsterdam (CASA), The Netherlands c BASF SE, Carl-Bosch-Strasse 38, Ludwigshafen am Rhein 67056, Germany d DSM Engineering Materials B.V., Urmonderbaan 22, Geleen, RD 6167 The Netherlands e Division of Bioanalytical Chemistry, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands d DSM Engineering Materials B.V., Urmonderbaan 22, Geleen, RD 6167 The Netherlands e Division of Bioanalytical Chemistry, Amsterdam Institute of Molecular and Life Sciences, Vrije Universite g , , , oanalytical Chemistry, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands a r t i c l e i n f o One ex- ample is size-exclusion chromatography (SEC), which is the cur- rent benchmark for the analysis of the MWD and is often cou- pled to various detectors to provide additional information such as on the change in average chemical composition across the molec- ular weight distribution [ 6 , 7 ] or to assess the degree of branch- ing [8] . To determine the CCD there is not a single, generally ac- cepted method. Gradient-elution LC methods, including reversed- phase liquid chromatography (RPLC) and normal-phase liquid chro- matography (NPLC) are most common, but isocratic LC methods such as temperature-gradient interaction chromatography (TGIC) ∗Corresponding author. E-mail address: L.E.Niezen@uva.nl (L.E. Niezen) . Disclaimer/Complaints regulations f UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) will be contacted as soon as possible. Download date:24 Oct 2024 Journal of Chromatography A 1679 (2022) 463386 Contents lists available at ScienceDirect https://doi.org/10.1016/j.chroma.2022.463386 0021-9673/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) 1. Introduction In ad- dition, peaks may be compressed thanks to the gradient, which causes the rear of the peak to travel faster than the front [35–37] . To properly determine the MWD or the CCD, both distributions must not simultaneously inﬂuence the separation. Typically this is not the case since the retention of a polymer increases approx- imately exponentially with molecular weight in the case of iso- cratic LC separations [15–17] . Both the MWD and CCD may be de- termined by using two-dimensional liquid chromatography (2D-LC) or comprehensive 2D-LC (LC ×LC), which can simultaneously pro- vide information on molar mass and chemical composition distri- butions if a method such as RPLC is coupled with SEC. However, in certain cases it can be desirable to have a one-dimensional method available that can provide information on solely the CCD, as this avoids the practical complexity of 2D-LC. Currently there are no easy-to-implement methods that do so, although examples of such separations exist [18–20] . One approach which may potentially be applied for this is recycling liquid chromatography (LC ⟳LC). This method, which was introduced several decades ago [ 21 , 22 ], aims to improve column performance by artiﬁcially increasing the col- umn length. Nowadays the method is primarily used for speciﬁc (preparative) puriﬁcation purposes, but has otherwise mostly been abandoned as a result of improvements in column and system per- formance [23–26] . However, the combination of gradient-elution and LC ⟳LC may prove especially beneﬁcial to obtain a separation less affected by the MWD. This is because it allows for a reduction of the molecular weight inﬂuence through an increase in the gra- dient steepness, which should reduce the inﬂuence of molar mass, by virtually increasing the column hold-up volume ( V 0 ) without being limited by pressure or requiring an increase in column di- ameter. However, retention in LAC is also strongly affected by analyte molecular weight. This causes broad and typically fronting peaks for polymers with a broad MWD. The ultimate elution pattern of the polymer depends on the actual gradient program and on the MWD. To understand the inﬂuence of the MWD during gradient elution, it must be known how the distribution of (local) reten- tion factors vary with the (local) mobile-phase composition. 1. Introduction Synthetic polymers play an important role in our current so- ciety. The use and applications of these materials is widespread; examples include polyurethane foam cushions, use of aramid in optical ﬁber cables and jet engine enclosures, the use of polyte- traﬂuoroethylene in low friction bearings or non-stick pans, and many more. To continue to develop new products tailored towards speciﬁc applications, the analysis of these materials and their un- derlying distributions is vital. For homopolymers these include dis- tributions in size or molecular weight (MWD), degree of branch- ing (DBD), functionality-type/end-group (FTD), or molecular archi- tecture (MAD). For copolymers additional distributions in terms of L.E. Niezen, B.B.P. Staal, C. Lang et al. Journal of Chromatography A 1679 (2022) 463386 [9–11] , barrier methods such as SEC-gradients (or gradient SEC, gSEC) [ 12 , 13 ], and thermal ﬁeld-ﬂow-fractionation (ThFFF) [14] are also used. on (or partitioning into) the stationary phase (i.e. liquid adsorption chromatography (LAC)); iii ) the polymer elutes without a signiﬁ- cant molecular-weight dependence, often attributed to a balance between enthalpic adsorption and entropic exclusion (but more accurately solely the balance between enthalpy and entropy) and termed liquid chromatography at critical conditions (LCCC) [27– 29] ; iv ) the polymer does not elute at all. For a homopolymer sub- jected to LAC the retention factor ( k ) increases approximately ex- ponentially with molar mass, so that Case ii can easily turn into Case iv. To avoid this, gradient-elution is generally preferred for the LAC analysis of high-molecular-weight analytes. In case of a gradi- ent, ϕ increases with time, which typically (if the initial k is suf- ﬁciently large) leads to a decrease in k with time [ 15–17 , 30–33 ]. When the initial mobile-phase composition is chosen such that k is large ( k init > 10 ) for all analytes and the injection solvent is not signiﬁcantly stronger than the starting eluent [34] , sample fo- cusing will occur at the top of the column. As the gradient pro- gresses, k decreases and the analyte’s velocity will increase as it is caught up by the gradient, until it leaves the column. At the time of leaving the column the local retention factor of the analyte has become (much) smaller compared to the starting conditions. This is the main reason why peaks in gradient-elution chromatograms are much narrower than well-retained peaks in isocratic LC. 1. Introduction With this knowledge one can describe the elution behaviour of the poly- mer distribution in a similar way as for small molecules by solving the differential gradient Eq. [ 15–17 , 28 , 30–33 , 38–42 ]. Many differ- ent models have been proposed to describe the variation of the retention factor with mobile-phase composition [43] . Examples in- clude models that are generally used for small molecules, such as the log-linear model, commonly referred to as the linear-solvent strength (LSS) model [ 16 , 17 , 44 ], quadratic-solvent strength (QSS) [40] and Neue-Kuss [45] models, but also polymer-speciﬁc mod- els that aim to incorporate entropic exclusion effects [ 28 , 39 ]. As has previously been shown by multiple authors [ 16 , 17 , 39 ], sim- pler models such as the LSS model can often adequately describe the retention of a polymer in gradient-LC, most likely as a re- sult of the typically (very) small range in ϕ across which high- molecular-weight analytes elute with reasonable retention factors ( e.g. 1 < k < 10 ). When using the log-linear (LSS) model it is as- sumed that the logarithm of the retention factor varies linearly with mobile-phase composition, Our objective in the present work was to investigate the appli- cability of gradient elution LC ⟳LC for achieving a separation that is dominated by the CCD, while minimizing the effect of the molec- ular weight. To lay the foundation for such an approach, several practical aspects of column selection ﬁrst needed to be considered and the approach was tested for narrow polystyrene standards, which were considered an ideal model system. The ultimate objec- tive was to obtain high-resolution separations of copolymers with very similar average composition and broad MWD and to clearly distinguish effects of the CCD and the MWD in the chromatogram. Challenging samples consisted of two (statistical) styrene/methyl methacrylate (S/MMA) copolymers and statistical copolymers of methyl methacrylate and butyl methacrylate (MMA/BMA). With this work we aim to explore the beneﬁts of LC ⟳LC, and to estab- lish when and how the method may be used for the analysis of synthetic (co-)polymers. 1.1. Theory To reduce the inﬂuence of a polymer’s molar mass in RPLC, one must have an indication of how the retention time ( t R ) of a polymer is inﬂuenced by its chemical composition and molecular weight. Under isocratic conditions the retention time increases lin- early with the analyte retention factor ( k ), which is governed by the distribution equilibrium of the analyte between the station- ary and the mobile phase. k varies with the (volume) fraction of strong solvent in the mobile phase ( ϕ). When the solubility of the analyte polymer in the mobile phase is not a limiting factor, one of four situations can occur, namely i ) the polymer elutes in or- der of high to low molecular weight before the void volume of the column without experiencing any interaction with the station- ary phase, and thus eluting primarily based on its hydrodynamic volume (i.e. size exclusion chromatography (SEC)); ii ) the polymer elutes in order of low to high molecular weight at a volume larger than the void volume of the column, due to differential adsorption ln k = ln k 0 −Sϕ (1) (1) ln k = ln k 0 −Sϕ in which k 0 is the retention factor extrapolated to ϕ = 0 and S is a parameter that captures the change in retention with mobile phase composition. Assuming a linear gradient and taking the above ap- proach to determine the dependence of t R on ϕ ′ (with ϕ ′ = dϕ dt ), one may deﬁne the intrinsic gradient steepness ( b, deﬁned as the rate of change in k during the gradient per volume of mobile phase passing through the column for a speciﬁc analyte). According to the linear-solvent-strength (LSS) concept of Snyder [44] b is de- ﬁned as in which k 0 is the retention factor extrapolated to ϕ = 0 and S is a parameter that captures the change in retention with mobile phase composition. Assuming a linear gradient and taking the above ap- proach to determine the dependence of t R on ϕ ′ (with ϕ ′ = dϕ dt ), one may deﬁne the intrinsic gradient steepness ( b, deﬁned as the rate of change in k during the gradient per volume of mobile phase passing through the column for a speciﬁc analyte). 1.1. Theory According to the linear-solvent-strength (LSS) concept of Snyder [44] b is de- ﬁned as b = −d( ln k ) dϕ dϕ dt t 0 = Sϕ V 0 V G = Sϕ t 0 t G = Sϕ V 0 t G F (2) (2) where V 0 and t 0 are the column hold-up volume and time, re- spectively, ϕ is the composition range spanned by the gradient, 2 L.E. Niezen, B.B.P. Staal, C. Lang et al. Journal of Chromatography A 1679 (2022) 463386 F is the volumetric ﬂowrate, and t G and V G are the duration and the volume of the gradient, respectively. Time and volume are re- lated by the ﬂow rate, i.e., t 0 = V 0 /F and V G = t G F . Therefore, b does not vary with F at constant V G , but does vary with F at constant t G . In Eq. 2 S depends on the molecular weight and the chemi- cal composition of the analyte. It has been shown that S increases with molecular weight for a homologues series [15] and, hence, for polymers of similar structure/composition. length to increase V 0 would cause an increase in the plate number and the peak capacity, but is limited by restrictions on the pres- sure and the analysis time. The above discussion suggests that it would be highly attractive to achieve the required high (effective) gradient steepness by increasing V 0 through lengthening the col- umn, without increasing the pressure drop. This is exactly what can be achieved by repeatedly recycling the gradient. From isocratic experiments performed on narrow polymer stan- dards it is known that at some particular ϕ (the so-called “criti- cal composition”, ϕ crit ) the inﬂuence of the molecular weight may vanish. At this mobile-phase composition the retention factor k is identical for all members of a homopolymeric series, irrespective of molecular weight [27–29] . Unless speciﬁc interactions occur, for example with end groups, the value of k at this critical composi- tion tends to be very small, resulting in elution close to t 0 . Per- forming an isocratic separation at this composition can give in- sights in end-group and block-length distributions. 1.2. Summary of potential advantages and disadvantages In the present work such an LC ⟳LC setup is realized by using a single ten-port valve, which allows for the initially created gra- dient to be alternated between two columns, increasing the gra- dient steepness by virtually increasing the column length. LC ⟳LC seems to be an effective method to achieve very small k e val- ues for analytes of divergent molecular weights, while potentially maintaining a high selectivity with regard to the chemical com- position. Furthermore, in LC ⟳LC the ﬂow rate does not have to be reduced, since the increase in (effective) column length does not result in an increase in pressure. Maintaining a high ﬂow rate reduces system-induced deformation of a low-volume gradi- ent caused by the mixer and avoids an increase in the dwell time [ 46 , 47 ]. LC ⟳LC is, therefore, expected to be considerably faster than a non-recycling approach where a low ﬂow rate must be used. However, LC ⟳LC is possibly not without disadvantages. Column- induced gradient deformation caused by adsorption or absorp- tion of mobile-phase components (“solvent de-mixing”) may play a larger role [ 4 8 , 4 9 ], as may a possible build-up of impurities (de- pending on their retention characteristics). LC ⟳LC requires fast col- umn equilibration. This is not expected to be a problem for RPLC, but it may be for other methods, such as hydrophilic-interaction liquid chromatography (HILIC) and ion-exchange chromatography (IEC). To remedy this, a larger initial ratio of V 0 V G , so that the gra- dient ﬁlls a smaller % of the column and allows for longer equi- libration of the stationary phase, would be required. Finally, be- cause very small values of k e are reached at the moment of elution, extra-column band broadening may become more signiﬁcant. The LSS model yields a simple approximation for the retention factor at the moment of elution ( k e ), k e = k 0 bk 0 + 1 (3) k e = k 0 bk 0 + 1 (3) which for very large values of k 0 , and not extremely shallow gra- dients, simpliﬁes to k e = 1 b . Because S values are large for high- molecular-weight analytes, b values are also large ( Eq. 2 ) and each analyte has a similarly small retention factor at the point of elution ( k e ). 1.2. Summary of potential advantages and disadvantages In contrast, the low-molecular-weight (oligomeric) members have much smaller S values and larger values of ϕ V 0 V G ( i.e. steeper gradients) are needed to minimize the effect of molecular weight on the elution composition (and, thus, on the elution time). For steep gradients (large values of b) the elution time depends solely on the chemical composition of the analyte and the selectivity de- pends primarily on ϕ. All copolymers created from monomers A and B are expected to elute between the respective critical com- positions of the two homopolymers, i.e. ϕ crit , A to ϕ crit , B . The high- est chemical selectivity for copolymers with a narrow chemical- composition distribution is obtained with steep gradients that span a narrow range in mobile phase composition ( ϕ) around the crit- ical point of the copolymer ϕ crit , AB . To compensate for the narrow range (small ϕ), V 0 V G must be made high, either by reducing the gradient volume ( e.g. by reducing the ﬂow rate, while keeping t G constant, or by shortening t G ), or by increasing the column vol- ume ( V 0 ). Reducing the ﬂow rate whilst keeping t G constant im- plies a reduction of the linear velocity, and an increase in analysis time. A lower gradient volume also increases the risk of system- induced gradient deformation, depending on the ratio of the gradi- ent volume to the system’s dwell volume ( V G V dwell ) [ 46 , 47 ]. It is gen- erally recommended that this ratio ( V G V dwell ) should remain around or above unity. Reducing t G would reduce the analysis time, but would lead to a decrease in peak capacity. An increase in column 1.1. Theory However, iso- cratic separations at the critical conditions are diﬃcult to perform and virtually impossible for separations of (high molecular weight) copolymers, because ϕ crit strongly depends on the composition of the copolymer. For statistical copolymers without strongly adsorb- ing end groups k varies due to chemical composition and molecu- lar weight. For high-molecular-weight molecules S is very large, so that analyte molecules do not migrate at ϕ values below the crit- ical composition ( i.e. weaker solvents). In case of gradient elution, large analytes are completely retained on the column until the crit- ical composition is reached. If an analyte molecule falls behind, it will catch up due to SEC effects; if it were to run ahead, it would immediately stop migrating, because of the weaker solvent com- position. Hence, all high-molecular-weight components of a series tend to be focussed at the critical composition. 2. Experimental Two different systems (A and B), in two different laboratories (referred to below as laboratory A and laboratory B), were used for different parts of this work for comparison and to demonstrate the transferability of the method. In case the utilized system is not indicated, system A was used. 2.1.1. Equipment and software 2.1.1. Equipment and software System A, located in Germany, consisted of an Acquity Quater- nary Solvent Manager, an Acquity Column Heater, an Acquity PDA Detector, equipped with a pressure-resistant UV cell (up to 413 bar), and an Acquity Sample Manager with ﬂow-through needle (FTN), all purchased from Waters (Milford, MA, USA). System con- trol and data acquisition was performed using WinGPC software purchased from PSS Polymer Standards Service GmbH (Mainz, Ger- many). 2.1.2. Chemicals and materials Acetonitrile (ACN, ≥99.9%, Acetonitrile (ACN, ≥99.9%, LC-MS Grade) was purchased from Honeywell Research Chemicals (Seelze, Germany) and tetrahydro- furan (THF, 99.9%, Isocratic grade, unstabilized) from Bernd Kraft (Oberhausen, Germany). Narrow polystyrene standards were ob- tained from Polymer Standards Service GmbH. 3 L.E. Niezen, B.B.P. Staal, C. Lang et al. Journal of Chromatography A 1679 (2022) 463386 2.2. Laboratory B 2.2. Laboratory B 2.4. Data analysis 2.2.1. Equipment and software All data analysis ( e.g. alignment, background correction, chro- matogram reshaping and peak analysis) was performed in MATLAB R2021a, purchased from Mathworks (Natick, MA, USA). System B, located in The Netherlands, included a (G1322A) 1100 degasser, (G1311A) 1100 quaternary pump, an (G1329A) 1100 auto-sampler, and an (G1316A) 1100 column oven, all purchased from Agilent (Waldbronn, Germany). An LC-10 AVvp UV detector, equipped with a pressure-resistant UV cell (up to 80 bar) was pur- chased from Shimadzu (Kyoto, Japan). 2.3. Material and methods common to Laboratory A and B The duration of the ﬁrst cycle was ( V 0 , 1 + V 0 , 2 )+ V dwell F ≈2 V 0 + V dwell F . In the present case two columns of (nearly) equal volume were used ( V 0 ≈V 0 , 1 ≈V 0 , 2 ). However, in principle any combination of columns (packed with the same particles) may be used when un- equal switching times are used, provided that the gradient vol- ume remains below the smallest of the two column volumes ( V G ≤ min { V 0 , 1 , V 0 , 2 } ). After the ﬁrst cycle, the gradient (with the ana- lytes positioned in it) was redirected to the ﬁrst column. The gra- dient was then alternated between columns for a number of n cy- cles with a constant recycle time of V 0 F . Folding the individual cy- cles ( Fig. 1 -C) reveals a few important aspects of LC ⟳LC. Firstly, it is possible to track the progression of an analyte within the gra- dient. Secondly, it shows that selecting the correct recycle timing is critical, especially when a very large number of cycles is to be performed. When the timing of each cycle is off, the gradient and the position of the analytes are not aligned in each run. In Fig. 1 -C the selected cycle time was about 1.2 s too short. The dotted line in Fig. 1 -C corresponds to a benchmark point (signal disturbance around the moment the valve is switched) in the chromatograms from each cycle. If the correct cycle time is used such a line be- comes vertical. In most cases the correct cycle timing could be ac- curately determined by aligning each cycle based on characteristic features in the background signal. Certain equipment and chemicals, as well as procedures, were transferred and therefore identical in both laboratories. These are included in this section. 2.2.2. Chemicals and Materials 2.2.2. Chemicals and Materials THF and non-stabilized THF (99.9%, LC-MS Grade, unstabilized) were obtained from VWR Chemicals (Darmstadt, Germany), ACN ( ≥99.9%, LC-MS Grade) and methanol (MeOH, 99.9%, LC-MS Grade) were obtained from Biosolve B.V. (Valkenswaard, the Nether- lands). 2,2 ′ -Azodi(2-MethylButyroNitrile) (AMBN, 98%) and Methyl- methacrylate monomers (MMA, 99%) were obtained from Sigma Aldrich (Steinheim, Germany). Styrene monomers (ST, 99%) was obtained from Fluka (Seelze, Germany). 1-Butanon (MEK, 99%) was obtained from Acros (Geel, Belgium). All water was puriﬁed in- house using a Satorius Arium 611VF at a resistivity of 18.2 M ·cm obtained from Sartorius (Göttingen, Germany). A polystyrene (PS) standards kit was obtained from Polymer Standards Service GmbH. 3.1.1. Design of the LC ⟳LC set-up To perform the recycling gradient experiments a ten-port valve and two identical columns were utilized. A scheme of the set-up is shown as Fig. 1 -A. For the experiment the gradient is only created a single time and is continuously recycled between two columns. Because it is not possible to recycle a gradient that exceeds a sin- gle column volume without losing part of the gradient to waste, the gradient volume was always kept below the void volume of one column. A pressure-resistant UV-detector was installed in-line to allow monitoring of the separation and the gradient during each cycle. Fig. 1 -B shows an example of the data obtained from this in-line UV detector when running LC ⟳LC of a test compound. A recurring signal is obtained that may be “folded” in a similar man- ner as is commonly done for modulations in LC ×LC or comprehen- sive two-dimensional gas chromatography (GC ×GC) ( Fig. 1 -C). The folded data can then be visualized as either a stacked plot (left) or as a surface plot (right). 3.1.2. Experimental evaluation of gradient deformation From previous work it is known that steep gradien 3.1.2. Experimental evaluation of gradient deformation i k i i k h di 3.1.2. Experimental evaluation of gradient deformation From previous work it is known that steep gradients come with a higher risk of strong column-induced gradient deformation [49] . To practically assess the magnitude of this effect and its conse- quences for LC ⟳LC, several initial tests were performed on a va- riety of columns. A reasonably large PS standard (113 kDa, PS6) was followed during a number of cycles. For all experiments the same gradient from 0-100% THF in ACN in 3 min was used. For the different columns the ﬂowrate was adjusted so that the gradi- ent volume remained below V 0 . For the 120 and 40 0 0 ˚A columns V 0 was about 3.1 mL, so a ﬂowrate of 1 mL ·min –1 was used. For the non-porous C18 columns V 0 was about 1.2 mL so a ﬂowrate of 0.4 mL ·min –1 was used. The results of these initial experiments are illustrated in Fig. 2 for several sets of columns with different 3.1. Design and initial experiments System control was performed using Agilent ChemStation. Data acquisition was performed using Shimadzu LabSolutions software. 2.3.1. Equipment and procedure 2.3.1. Equipment and procedure For the recycling experiments two sets of two 250 × 4.6 mm Nucleosil columns (C18 and bare silica), both containing 5-μm par- ticles with a pore size of 40 0 0 ˚A were obtained from Macherey Nagel (Düren, Germany). Two 250 × 4.6 mm C18 columns contain- ing 5-μm particles with a pore size of 120 ˚A were obtained from YMC (Kyoto, Japan). Additionally, two 250 × 4.6 mm Imtakt Presto FF-C18 columns from Imtakt (Kyoto, Japan), containing non-porous 2-μm particles, were also evaluated. For the SEC experiments three 150 × 4.6 mm Acquity APC XT columns containing 1.7-μm particles with a pore size of 45 ˚A were used. Non-stabilized THF was used as eluent. A 10-port 2-position UHPLC valve (MXT715-102) was purchased from Rheodyne, IDEX Corporation (Lake Forest, IL, USA). An Ar- duino Uno Rev 3 was purchased from a local electronics supplier and was used to control the timing of the 10-port valve, irrespec- tive of the system used. In all cases the approximate cycle timing was determined from a blank THF injection and a 0-100% gradient of THF in ACN was run to determine the dwell volume. Unless otherwise mentioned, the temperature of the column oven was set to 30 ºC. 2.3.2. Chemicals Due to the inadequate equilibration of the column and an apparent saturation of the sta- tionary phase with THF no useful results were obtained After Fig. 1. A) Schematic illustration of the recycling-gradient set-up, B) Trace from the in-line DAD resulting from the recycling gradient with the switching moments of the valve indicated by the dotted lines, C) Data folded and aligned, displayed as stacked individual cycles (left) or as a surface plot (right). Fig. 1. A) Schematic illustration of the recycling-gradient set-up, B) Trace from the in-line DAD resulting from the recycling gradient with the switching moments of the valve indicated by the dotted lines, C) Data folded and aligned, displayed as stacked individual cycles (left) or as a surface plot (right). Fig. 2. LC ⟳LC of PS6 (113 kDa) using recycling of a 3-min 0-100% THF in ACN gradient for a couple of A) 120 ˚A, 5-μm C18 columns, B) 40 0 0 ˚A, 5-μm C18 columns, C) 40 0 0 ˚A, 5-μm bare silica columns and D) non-porous 2-μm C18 columns Fig. 2. LC ⟳LC of PS6 (113 kDa) using recycling of a 3-min 0-100% THF in ACN gradient for a couple of A) 120 ˚A, 5-μm C18 columns ˚A, 5-μm bare silica columns and D) non-porous 2-μm C18 columns cycling of a 3-min 0-100% THF in ACN gradient for a couple of A) 120 ˚A, 5-μm C18 columns, B) 40 0 0 ˚A, 5-μm C18 columns, C) 40 0 0 -porous 2-μm C18 columns Fig. 2. LC ⟳LC of PS6 (113 kDa) using recycling of a 3-min 0-100% THF in ACN gradient for a couple of A) 120 ˚A, 5-μm C18 columns, B) 40 0 0 ˚A, 5-μm C18 columns, C) 40 0 0 ˚A, 5-μm bare silica columns and D) non-porous 2-μm C18 columns stationary-phase chemistries, pore sizes, and particle sizes. The de- cision to recycle the entirety of the gradient ( ϕ = 1, V G = V 0 ) was based on the desire to cover a wide range of possible critical com- positions ( ϕ crit ). This is especially relevant when little or no infor- mation is available on the retention characteristics of the sample ( i.e. no known information on the distributions of ln k 0 and S, or on ϕ crit ). This will often be the case when analysing (co-)polymers. 2.3.2. Chemicals Five (statistical) copolymer samples consisting of styrene and methyl methacrylate (S/MMA), with average compositions of: 84/16; 71/29; 57/43; 42/58; 25/75, were synthesized in-house in laboratory B using thermally-initiated free-radical polymerization. The full procedure is included in the supplementary information (section S1). Six different (statistical) copolymer samples consisting of methyl methacrylate and butyl methacrylate (MMA/BMA) were ob- tained from DSM (Waalwijk, The Netherlands). A block copoly- mer from MMA/BMA was obtained from Polymer Standards Service GmbH. 4 L.E. Niezen, B.B.P. Staal, C. Lang et al. Journal of Chromatography A 1679 (2022) 463386 Fig. 1. A) Schematic illustration of the recycling-gradient set-up, B) Trace from the in-line DAD resulting from the recycling gradient with the switching moments of the valve indicated by the dotted lines, C) Data folded and aligned, displayed as stacked individual cycles (left) or as a surface plot (right). Journal of Chromatography A 1679 (2022) 463386 L.E. Niezen, B.B.P. Staal, C. Lang et al. L.E. Niezen, B.B.P. Staal, C. Lang et al. E. Niezen, B.B.P. Staal, C. Lang et al. L.E. Niezen, B.B.P. Staal, C. Lang et al. Journal of Chromatography A 1679 (2022) 463386 Fig. 1. A) Schematic illustration of the recycling-gradient set-up, B) Trace from the in-line DAD resulting from the recycling gradient with the switching moments of the valve indicated by the dotted lines, C) Data folded and aligned, displayed as stacked individual cycles (left) or as a surface plot (right). Fig. 2. LC ⟳LC of PS6 (113 kDa) using recycling of a 3-min 0-100% THF in ACN gradient for a couple of A) 120 ˚A, 5-μm C18 columns, B) 40 0 0 ˚A, 5-μm C18 columns, C) 40 0 0 ˚A, 5-μm bare silica columns and D) non-porous 2-μm C18 columns stationary-phase chemistries, pore sizes, and particle sizes. The de- cision to recycle the entirety of the gradient ( ϕ = 1, V G = V 0 ) was based on the desire to cover a wide range of possible critical com- positions ( ϕ crit ). This is especially relevant when little or no infor- mation is available on the retention characteristics of the sample ing part of the gradient is indicative of solvent de-mixing caused by the preferential adsorption of the more-UV-active and most non-polar solvent (THF) on the column. 2.3.2. Chemicals ing part of the gradient is indicative of solvent de-mixing caused by the preferential adsorption of the more-UV-active and most non-polar solvent (THF) on the column. Due to the inadequate equilibration of the column and an apparent saturation of the sta- tionary phase with THF, no useful results were obtained. After only three cycles the peak corresponding to PS6 completely over- laps with a “breakthrough peak” of THF. In contrast, for both the columns containing 40 0 0 ˚A particles ( Fig. 2 -B for C18 particles and Fig. 2 -C for bare-silica particles), as well as the columns contain- ing non-porous C18 particles ( Fig. 2 -D) the traces for each cycle are much more consistent and the PS6 standard readily assumes its position around the critical composition for polystyrene in the gra- dient (which is expected considering its relatively large molecular weight). For all columns other than the 120 ˚A C18 columns, a grad- ual increase in the pressure was consistently observed during each stationary-phase chemistries, pore sizes, and particle sizes. The de- cision to recycle the entirety of the gradient ( ϕ = 1, V G = V 0 ) was based on the desire to cover a wide range of possible critical com- positions ( ϕ crit ). This is especially relevant when little or no infor- mation is available on the retention characteristics of the sample ( i.e. no known information on the distributions of ln k 0 and S, or on ϕ crit ). This will often be the case when analysing (co-)polymers. From Fig. 2 it may be concluded that the worst result was ob- tained for the 120 ˚A C18 columns. The shape of the background absorbance signal due to the gradient is seen to drastically change and the PS6 peak (indicated by the asterisk) in the gradient be- comes eventually obscured ( Fig. 2 -A). Apparently, the column is not suﬃciently equilibrated between cycles. Also, a spurious peak appears in the ﬁrst cycle, and can be more clearly seen in the sec- ond cycle (indicated by the red arrow). A convex shape of the lead- stationary-phase chemistries, pore sizes, and particle sizes. The de- cision to recycle the entirety of the gradient ( ϕ = 1, V G = V 0 ) was based on the desire to cover a wide range of possible critical com- positions ( ϕ crit ). 2.3.2. Chemicals A) non-porous C18 columns using a 3-min gradient of 20-80% THF in ACN at a ﬂow rate of 0.4 mL.min –1 ; B) 40 0 0 ˚A C18 columns using a 3-min gradient of 20-80% THF in ACN at a ﬂow rate of 1 mL.min –1 ; C) 40 0 0 ˚A bare-silica columns using a 3-min gradient of 0-100% THF in ACN at a ﬂow rate of 1 mL.min –1 . The ﬁrst-cycle chromatograms are shown in the bottom panel; the last (20 th or 10 th ) cycle chromatograms are shown in the top panel. The central panel displays the surface plots for all cycles. dent of molecular weight. A comparison of Fig. 3 -A and Fig. 3 -B also demonstrates that, in case of gradient elution, the presence of pores does not determine whether a (pseudo) critical composi- tion exists. For the bare-silica columns ( Fig. 3 -C), only a marginal reduction in the molecular-weight inﬂuence was observed, which indicates the absence of critical conditions on these columns and with this combination of solvents. The separation obtained using the bare-silica columns ( Fig. 3 -C) is nearly independent of the ef- fective column length and there is little or no variation in the re- tention factor at the moment of elution ( k e ) with b. This demon- strates that LC ⟳LC may, within one experiment, also provide infor- mation on the underlying elution behaviour, as the minor inﬂuence of an increase in column length indicates that elution is governed more so by solubility (ACN to THF corresponding to a non-solvent to solvent gradient) than by interaction with the column. This re- sults in another potential practical application of LC ⟳LC, namely the ability to determine approximate critical conditions when nar- row standards are not available, as is very often the case ( e.g. for copolymers). cycle, due to an increase in the fraction of the more-viscous THF. In conclusion, successful recycling of the full gradient ( ϕ = 1, V G = V 0 ) could not be achieved in columns that contained parti- cles with small pores (120 ˚A), likely because the required equi- libration time for these columns was much longer than for the wide pore packings [50] . 2.3.2. Chemicals This is especially relevant when little or no infor- mation is available on the retention characteristics of the sample ( i.e. no known information on the distributions of ln k 0 and S, or on ϕ crit ). This will often be the case when analysing (co-)polymers. From Fig. 2 it may be concluded that the worst result was ob- tained for the 120 ˚A C18 columns. The shape of the background absorbance signal due to the gradient is seen to drastically change and the PS6 peak (indicated by the asterisk) in the gradient be- comes eventually obscured ( Fig. 2 -A). Apparently, the column is not suﬃciently equilibrated between cycles. Also, a spurious peak appears in the ﬁrst cycle, and can be more clearly seen in the sec- ond cycle (indicated by the red arrow). A convex shape of the lead- From Fig. 2 it may be concluded that the worst result was ob- tained for the 120 ˚A C18 columns. The shape of the background absorbance signal due to the gradient is seen to drastically change and the PS6 peak (indicated by the asterisk) in the gradient be- comes eventually obscured ( Fig. 2 -A). Apparently, the column is not suﬃciently equilibrated between cycles. Also, a spurious peak appears in the ﬁrst cycle, and can be more clearly seen in the sec- ond cycle (indicated by the red arrow). A convex shape of the lead- 5 Journal of Chromatography A 1679 (2022) 463386 L.E. Niezen, B.B.P. Staal, C. Lang et al. L.E. Niezen, B.B.P. Staal, C. Lang et al. Journal of Chromatography A 1679 (2022) 463386 Fig. 3. LC ⟳LC of PS1-6. A) non-porous C18 columns using a 3-min gradient of 20-80% THF in ACN at a ﬂow rate of 0.4 mL.min –1 ; B) 40 0 0 ˚A C18 columns using a 3-min gradient of 20-80% THF in ACN at a ﬂow rate of 1 mL.min –1 ; C) 40 0 0 ˚A bare-silica columns using a 3-min gradient of 0-100% THF in ACN at a ﬂow rate of 1 mL.min –1 . The ﬁrst-cycle chromatograms are shown in the bottom panel; the last (20 th or 10 th ) cycle chromatograms are shown in the top panel. The central panel displays the surface plots for all cycles. Fig. 3. LC ⟳LC of PS1-6. 2.3.2. Chemicals However, if an application is run across a narrower range of compositions (smaller ϕ), small-pore parti- cles with large available surface areas may still feasibly be used. In the present study all further experiments were conducted us- ing the stationary phases with 40 0 0 ˚A pores and the non-porous particles. 3.1.3. LC ⟳LC of PS standards on various columns 3.1.3. LC ⟳LC of PS standards on various columns Using gradi- ent elution the peak width (in volume units, σV ) may be described using Eq. 4 : differences in the pressure for the two columns. The latter effect is a less likely explanation, because LC ⟳LC requires only moderate pressures. An eventual pressure effect may be expected to be more pronounced for high-molecular-weight analytes, which from pre- vious studies are known to experience relatively large changes in partial molar volume with a change in pressure compared to small analytes [51–53] , which cannot be discerned from Fig. 4 . Concern- ing the shape of the peak, two processes can be observed. Firstly, the peak fronting decreased signiﬁcantly with cycle number, most noticeably for the low-molecular-weight analytes and marginally for PS5 and PS6. Secondly, the peak tailing increased with cycle number, again less strongly for the high-molecular-weight stan- dards. The ﬁrst process is likely a result of the selectivity with re- spect to molecular weight, which is much larger for PS1 than for PS6, as a result of the much shallower effective gradient that this standard experiences ( i.e. lower value of b, because of smaller S values). The second process may be a result of either chromato- graphic peak broadening or an inversion of the molecular weight dependence around the “pseudo” critical composition. Using gradi- ent elution the peak width (in volume units, σV ) may be described using Eq. 4 : Small differences in elution time (and thus molecular weight) are found to remain after 10 cycles, especially for fractions 3 and 4 ( M p ≈1.1 kDa). Additionally, the average M p (as deter- mined by calibration relative to a different set of PS standards) differed slightly from the listed value. Irrespective of these differ- ences, all later fractions showed nearly consistent peak molecular weights. This conﬁrms that the observed peak tailing is a result of chromatographic and extra-column dispersion, rather than se- lectivity. Chromatographic peak broadening occurs predominantly at the trailing edge of the peak. This can be explained by the fact that, after the molecular-weight effect on retention is fully di- minished (no remaining selectivity as observed in Fig. 5 ), a peak- sharpening effect due to the gradient likely prevails at the front of the peaks. Molecules that run ahead of the peak (and thus the gra- dient) will slow down due to the increase in weak solvent and get back in line. 3.1.3. LC ⟳LC of PS standards on various columns f To investigate the applicability of the method for reducing the molecular-weight inﬂuence on retention, PS standards of different molecular weight were used as a model system. Peak molecular weights ( M p ) and polydispersity indices (PDI, in brackets) were 4.29 kDa (1.05), 10.4 kDa (1.03), 19.6 kDa (1.03), 43.3 kDa (1.03), 70.9 kDa (1.03), and 113 kDa (1.03), respectively, henceforth re- ferred to as PS1 through PS6. The separation obtained for these standards on the non-porous C18, the 40 0 0 ˚A C18, and the 40 0 0 ˚A bare-silica columns is illustrated in Fig. 3 . Examples of the non- aligned signals are included in the supplementary material (Fig. S- 1, section S2). For all analytes the changes in peak width and shape as a func- tion of cycle number were assessed for both the non-porous and 40 0 0- ˚A C18 packings ( Fig. 4 ). The obtained peak-width parameters on the columns packed with non-porous particles was, in most cases, a factor two to three smaller than those obtained for the 40 0 0 ˚A C18 columns, likely thanks to faster mass-transfer in these columns, because of the smaller particle size (2-μm vs. 5-μm) and the absence of pores. Ad- ditionally, irrespective of the column used, the shape of the peak depends on the molecular weight of the analyte and small dif- ferences can be observed in the peak widths between successive cycles (“zig-zag” effect). Apparently, the chromatogram depends slightly on which of the two columns the gradient has passed through before entering the in-line DAD. This may be explained by differences in the packing, the stationary phase itself, or small These experiments conﬁrm that the inﬂuence of the molecular weight is progressively reduced with an increasing number of cy- cles in case of the C18 columns (for both the non-porous particles, Fig. 3 -A, and the 40 0 0 ˚A particles, Fig. 3 -B). The mitigation of the molecular-weight effect concurs with an increase in the effective gradient steepness ( b). On the non-porous columns ( Fig. 3 -A), the difference in elution composition between PS1 (4.29 kDa) and PS6 (113 kDa) is reduced from ϕ = 17% (ﬁrst cycle, i.e. no recycling) to ϕ < 0 . 1% (20 cycles). Evidently, when the gradient steepness is suﬃciently large, the elution order becomes essentially indepen- 6 L.E. Niezen, B.B.P. Staal, C. Lang et al. 3.1.3. LC ⟳LC of PS standards on various columns Journal of Chromatography A 1679 (2022) 463386 Fig. 4. Front and tail peak widths (in mL) obtained during LC ⟳LC of PS1-6; widths are measured to the peak center line at 10% of the maximum peak height, and depicted as function of cycle number. Blue circles: front peak widths; red diamonds: tail peak widths. Gradient: 3-min 20-80% THF in ACN. A) non-porous C18 particles; ﬂow rate, 0.4 mL.min –1 ; B) 40 0 0 ˚A C18 particles; ﬂow rate, 1 mL.min –1 . Journal of Chromatography A 1679 (2022) 463386 L.E. Niezen, B.B.P. Staal, C. Lang et al. Fig. 4. Front and tail peak widths (in mL) obtained during LC ⟳LC of PS1-6; widths are measured to the peak center line at 10% of the maximum peak height, and depicted as function of cycle number. Blue circles: front peak widths; red diamonds: tail peak widths. Gradient: 3-min 20-80% THF in ACN. A) non-porous C18 particles; ﬂow rate, 0.4 mL.min –1 ; B) 40 0 0 ˚A C18 particles; ﬂow rate, 1 mL.min –1 . of these experiments, as performed on the non-porous-particle C18 columns, are illustrated in Fig. 5 . differences in the pressure for the two columns. The latter effect is a less likely explanation, because LC ⟳LC requires only moderate pressures. An eventual pressure effect may be expected to be more pronounced for high-molecular-weight analytes, which from pre- vious studies are known to experience relatively large changes in partial molar volume with a change in pressure compared to small analytes [51–53] , which cannot be discerned from Fig. 4 . Concern- ing the shape of the peak, two processes can be observed. Firstly, the peak fronting decreased signiﬁcantly with cycle number, most noticeably for the low-molecular-weight analytes and marginally for PS5 and PS6. Secondly, the peak tailing increased with cycle number, again less strongly for the high-molecular-weight stan- dards. The ﬁrst process is likely a result of the selectivity with re- spect to molecular weight, which is much larger for PS1 than for PS6, as a result of the much shallower effective gradient that this standard experiences ( i.e. lower value of b, because of smaller S values). The second process may be a result of either chromato- graphic peak broadening or an inversion of the molecular weight dependence around the “pseudo” critical composition. 3.1.3. LC ⟳LC of PS standards on various columns Such gradient-sharpening is absent at the back side of the peaks, where all k values are low. Such an explanation is in agreement with the observation that the broadening is great- est for low-molecular-weight standards, while higher-molecular- weight standards show less broadening. Contrarily, extra-column band broadening is expected to be more severe for high-molecular- weight standards, as a result of their much smaller diffusion coef- ﬁcients. However, SEC or hydrodynamic effects could help sharpen the peaks, as this would allow large molecules that have fallen be- hind to catch up. For the 40 0 0 ˚A columns a brief assessment of the inﬂuence of ﬂowrate and the range of mobile-phase composi- tion covered by the gradient ( ϕ) on peak width was performed across 10 cycles for a narrow and broad PS standard. The results of these experiments are included in the supplementary material (Fig. S-2, section S3) and indicated that broad and narrow stan- dards reach nearly equal peak width at high number of cycles for the same gradient. Gradients spanning smaller ϕ and higher ﬂow rates generally resulted in broader peaks. σV = G V 0 √ N ( 1 + k e ) (4) σV = G V 0 √ N ( 1 + k e ) (4) In which G is a band compression factor, which for very steep gradients (large b) and an unretained mobile-phase modiﬁer should reach a (supposedly limiting) value of about 0.58 [ 36 , 37 ]. Because in our case large b values can likely be reached and the resulting k e values are small (and likely similar) for all analytes, the peak width after a given number of cycles should depend pri- marily on N and V 0 . When such conditions are reached σV is ex- pected to increase with the square root of the number of cycles. Given the small k e values, extra-column band broadening is also a point of concern. In this work the peak broadening seemed to manifest itself primarily in the form of peak tailing, rather than as an increase in overall peak width. This effect was largest for PS1. To inves- tigate this effect, an LC ⟳LC analysis of PS1 on the non-porous column was ended after the 10 th cycle. Fractions of the eﬄuent were collected and subsequently measured with SEC. The results 3.2. 3.1.3. LC ⟳LC of PS standards on various columns LC ⟳LC for the analysis of chemical-composition distributions 3.2.1. Separations of S/MMA copolymers 3.2.1. Separations of S/MMA copolymers Because LC ⟳LC could successfully suppress the inﬂuence of the molecular weight in case of PS, it was deemed to be a good tech- 7 Journal of Chromatography A 1679 (2022) 463386 L.E. Niezen, B.B.P. Staal, C. Lang et al. Fig. 5. A) Fractionation of PS1 after analysis by LC ⟳LC (10 cycles) using non-porous C18 particles with a 3-min 20-80% THF gradient in ACN at a ﬂowrate of 0.4 mL.min –1 ; fraction numbers are indicated. B) SEC chromatograms of the fractions indicated in A, measured using Acquity APC XT columns, with unstabilized THF at a ﬂowrate of 0.5 mL ·min –1 and a column oven temperature of 60 ºC. Fig. 5. A) Fractionation of PS1 after analysis by LC ⟳LC (10 cycles) using non-porous C18 particles with a 3-min 20-80% THF gradient in ACN at a ﬂowrate of 0.4 mL.min –1 ; fraction numbers are indicated. B) SEC chromatograms of the fractions indicated in A, measured using Acquity APC XT columns, with unstabilized THF at a ﬂowrate of 0.5 mL ·min –1 and a column oven temperature of 60 ºC. Fig. 6. LC ⟳LC of S/MMA copolymers SM1-2 (A) and SM1-5 (B) performed on two 40 0 0 ˚A C18 columns using a ﬂow rate of 1 mL.min –1 . Gradient, A) 30-50% THF in ACN in 2.5 min, B) 0-60% THF in ACN in 2.5 min. Average S/MMA compositions: SM1, 84/16; SM2, 71/29; SM3, 57/43; SM4, 42/58; SM5, 25/75. Experiments were performed on System B. Fig. 6. LC ⟳LC of S/MMA copolymers SM1-2 (A) and SM1-5 (B) performed on two 40 0 0 ˚A C18 columns using a ﬂow rate of 1 mL.min –1 . Gradient, A) 30-50% THF in ACN in 2.5 min, B) 0-60% THF in ACN in 2.5 min. Average S/MMA compositions: SM1, 84/16; SM2, 71/29; SM3, 57/43; SM4, 42/58; SM5, 25/75. Experiments were performed on System B. compositions (broad CCD) is considered (SM1-5), a gradient with a larger ϕ is required ( Fig. 6 -B). This increases the value of b and reduces the inﬂuence of the MWD for all copolymers, even in the ﬁrst cycle. 3.1.3. LC ⟳LC of PS standards on various columns Separations of MMA/BMA copolymers 3.1.3. LC ⟳LC of PS standards on various columns Because the difference in the critical compositions of SM1 and SM2 ( ϕ crit = ϕ crit , SM 2 −ϕ crit , SM 1 ) is about 4.8%, and is independent of the slope of the gradient, a higher resolution in terms of chemical composition is obtained when the gradient cov- ers a smaller range of eluent compositions, within the same time frame. This conﬁrms that the retention of these copolymers fol- lows the same basic rules as the PS homopolymers, with a strong correlation between the molecular-weight dependent slope ( S) and intercept ( ln k 0 ) of Eq. (1) . Peaks are seen to remain broader in time units at smaller ϕ even after recycling of the gradient. In terms of volume-fraction units (at the elution composition) peaks are narrower for narrow range gradients. This may be the best re- ﬂection of the actual CCD, because the chemical-composition se- lectivity of the separation is maximized and overshadows the con- tribution of the chromatographic dispersion. nique for determining chemical-composition distributions (CCD), without a confounding effect of molecular weight. Experiments were performed on ﬁve statistical copolymers consisting of S/MMA (SM1-5), as well as on seven MMA/BMA copolymers (MB1-7), to assess whether the approach could be applied to achieve higher resolution between samples differing only slightly in composi- tion. For SM1-2 a gradient spanning a narrow range in compo- sition (small ϕ) was used. This caused a pronounced inﬂuence of the underlying broad MWD ( M w = 54 kDa (PDI = 2.3) and 64 kDa (PDI = 2.1) for copolymer SM1 and SM2, respectively) of these samples on the elution proﬁle obtained with conventional gradient-elution LC, as is clear from the ﬁrst-cycle trace in Fig. 6 -A where distinctly fronting peaks are obtained. The underlying MWD jeopardizes the determination of the CCD when a shallow gradient is used. In subsequent cycles the effec- tive gradient slope ( b ) gradually increases causing the proﬁle to reﬂect the CCD, with little or no inﬂuence of the broad MWD. Much sharper peaks were obtained after ten cycles, as a result of the narrow CCD of both copolymers. The signal-to-noise ratio im- proved by more than a factor of three for both distributions and the their resolution improved from 0.66 to 1.5 (determined after deconvoluting the two distributions). If a broader range of polymer 3.2.2. Separations of MMA/BMA copolymers 3.2.2. 3.2.2. Separations of MMA/BMA copolymers 5 ϕ crit ,AB = p A ( 1 −X B ) + p B X B q A p A ( 1 −X B ) + q B p B X B (5) (5) in which the subscripts A and B indicate monomer A and B, re- spectively, X is the mass fraction of the respective monomer in the copolymer AB, q is the slope obtained by assuming a linear correlation between S and ln k 0 , and corresponds to the approxi- mate critical composition as ϕ crit = 1 q , p is the slope obtained by assuming a linear correlation between ln k 0 and molecular weight, and ϕ crit ,AB is the approximate critical composition of copolymer AB with mass fraction X B . Determining p A and p B individually for both homopolymers may require multiple experiments and can be tedious. However, since ϕ crit ,AB can be shown to depend on p A p B by dividing Eq. 5 by p B it can be easier to rewrite Eq. 5 to: in which the subscripts A and B indicate monomer A and B, re- spectively, X is the mass fraction of the respective monomer in the copolymer AB, q is the slope obtained by assuming a linear correlation between S and ln k 0 , and corresponds to the approxi- mate critical composition as ϕ crit = 1 q , p is the slope obtained by assuming a linear correlation between ln k 0 and molecular weight, and ϕ crit ,AB is the approximate critical composition of copolymer AB with mass fraction X B . Determining p A and p B individually for both homopolymers may require multiple experiments and can be tedious. However, since ϕ crit ,AB can be shown to depend on p A p B by dividing Eq. 5 by p B it can be easier to rewrite Eq. 5 to: As seen in Fig. 8 -A, the peak fronting decreases during the cy- cles, until it seems to converge after 20 cycles, indicating that the confounding effect of the underlying MWD has been diminished. However, signiﬁcant fronting remains, even after 20 cycles ( Fig. 8 - C), the underlying gradient is indicated in the FIG. to better high- light the remaining extent of peak fronting. An analysis of the frac- tions taken from the 20 th cycle ( Fig. 3.2.2. Separations of MMA/BMA copolymers The differences be- tween the measured elution compositions and the elution com- positions predicted in this way (calculated as: \| ϕ e −ϕ crit ,AB \| ∗100 ) for MB1 and MB4 decreased from 7.9% and 2.0% in the ﬁrst cycle, to 1.4% and 0.092% after the ﬁnal cycle, respectively. Assuming in- stead that ϕ crit ,AB varied linearly with X BMA between ϕ crit , PMMA and ϕ crit , PBMA led to an overestimation in all cases. A full overview is given in the supplementary information (Fig. S-3, section S4). The largest shift in elution composition after recycling of the gradient occurred for copolymer MB1. This is not surprising, since this is a low-molecular-weight copolymer ( M w = 4.2 kDa). Additionally, because it is a block copolymer, the peak remains broad even af- ter recycling. Block copolymers tend to have a much broader CCD than statistical copolymers, due to the block-length distributions of the two blocks. The peak of copolymer MB4 showed signiﬁcant fronting, even after 10 cycles. To evaluate whether this fronting oc- curred due to the remaining inﬂuence of the MWD or was the re- sult of the underlying CCD, peak fractions were taken after 1 and 20 cycles. The MWD of each fraction was subsequently determined using SEC and also the change in peak asymmetry during the re- cycling experiment was evaluated ( Fig. 8 ). In this case a broader range of composition ( ϕ) was used. Again we observed that the separation with respect to polymer composition, once obtained, can be maintained in subsequent cy- cles. Unlike the above example of the S/MMA copolymers, most peaks show the characteristic fronting due to the confounding MWD in the ﬁrst cycle (upper panels in Fig. 7 ). The fronting is re- duced or disappears for many peaks with an increasing number of cycles, as the effect of the MWD is increasingly suppressed. An ad- ditional method to illustrate the effect of the recycling is to predict the approximate critical compositions of the copolymers and com- paring these with the obtained elution compositions before and af- ter a recycling of the gradient. Previous work has shown that the approximate critical composition of a statistical copolymer can be calculated using data obtained for the corresponding homopoly- mers [16] , by using Eq. 3.2.2. Separations of MMA/BMA copolymers To further illustrate the effect of gradient recycling the method was also applied to a separation of MMA/BMA copolymers (MB1- 8 Journal of Chromatography A 1679 (2022) 463386 L.E. Niezen, B.B.P. Staal, C. Lang et al. Fig. 7. LC ⟳LC of MMA/BMA copolymers MB1-7 performed on A) non-porous C18 particles using a gradient of 0-60% THF in ACN in 3 min at a ﬂowrate of 0.4 mL.min –1 , and B) 40 0 0- ˚A C18 particles using a gradient of 0-60% THF in ACN in 2.5 min at a ﬂowrate of 1 mL.min –1 . Average MMA/BMA compositions (as determined by 1 H-NMR) and M w : MB1, 50/50 (4.2 kDa); MB2, 76/24 (80 kDa); MB3, 58/42 (20 kDa); MB4, 32/68 (15 kDa); MB5, 30/70 (50 kDa); MB6, 85/15 (100 kDa); MB7, 0/100 (160 kDa). Fig. 7. LC ⟳LC of MMA/BMA copolymers MB1-7 performed on A) non-porous C18 particles using a gradient of 0-60% THF in ACN in 3 min at a ﬂowrate of 0.4 mL.min –1 , and B) 40 0 0- ˚A C18 particles using a gradient of 0-60% THF in ACN in 2.5 min at a ﬂowrate of 1 mL.min –1 . Average MMA/BMA compositions (as determined by 1 H-NMR) and M w : MB1, 50/50 (4.2 kDa); MB2, 76/24 (80 kDa); MB3, 58/42 (20 kDa); MB4, 32/68 (15 kDa); MB5, 30/70 (50 kDa); MB6, 85/15 (100 kDa); MB7, 0/100 (160 kDa). 7), using both the columns containing non-porous and 40 0 0 ˚A C18 particles ( Fig. 7 ). proximate critical composition. Therefore, it is expected that the difference between the measured elution composition ( ϕ e ) and the predicted critical composition ( ϕ crit ,AB ) is minimized with an in- crease in the number of cycles (or gradient steepness), especially for the lowest-molecular-weight analytes (MB1 and MB4). The ap- proximate critical compositions were calculated in this way us- ing ϕ crit , PMMA = 0 . 09 , ϕ crit , PBMA = 0 . 47 , and ϕ crit , MB 5 = 0 . 34 (with X BMA = 0 . 70 , as determined from 1 H-NMR). [2] A.M. Striegel, W.W. Yau, J.J. Kirkland, D.D. Bly, Modern Size-Exclusion Liquid Chromatography: Practice of Gel Permeation and Gel Filtration Chromatogra- phy: Second Edition, 2009. 10.1002/9780470442876. 3.2.2. Separations of MMA/BMA copolymers 8 -D) shows that the underly- ing MWD within all fractions after the ﬁrst two is the same, in- dicating that even for a relatively low molecular weight polymer ( M p = 15 kDa) a good reﬂection of the true CCD of the polymer can be obtained. This case underlines the value of LC LC. Without recycling there is a strong confounding effect of the MWD and the CCD, which prevents correct interpretation of the results. p A p B = X B 1 −ϕ crit ,AB ϕ crit ,B ( 1 −X B ) ϕ crit ,AB ϕ crit ,A −1 (6) (6) This equation allows one to determine p A p B provided that the approximate critical conditions are determined for two high- molecular-weight homopolymers A and B, and one high-molecular- weight copolymer AB of known average composition, given by X B . In our case recycling of the gradient promotes elution at the ap- 9 Journal of Chromatography A 1679 (2022) 463386 L.E. Niezen, B.B.P. Staal, C. Lang et al. L.E. Niezen, B.B.P. Staal, C. Lang et al. Journal of Chromatography A 1679 (2022) 463386 Fig. 8. LC ⟳LC of copolymer MB4 using non-porous C18 particles with a 3-min 0-60% THF gradient in ACN at a ﬂowrate of 0.4 mL.min –1 . A) Front (blue) and tail (red) peak widths (in mL) as function of cycle number (calculation, see Fig. 4 ). B and C) Peak proﬁles after 1 st and 20 th cycle, respectively, with fractions taken indicated; dashed line under the peak indicates the background signal of the gradient. D and E) SEC chromatograms of the fractions indicated in B and C, respectively, measured using Acquity APC XT columns at a ﬂowrate of 0.5 mL.min –1 and a column oven temperature of 60 ºC. ticles with a 3 min 0 60% THF gradient in ACN at a ﬂowrate of 0 4 mL min–1 A) Front (blue) and tail (red) peak Fig. 8. LC ⟳LC of copolymer MB4 using non-porous C18 particles with a 3-min 0-60% THF gradient in ACN at a ﬂowrate of 0.4 mL.min –1 . A) Front (blue) and tail (red) peak widths (in mL) as function of cycle number (calculation, see Fig. 4 ). Acknowledgements LN acknowledges the UNMATCHED project, which is supported by BASF, DSM and Nouryon and receives funding from the Dutch Research Council (NWO) in the framework of the Innovation Fund for Chemistry (CHIPP Project 731.017.303) and from the Ministry of Economic Affairs in the framework of the “TKI-toeslagregeling”. BP acknowledges the Agilent UR grant #4354. Chromatographic dispersion remains, but gradient conditions and column dimensions may be chosen such that the chemical- composition selectivity is dominant. Columns packed with large- pore particles or non-porous particles can be used for LC ⟳LC, but small-pore particles give rise to column-induced gradient deforma- tion. This was ascribed to adsorption of mobile-phase components on packings with large surface areas. This work was performed in the context of the Chemometrics and Advanced Separations Team (CAST) within the Centre for An- alytical Sciences Amsterdam (CASA). The valuable contributions of the CAST members are gratefully acknowledged. An LC ⟳LC experiment may be ended after any number of cy- cles and combined with any detector suitable for gradient LC. Also, LC ⟳LC may be coupled on-line with other methods, such as size- exclusion chromatography, to better highlight potential differences between samples. A comprehensive coupling of LC ⟳LC and SEC may provide clearly interpretable results, and the orthogonality be- tween RPLC or NPLC and SEC will be increased. Even without addi- tion of another method LC ⟳LC was shown to be capable of a more direct determination of the CCD. 3.2.2. Separations of MMA/BMA copolymers B and C) Peak proﬁles after 1 st and 20 th cycle, respectively, with fractions taken indicated; dashed line under the peak indicates the background signal of the gradient. D and E) SEC chromatograms of the fractions indicated in B and C, respectively, measured using Acquity APC XT columns at a ﬂowrate of 0.5 mL.min –1 and a column oven temperature of 60 ºC. 4. Conclusion Leon E. Niezen: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Visualization. Bastiaan B.P. Staal: Conceptualization, Methodology, Writing – review & editing, Resources, Supervision. Christiane Lang: Resources, Writing – review & editing. Harry J.A. Philipsen: Resources, Project administration, Writing – review & editing. Bob W.J. Pirok: Resources, Supervision, Funding acquisition, Project administration, Writing – review & editing. Govert W. Somsen: Funding acquisition, Project administration, Writing – review & editing. Peter J. Schoenmakers: Resources, Supervision, Funding acquisition, Project administration, Writing – review & editing. In this work the use of LC ⟳LC for the analysis of the CCD of copolymers is introduced and demonstrated. The entirety of the gradient is continuously recycled to achieve extremely steep gra- dients, so as to minimize the effect of the MWD on the elution proﬁle. Conventionally, very fast gradients require short durations, in combination with long columns and low ﬂow rates, resulting in decreased peak capacities, long analysis times, and an increased risk of system-induced gradient deformation. Such issues can be avoided with LC ⟳LC. It is demonstrated that a set of polystyrene standards of greatly different molecular weights can be made to (nearly) completely co-elute. LC ⟳LC was used to determine the CCD of two sets of copolymers (S/MMA and MMA/BMA), with the confounding effect of the MWD being successfully suppressed. Based on the results presented, LC ⟳LC appears suitable for the ac- curate determination of the CCD of a wide range of copolymers with narrow or broad CCDs and MWDs. 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https://openalex.org/W2105193178	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0107012&type=printable	English	null	Altered Serum MicroRNAs as Novel Diagnostic Biomarkers for Atypical Coronary Artery Disease	PloS one	2,014	cc-by	7,756	Abstract Background and Aim: Atypical coronary artery disease (ACAD) is characterized by atypical angina pectoris or silent myocardial ischemia. However, conventional diagnostic techniques are insufficient to identify this subtype of coronary atherosclerotic pathology, and specific and sensitive markers for diagnosing ACAD are still currently lacking. The aim of the present study is to identify a novel serum microRNA (miRNA) expression profile of ACAD patients and evaluate its clinical diagnostic value. Patients and Methods: 127 patients who were diagnosed with ACAD and 54 age-matched controls were enrolled in this study. An initial screen of miRNA expression was performed in serum samples from 35 patients and 20 controls using TaqMan Low Density Array. A stem-loop quantitative reverse-transcription PCR (RT-qPCR) assay was conducted in the training and validation sets to confirm the levels of the altered miRNAs in 122 patients with ACAD and 68 controls. In addition, the potential target genes of the altered miRNAs were predicted using bioinformatics methods. Results: The TaqMan low density array and RT-qPCR analysis identified four serum miRNAs including miR-487a, miR-502, miR-208 and miR-215 that were significantly increased, while one miRNA, miR-29b, that was significantly decreased in ACAD patients compared with normal controls (P,0.05). The area under the receiver-operating-characteristic (ROC) curve (AUC) for the combined 5 serum miRNAs were 0.850 (95% CI, 0.734–0.966, P,0.001) and 0.909 (95% CI, 0.858–0.960, P,0.001) for the training set and validation set, respectively. In addition, target gene prediction showed that these five altered miRNAs are involved in affecting various aspects of cardiac or vascular remodeling, especially in the pathway involving inflammation and fibrosis. Conclusion: Our findings indicate that the five altered serum miRNAs could be novel non-invasive biomarkers for ACAD and may also represent potential therapeutic targets for atherosclerosis and myocardial ischemia. Citation: Wang J, Pei Y, Zhong Y, Jiang S, Shao J, et al. (2014) Altered Serum MicroRNAs as Novel Diagnostic Biomarkers for Atypical Coronary Artery Disease. PLoS ONE 9(9): e107012. doi:10.1371/journal.pone.0107012 Received April 29, 2014; Accepted August 7, 2014; Published September 8, 2014 Received April 29, 2014; Accepted August 7, 2014; Published September 8, 2014 Copyright: 2014 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. ang1., Yinghao Pei1., Yong Zhong2, Shisen Jiang1, Jiaqing Shao3, Jianbin Gong1 1 Department of Cardiology, Jinling Hospital, School of Medicine, Nanjing University/Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu Province, P.R. China, 2 Department of Health-Care, Jinling Hospital, School of Medicine, Nanjing University/Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu Province, P.R. China, 3 Department of Endocrinology, Jinling Hospital, School of Medicine, Nanjing University/Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu Province, P.R. China Abstract All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by the Natural Science Foundation of China (81000352, 30900697, 81100568), the Natural Science Foundation of Jiangsu Province (BK2011661), the Postdoctoral Scientific Foundation of China (20100471843), and the Postdoctoral Scientific Foundation of Jiangsu Province (1001027C). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: zero991127@hotmail.com (JG); shaojiaq@hotmail.com (JS) * Email: zero991127@hotmail.com (JG); shaojiaq@hotmail.com (JS) . These authors contributed equally to this work. September 2014 \| Volume 9 \| Issue 9 \| e107012 Serum MiRNA Profile of Atypical Coronary Artery Disease Serum MiRNA Profile of Atypical Coronary Artery Disease Myocardial perfusion imaging etc.), other than chest pain or dyspnea. The exclusion criteria for all subjects were a previous history of cardiac disease (e.g., myocardial infarction, heart failure, cardiac arrhythmias, pacing, and cardiomyopathy), known history of leukopenia, thrombocytopenia, malignancy, severe hepatic or renal dysfunction, surgery or skeletal muscle damage, and evidence for inflammatory disease. Each patient was interviewed to collect demographic characteristics and medical history and underwent coronary angiography. Coronary angiograms were obligatorily combined with intravascular ultrasound and were evaluated independently by two cardiologists who made a visual estimation of luminal narrowing in multiple segments. According to the American Heart Association/American College of Cardi- ology classification of the coronary tree, a significant atheroscle- rotic lesion is defined as at least one major epicardial vessel with . 50% stenosis as assessed by quantitative coronary angiography. Patient characteristics are summarized in Table 1. 45 normal controls were randomly selected from healthy individuals who had contemporaneously visited Jinling Hospital for a routine physical examination who were found to be normal on physical examina- tion, electrocardiographic evaluation and laboratory tests without evidence of diseases such as hyperlipemia, hypertension, acute cardiovascular or cerebrovascular disease, diabetes mellitus, respiratory tract infections or any clinical evidence of atheroscle- rosis. pathological processes including cardiovascular disease, angiogen- esis and inflammation. For instance, some muscle-specific miRNAs were found to be up-regulated in response to ischemia/reperfusion injury in the rat heart [5] and in the myocardial infarction rat model [6]. Subsequent studies demon- strated that these miRNAs are involved in apoptotic cell death induced by cardiac ischemia through the post-transcriptional repression of the anti-apoptotic proteins B-cell lymphoma-2 and insulin-like growth factors [5,6]. Conversely, other miRNAs were discovered to produce opposing effects on apoptosis by targeting heat shock protein 60, heat shock protein 70 and caspase-9 in cardiomyocytes [7]. Very recently, a series of studies have demonstrated that miRNAs are remarkably stable and can be readily quantified in human or animal serum and plasma. More importantly, circulating miRNAs demonstrate significant dynamic change in some pathological conditions and can partly reflect tissue damage [8,9]. These discoveries have laid the ground work for potentially using circulating miRNAs as non-invasive biomarkers of cardiac disease. Towards that aim, efforts have been made to search for appropriate circulating miRNAs that could be novel and useful biomarkers for CAD [10,11]. Sample collection and processing A total of 5 mL of venous blood was obtained by venipuncture from each donor after a 12 h overnight fast. The sample was immediately centrifuged at 1,500 g for 10 min at room temper- ature, followed by a 10-min high-speed centrifugation at 10,000 g at 4uC to completely remove cell debris. The samples were stored at 280uC until analysis. RNA extraction For the TaqMan Low Density Array, equal volumes of serum from 35 ACAD patients and 20 normal subjects were pooled separately to form patient and control sample pools (total volume for each pool is ,10 mL). RNA was extracted from each pooled sample according to a previously described protocol [13]. Briefly, 10 mL pooled serum was mixed with Trizol Reagent (Invitrogen, Carlsbad, Calif., USA) with a 1:2 ratio and the samples were homogenized by vortexing vigorously for 1 min. After incubation in room temperature for 10 min, the mixture was centrifuged at 4uC for 15 min. After phase separation, the aqueous layer was transferred to a new tube and mixed with 1.5 volumes of isopropyl alcohol. This solution was stored at 220uC for 1 h. The RNA pellet was collected by centrifugation at 10,000 g for 20 min at 4uC. The resulting RNA pellet was washed once with 75% ethanol and dried for 10 min at room temperature. Finally, the pellet was dissolved in 20 mL of RNase-free water and stored at 280uC until further analysis. Ethics Statement The present study was approved by the ethics committee board of Jinling hospital. All the patients and healthy volunteers from whom serum samples were obtained provided written informed consent prior to the study. Serum MiRNA Profile of Atypical Coronary Artery Disease Thus far, at least 40 non-invasive, blood-based studies have examined miRNA expression profiles in order to identify miRNA biomarkers for the diagnosis of CAD. Most of them have focused on miRNAs in the serum or plasma of acute myocardial infarction patients and discovered some cardio- specific miRNAs including miR-1, miR-133 and miR-208 in acute myocardial infarction patients which were significantly increased compared to control subjects. These findings suggest that these circulating miRNAs might be potential diagnostic markers for acute myocardial infarction [10,11]. These studies have shown promising results and have suggested that circulating miRNAs represent interesting candidates that could potentially serve as non-invasive biomarkers of cardiovascular disease. However, most of the results are based on a limited number of patients and few specific miRNAs, which may partially explain some the discrep- ancies observed. Moreover, these studies failed to explore the potential diagnostic value of circulating miRNAs in screening ACAD patients. We therefore hypothesized that circulating miRNAs signatures may be used as a novel tool to stratify ACAD. In the present study, using blood samples obtained from ACAD patients, we aimed to find a cluster of miRNAs that could be used as non-invasive and new risk-markers for the diagnosis of ACAD. Introduction ability to identify patients who present with uncommon symptoms or silent myocardial ischemia in its early stages [3]. Therefore, identification of simple, specific and non-invasive biomarkers that can help diagnose ACAD in its early phase is urgently needed. MicroRNAs (miRNAs) are a class of small non-coding RNAs approximately 22 nucleotides in length that regulate gene expression by accelerating mRNA degradation or inhibiting translation at the posttranscriptional level [4]. Numerous studies have revealed that miRNAs play a pivotal role in the control of most biological processes including cell differentiation, prolifera- tion, migration, development and apoptosis. Importantly, there is increasing evidence that miRNAs are involved in various ability to identify patients who present with uncommon symptoms or silent myocardial ischemia in its early stages [3]. Therefore, identification of simple, specific and non-invasive biomarkers that can help diagnose ACAD in its early phase is urgently needed. Atypical coronary artery disease (ACAD) is a type of peripheral arterial disease characterized as atypical angina pectoris or silent myocardial ischemia. Numerous studies have shown that the presence of ACAD predicts a much higher risk of major adverse cardiovascular events [1]. Because both the necessity and the benefit of treating ACAD remains in question, particularly in asymptomatic patients without a history of coronary artery disease (CAD), useful biomarkers for ACAD are needed [2,3]. Currently available diagnostic techniques such as the use of traditional cardiac biomarkers or electrocardiogram are limited in their MicroRNAs (miRNAs) are a class of small non-coding RNAs approximately 22 nucleotides in length that regulate gene expression by accelerating mRNA degradation or inhibiting translation at the posttranscriptional level [4]. Numerous studies have revealed that miRNAs play a pivotal role in the control of most biological processes including cell differentiation, prolifera- tion, migration, development and apoptosis. Importantly, there is increasing evidence that miRNAs are involved in various September 2014 \| Volume 9 \| Issue 9 \| e107012 1 PLOS ONE \| www.plosone.org Study population For the RT-qPCR assay, an amount of 100 mL of serum was mixed with 200 mL of acid phenol, 200 mL of chloroform, and 300 mL of RNase-free water. The mixture was vortex-mixed vigorously and centrifuged at room temperature for 15 min. After phase separation, the aqueous layer was mixed with 1.5 volumes of isopropyl alcohol and 0.1 volumes of 3 mol/L sodium acetate (pH 5.3). This solution was stored at 220uC for 1 h. The RNA pellet was collected by centrifugation at 16,000 g for 20 min at 4uC. The resulting RNA pellet was washed once with 75% ethanol and dried for 10 min at room temperature. Finally, the pellet was dissolved in 20 mL of RNase-free water and stored at 280uC until further analysis. A total of 127 consecutive patients who were admitted to the department of cardiology, Jinling Hospital (Nanjing, China) between November 2011 and June 2012 were enrolled in this study. The inclusion criteria for the ACAD patient population were as follows: 1) #1–2 of the 5 characteristics based on the Duke Clinical Score [12], including none; 2) normal level of the myocardial enzyme, high-sensitivity troponin I (hsTnI), and other biochemical indicators; 3) at least one coronary artery with significant stenosis; 4) uncommon reasons for referral such as CAD risk factors, atrial fibrillation, and pre-operative evaluation (e.g. Electrocardiogram, Treadmill Test, Ultrasonic cardiograph, September 2014 \| Volume 9 \| Issue 9 \| e107012 2 PLOS ONE \| www.plosone.org Serum MiRNA Profile of Atypical Coronary Artery Disease Table 1. Demographic and clinical features of the Atypical coronary artery disease (ACAD) patients and controls in the training set and validation seta. Table 1. Demographic and clinical features of the Atypical coronary artery disease (ACAD) patients and controls in the training set and validation seta. Table 1. Demographic and clinical features of the Atypical coronary artery disease (ACAD) patients and controls in the training set and validation seta. Table 1. Demographic and clinical features of the Atypical coronary artery disease (ACAD) patients and controls in the training set and validation seta. Study population Briefly, 2 mL of total RNA was reverse-transcribed to cDNA using the AMV reverse transcriptase (TaKaRa, Dalian, China) for synthesis of cDNA, the reaction mixtures were incubated at 16uC for 30 min, at 42uC for 30 min, at 85uC for Study population Training set Validation set Control ACAD p-value Control ACAD p-valueb (n = 10) (n = 30) (n = 34) (n = 92) Age (years) 59.0613.4 67.0610.7 0.589 59.4613.1 65.2610.5 0.122 Male sex, n (%) 6 (60) 12 (40) 0.463 15 (44.1) 53 (57.6) 0.177 Clinical features Height (cm) 170.3367.24 168.6565.03 0.422 167.8866.21 16965.89 0.876 Body weight (kg) 71.67612.06 71.23610.82 0.856 70.23610.82 71.88611.56 0.556 Body mass index 25.5364.02 24.9962.56 0.453 24.8262.72 25.8361.48 0.466 Blood parameter AST (IU/L) 39.27620.83 30.80619.83 0.444 33.30613.83 27.92626.71 0.331 ALT (IU/L) 29.55616.92 26.7269.68 0.442 21.4164.95 26.57621.08 0.096 CK (IU/L) 79.00685.44 87.33658.34 0.772 68.48624.50 85.23642.59 0.063 CK-MB (IU/L) 15.55611.84 10.6763.09 0.205 12.0067.44 11.5864.08 0.748 Serum creatinine (mg/dl) 78.82610.35 79.60626.22 0.924 73.02614.79 76.11610.36 0.335 LDH (IU/L) 179.45614.86 185.53647.37 0.547 169.30653.13 186.22625.12 0.122 cTnI (ng/ml) 0.0160.01 0.1360.38 0.302 0.0260.02 0.0860.34 0.386 Platelets (6103/mL) 180.91672.04 184.87660.42 0.861 192.96651.97 171.60650.99 0.082 RBC (6104/mL) 4.5960.441 4.4060.485 0.272 4.5260.38 4.4160.54 0.273 WBC (mL) 6.1962.548 6.2161.543 0.779 6.1862.01 6.6861.51 0.027 Ca2+ (mmol/L) 2.2260.06 2.2860.32 0.512 2.1660.10 2.1260.11 0.091 Cl2 (mmol/L) 103.0063.29 105.0368.21 0.45 104.9363.20 106.9662.31 0.05 K+ (mmol/L) 4.0060.31 3.9960.47 0.966 4.1760.40 3.9260.33 0.004 Na+ (mmol/L) 142.0062.61 141.7364.23 0.846 141.8562.66 143.7562.72 0.004 Cholesterol (mmol/L) 4.2360.79 4.5461.57 0.306 4.3661.07 4.6961.07 0.204 Total glycerin (mmol/L) 1.2860.21 1.5660.69 0.056 1.3360.25 1.4460.22 0.51 HDL (mmol/L) 1.0160.24 0.8960.21 0.167 1.1360.70 1.2760.99 0.67 LDL (mmol/L) 2.6060.68 3.1861.41 0.218 2.5860.93 2.9960.98 0.071 Other disease Hypertension, n (%) 4 (40) 19 (63.3) 0.356 16 (47.1) 62 (67.4) 0.512 Diabetes mellitus, n (%) 4 (40) 16 (53.3) 0.465 19 (55.9) 55 (59.8) 0.693 Hyperlipidaemia, n (%) 3 (30) 16 (53.3) 0.360 9 (26.5) 59 (64.1) 0.36 Cerebral vascular event, n (%) 0 (0) 5 (16.7) 0.408 6 (17.6) 11 (12.0) 0.592 Arrhythmia, n (%) 4 (40) 9 (30) 0.845 9 (26.5) 36 (39.1) 0.268 Smoking status, n (%) 1 (10) 11 (36.7) 0.231 17 (50) 35 (38.0) 0.226 Alcohol consumption, n (%) 1 (10) 8 (26.7) 0.512 12 (35.3) 32 (34.8) 0.957 aAge data are presented as the mean (SD) TaqMan probe-based RT-qPCR on a 7300 Real-Time PCR Sequence Detection System (Applied Biosystems). Because U6 and 5S rRNA are degraded in serum samples and the lack of a consensus housekeeping miRNA for the RT-qPCR analysis of serum miRNAs, miRNA expression was normalized to serum volume. TaqMan probe-based RT-qPCR on a 7300 Real-Time PCR Sequence Detection System (Applied Biosystems). Because U6 and 5S rRNA are degraded in serum samples and the lack of a consensus housekeeping miRNA for the RT-qPCR analysis of serum miRNAs, miRNA expression was normalized to serum volume. Briefly, 2 mL of total RNA was reverse-transcribed to cDNA using the AMV reverse transcriptase (TaKaRa, Dalian, China) for synthesis of cDNA, the reaction mixtures were incubated at 16uC for 30 min, at 42uC for 30 min, at 85uC for TaqMan low-density array and RT-qPCR analysis TaqMan low-density array and RT-qPCR analysis TaqMan Human MicroRNA A and B Arrays, version 3.0 (Applied Biosystems, Foster City, CA, USA), were used for miRNA expression screening of 754 serum miRNAs on an ABI PRISM 7900HT instrument. To increase the sensitivity of the TaqMan Low Density Array, a pre-amplification was performed after the reverse transcription. All reactions were performed as specified in the manufacturer’s protocol. The serum miRNA expression levels in individual samples were determined by a September 2014 \| Volume 9 \| Issue 9 \| e107012 PLOS ONE \| www.plosone.org 3 Serum MiRNA Profile of Atypical Coronary Artery Disease ure 1. Overview of the experimental design. Overview of the experimental design Figure 1. Overview of the experimental design. doi:10.1371/journal.pone.0107012.g001 between the two groups. A p-value,0.05 was considered statistically significant. The receiver operating characteristic (ROC) curve was generated and the area under the curve (AUC) was calculated to evaluate the specificity and sensitivity of ACAD prediction for each serum miRNA. Risk score analysis was performed to evaluate the associations between ACAD and the expression levels of the serum miRNAs as previously described [14]. In brief, the risk score of the upregulated miRNA, denoted as ‘‘s’’, was set to 1 if the expression level was greater than the upper 95% reference interval for the corresponding miRNAs level in controls and was set to 0 in all other cases. For the downregulated miRNAs, the ‘‘s’’ was set to 1 if the expression level was lower than the lower 5% reference interval for the corresponding miRNAs level in controls and was set to 0 in all other cases. When taking into account the correlation of each miRNA with ACAD risk, each patient was assigned a risk score function (RSF) according to a linear combination of the expression level of the miRNA. The RSF for sample i using the information from the five miRNAs was: rsfi = g5 j-1Wj.sij. 5 min, and then held at 4uC. Real-time PCR was performed using a TaqMan PCR kit according to the manufacturer’s instructions with a minor modification as described in a previous study. The Real-Time PCR cycles consisted of pre-denaturation at 1 cycle of 95uC for 5 min, and 40 cycles of 95uC for 15 sec and 60uC for 1 min. All reactions, including no-template controls, were performed in triplicate. The resulting Cq values were determined using fixed threshold settings. TaqMan low-density array and RT-qPCR analysis We assessed the detection limits of the RT-qPCR assay, dynamic range and calculated the absolute concentration of target miRNAs based on a calibration curve developed by synthetic miRNA oligonucleotides with known concentrations. miRNA Target gene prediction Targets of the five altered miRNAs were created by combining predicted results from the public database including TargetScan, miRanda, and PicTar. Potential targets were selected based on gene function, the number of predicted target sites, and target prediction by multiple algorithms. The NCBI DAVID server (http://david.abcc.ncifcrf.gov/tools.jsp) was used to provide fur- ther information about the corresponding genes’ functions. In the above equation, sij is the risk score for miRNA j on sample i, and Wj is the weight of the risk score of miRNA j. To determine the Ws, five univariate logistic regression models were fitted using the disease status with each of the risk scores. The regression coefficient of each risk score was used as the weight to indicate the contribution of each miRNA to the RSF. The frequency table and ROC curves were then used to evaluate the diagnostic effects of the profiling and to find the appropriate cutoff point. September 2014 \| Volume 9 \| Issue 9 \| e107012 Statistical analysis Statistical analysis was performed with SPSS 16.0 software (SPSS, Inc., Chicago, USA). The data are presented as the means 6 SEM for serum miRNA levels or means 6 SD for other variables. Non-parametric Mann-Whitney tests were used to compare the differences in serum miRNA expression levels between the ACAD group and healthy controls group. Student’s t-test was used to compare the differences in other variables September 2014 \| Volume 9 \| Issue 9 \| e107012 PLOS ONE \| www.plosone.org 4 Serum MiRNA Profile of Atypical Coronary Artery Disease 2. Differential expression of the five miRNAs (A) miR-487a, (B) miR-29b, (C) miR-502, (D) miR-208 and (E) miR-215 in the between Atypical Coronary Artery Disease (ACAD) cases and controls. The expression levels of the five miRNAs are detected by RT- ndividually. Serum levels of the five miRNAs were measured in 122 AD cases and in 44 controls of training set and validation set using a sis probe-based RT-qPCR assay. Cq values were converted to absolute values based on the standard curves. Each point represents the mean cate samples. Each p-value was derived from a nonparametric Mann–Whitney U-test. p,0.01; p,0.001. 371/journal.pone.0107012.g002 Figure 2. Differential expression of the five miRNAs (A) miR-487a, (B) miR-29b, (C) miR-502, (D) miR-208 and (E) miR-215 in the serum between Atypical Coronary Artery Disease (ACAD) cases and controls. The expression levels of the five miRNAs are detected by RT- qPCR individually. Serum levels of the five miRNAs were measured in 122 AD cases and in 44 controls of training set and validation set using a hydrolysis probe-based RT-qPCR assay. Cq values were converted to absolute values based on the standard curves. Each point represents the mean of triplicate samples. Each p-value was derived from a nonparametric Mann–Whitney U-test. p,0.01; **p,0.001. doi:10.1371/journal.pone.0107012.g002 volunteers (29 men and 25 women, mean age 61.3614.5 years). All ACAD patients were selected on the basis of clinical parameters (e.g. Signs and symptoms, history, exam and lab value, etc.) combined with angiographic documentation. The Results Baseline clinical characteristics of the study population We recruited 166 participants including 127 ACAD patients (69 men and 58 women; mean age, 66.8610.3 years) and 54 healthy Table 2. Differentially-expressed miRNAs in Atypical coronary artery disease (ACAD) serum samples compared to control samples in training set and validation seta. Training set Validation set miRNA Control ACAD P valueb Control ACAD P valueb (n = 10) (n = 30) (n = 34) (n = 92) miR-487a 184.27623.54 400.95641.28 0.002 251.31623.03 460.59637.71 0.027 miR-29b 2290.916410.84 635.316145.71 ,0.001 1890.086251.39 520.97642.02 ,0.001 miR-502 1229.976232.18 3186.686368.78 ,0.001 1116.996102.41 2772.156412.40 0.016 miR-208 1551.196167.21 3839.486452.85 ,0.001 2051.026330.05 5574.026293.53 ,0.001 miR-215 86.76624.00 385.73637.45 ,0.001 198.80623.97 418.96628.89 ,0.001 aThe absolute concentrations of miRNAs are presented as mean 6 SEM (fM/L). bMann-Whitney unpaired test for rank sum. doi:10.1371/journal.pone.0107012.t002 Table 2. Differentially-expressed miRNAs in Atypical coronary artery disease (ACAD) serum samples compared to control samples in training set and validation seta. miRNAs in Atypical coronary artery disease (ACAD) serum samples compared to control samples September 2014 \| Volume 9 \| Issue 9 \| e107012 PLOS ONE \| www.plosone.org Serum MiRNA Profile of Atypical Coronary Artery Disease Figure 3. The receiver operating characteristic (ROC) curve analysis for discriminative ability between the Atypical Coronary A Disease (ACAD) cases and noncancer controls by the 5 miRNAs and their panel. ROC curves for the ability of the the 5 miRNAs and panel to differentiate the 30 ACAD cases from the 10 controls in the training set (A–F), 92 ACAD cases from the 30 controls in the validation set oi:10.1371/journal.pone.0107012.g003 Figure 3. The receiver operating characteristic (ROC) curve analysis for discriminative ability between the Atypical Coronary Artery Disease (ACAD) cases and noncancer controls by the 5 miRNAs and their panel. ROC curves for the ability of the the 5 miRNAs and their panel to differentiate the 30 ACAD cases from the 10 controls in the training set (A–F), 92 ACAD cases from the 30 controls in the validation set (G–L). doi:10.1371/journal.pone.0107012.g003 TaqMan low-density array analysis of serum miRNA in ACAD patients Furthermore, the ROC curve for the panel revealed a pronounced diagnostic accuracy, evidenced by the AUC of 0.909 (95% CI, 0.858–0.960, P,0.0001), which was much better than that for five individual miRNA (Figure 3). Moreover, we also investigated the five miRNAs and their different panels in all the ACAD cases and controls of training set and validation set, and found that both panels of the five-serum miRNAs (miR-487a, miR-502, miR-208, miR-215 and miR-29b) and other different panels as well as individual miRNA (Table S2 and Table S3) could reliably discriminate ACAD from controls, and the panel of five-serum miRNAs had a significant higher AUC when compared with the other panels, which indicated that the five-miRNA panel is really a comprehensive and specific indicator than other panels for ACAD. expression levels between the two groups, we used two criteria: (1) Cq values ,30 and (2) miRNA levels showed at least 100-fold difference. These two criteria yielded a list of 44 differentially expressed miRNAs, 22 of which were up-regulated and 22 down- regulated in ACAD patients compared with healthy controls (Table S1). TaqMan low-density array analysis of serum miRNA in ACAD patients demographics and clinical features of the patients enrolled in training set and validation set are listed in Table 1. Healthy controls were recruited from a large pool of individuals seeking a routine health check-up at the Healthy Physical Examination Centre of Jinling Hospital. People without evidence of any disease were selected as control subjects. Control subjects were matched to the patients by age, sex and ethnicity. demographics and clinical features of the patients enrolled in training set and validation set are listed in Table 1. Healthy controls were recruited from a large pool of individuals seeking a routine health check-up at the Healthy Physical Examination Centre of Jinling Hospital. People without evidence of any disease were selected as control subjects. Control subjects were matched to the patients by age, sex and ethnicity. A two-phase case-control study was designed to assess serum miRNAs as a surrogate marker for ACAD (Figure 1). We first performed a TaqMan low-density array analysis to screen and select candidate miRNAs that showed markedly alterations in pooled serum samples between ACAD patients and healthy controls. Of the 766 miRNAs scanned, 315 and 286 miRNAs could be detected in the sera of ACAD patients and healthy controls, respectively. To further identify miRNAs with differential PLOS ONE \| www.plosone.org September 2014 \| Volume 9 \| Issue 9 \| e107012 6 Serum MiRNA Profile of Atypical Coronary Artery Disease Table 3. Risk score analysis of ACAD cases and control donors. 0,2.1305 2.1305,10.478 PPVa NPVb Training set Control 10 0 100% 50% ACAD 10 20 Validation set Control 28 6 92.60% 62.20% ACAD 17 75 aPPV, positive predictive value; bNPV, negative predictive value. doi:10.1371/journal.pone.0107012.t003 markers for ACAD detection were 66.7% and 100%, respectively, and the AUCs were 0.850 (95% CI, 0.734–0.966, P,0.001) (Figure 3). The diagnostic values of these five miRNAs were further evaluated in the biomarker validation set. As shown in the Figure 3, the AUCs for these five miRNAs ranged from 0.628 to 0.886. When using the same risk score formula to calculate the risk score of samples from the verification set and constructed ROC curves using these RSFs to estimate the diagnostic sensitivity and specificity of the 5-miRNA–based biomarker. The diagnostic sensitivity and specificity of the five-miRNA panel for ACAD detection in the validation set were 83.7% and 82.4%, respec- tively. Confirmation of the altered serum miRNAs levels in ACAD patients by RT-qPCR analysis p y q y We next employed a TaqMan probe-based RT-qPCR assay to confirm the expression of the candidate miRNAs selected from the TaqMan low-density array analysis. The serum samples were arranged in two sets including a biomarker screening set (training set) and biomarker verification set (validation set). In the training set, miRNAs were detected in a set of individual serum samples including 30 ACAD patients and 10 normal controls which from the same cohort of TaqMan low-density array screening phase. Only those miRNAs with a mean fold-change $2.0 and a P-value ,0.05 were chosen for further analysis. Moreover, miRNAs with a Cq value .35 and a detection rate ,75% in either the ACAD or control group were excluded. Based on this criteria, four miRNAs including miR-487a, miR-502, miR-208 and miR-215 were markedly increased in serum from ACAD patients compared to control subjects (p,0.05), whereas miR-29b were significantly lower than in control subjects (p,0.001) (Table 2). Expression levels of the five altered miRNAs were then chosen from the training set and further confirmed in a large cohort samples set (validation set) composed of 92 ACAD patients and 34 age-gender matched controls. Consistent with the results from the training set, serum levels of miR-487a, miR-502, miR-208 and miR-215 were significantly higher in the ACAD patients compared to the control subjects, and miR-29b was significantly decreased with a similar trend to the previous results (Table 2). The differences in concentration for these seven miRNAs in all ACAD patients and the control individuals enrolled in the training and validation sets are shown in Figure 2. To further evaluate the diagnostic value of the 5-miRNA profiling system, we performed a risk score formula to calculate the RSF for ACAD patients and control samples according to above description and previous study [14,15]. Based on their risk scores and at a set cutoff, samples could be divided into a high-risk group representing the predicted ACAD cases, or a low risk group representing the predicted controls. When using the optimal cutoff value (RSF = 2.1305) at which the sum of the sensitivity and specificity was maximal, the specificity and sensitivity were 78.7% and 86.4%, respectively. At this cutoff, 38 of the 44 controls had RSF values ,2.1305, while 95 of the 122 ACAD samples had a risk score .2.1305 (Table 3). Prediction of the five altered miRNAs mediated functional gene regulation for their detection [8,9]. For some time, peripheral blood has been attracting increasing attention from the scientific community for its use as a source for detecting clinical biomarkers of cardiovascular disease because it can be easily obtained in the clinical setting. Because miRNAs have been found to be stably expressed in blood and can be readily quantified by RT-qPCR, studies have highlighted the crucial role of serum miRNAs in diagnosing cardiovascular diseases such as acute myocardial infarction, CAD and ischemia. In our study, significant differences in the expression of 5 novel miRNAs were observed between the ACAD cases and controls. Of the five miRNAs, miR-208 has also been reported in multiple studies as being differentially expressed between normal and patient group. The biological basis for alterations of miRNAs in ACAD remains unclear. It is possible that it involves, at least to some degree, the body’s systemic response and/or genetic susceptibility to CAD. To explore the possible regulatory role of miR-487a, miR-502, miR-208, miR-215 and miR-29b in ACAD, gene targets were predicted by using miRNA target prediction databases, namely, Target Scan, miRanda and PicTar. The predicted targets were then compared with a list of upregulated and downregulated genes identified in other earlier studies. Functional classification of the target genes was carried out with gene ontology analysis using the DAVID tool. Our analysis showed that some genes such as transforming growth factor-b activated kinase-1 binding (TAB) protein 3 and catenin-beta interacting protein 1 targeted by miR- 487a and miR-215 were involved in the inflammatory process. Among the predicted genes (such as vascular endothelial growth factor A, microfibrillar-associated protein 3, Kv channel interact- ing protein 1 and TNF receptor-associated factor 3) that were targeted by miR-361-5p, vascular endothelial growth factor A was found to be a key point in a signaling pathway for vasculogenesis and endothelial cell growth, cell migration, cell apoptosis and arteriosclerosis. Consistent with the earlier published articles, miR- 208 and miR-29b apparently play a role in the process of vascular or myocardial remodeling. The identification of miRNAs’ targets is crucial for elucidating their function. However, due to the complexity of the miRNA– target interactions, this step has proven computationally difficult. At present, several target prediction algorithms have been developed, but they show a poor overlap between their outputs suggesting that there are a number of false-negatives and as well as false-positive predictions [17]. Serum MiRNA Profile of Atypical Coronary Artery Disease Serum MiRNA Profile of Atypical Coronary Artery Disease Prediction of the five altered miRNAs mediated functional gene regulation The mechanisms responsible for ACAD are not well understood, and individual differences in pain threshold may only partially explain the variability in pain perception [18–20]. Pain perception may result from microenvi- ronmental balances between proinflammatory cytokines (including interleukin-1b, tumor necrosis factor-a, interleukin-6, and inter- feron-c) and anti-inflammatory cytokines (including interleukin-4, interleukin-10) [21,22]. The proinflammatory activation seems to intensify nociception, whereas Th2 lymphocyte production seems to abolish the pain perception [23]. The predicted targets of most miRNAs uncovered in our study pointed towards a significant role in local inflammation. Therefore, we suspected that the miRNAs were involved in the imbalance between pro-inflammatory and anti-inflammatory cytokines and played a role in the atheroscle- rosis of ACAD. Of the five selected serum miRNAs in the present study, miR-215 has been reported to promote b-catenin activation and upregulate a-SMA and fibronectin expression in Transform- ing growth factor-b1 treated mouse mesangial cell by targeting catenin-beta interacting protein 1. All of these genes were reported to be participated in neointimal lesion formation [24]. Since neointimal lesions often occur at the site of subclinical atheroscle- rosis, we presumed subclinical atherosclerosis to be one possible contributor to silent myocardial ischemia. Furthermore, altered miR-215 could stimulate subclinical atherosclerosis via neointimal lesion formation. TGF-b has been found to be an atheroprotective factor in arteriosclerosis [25], and the TGF-b/Smad pathway can suppress NF-kB activation-induced inflammation by blocking TAB2 or TAB3 [26]. Our bioinformatics analysis showed that TAB3 is a potential target of miR-487a. This result in combination with existing reports led us to speculate that miR-487a may be involved in the occurrence of atherosclerosis by regulating TAB3 expression. Taken together, the above evidences may fuel the notion that miRNAs are associated with the TGF-b cascade and may be a potential therapeutic target for ACAD. Of the five selected miRNAs, miR-29b and miR-208 have been shown to be useful biomarkers for myocardial injury [27]. In our study, we found that miR-29b was significantly decreased in silent myocar- dial ischemia patients. The precise mechanism by which miR-29b participates in myocardial ischemia is still unclear; however, the predicted targets of miR-29b, including 20 collagens and several extracellular matrix genes, may explain the role of this miRNA in myocardial ischemia [28]. MiR-208 has been associated with Receiver Operating Characteristic Analysis We next performed an ROC analysis for traditional CAD protein marker plasma hsTnI between all ACAD patients and normal subjects in order to further weigh the usefulness of the selected miRNAs. The AUC for plasma hsTnI was 0.627 (95% confidence interval; CI = 0.536–0.718), significantly lower than AUC for the five miRNA and their panel (Table S2 and Table S3). These data implied that the five-serum miRNA signature could represent a suitable biomarker that allowed for efficient differentiation of ACAD patients from other subjects. To evaluate the usefulness of the five altered circulating miRNA as potential biomarkers for ACAD, we conducted a ROC analysis. When a comparison was made between ACAD patients and healthy controls of biomarker training set, the AUC for these miRNAs ranged from 0.817 to 0.930 (Figure 3). To determine the diagnostic value of the combination of the five miRNAs, we performed a risk score analysis on the data set and used it to predict ACAD case and control status. The frequency table and the ROC curves were then used to evaluate the diagnostic effect of the five-miRNA panel. When using the optimal cut off value, where the sum of sensitivity and specificity was maximal, the diagnostic sensitivity and specificity of the five miRNA–based September 2014 \| Volume 9 \| Issue 9 \| e107012 7 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org References 1. Canto JG, Shlipak MG, Rogers WJ, Malmgren JA, Frederick PD, et al. 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J Clin Invest 119: 2772–2786. 12. Pryor DB, Harrell FE Jr, Lee KL, Califf RM, Rosati RA (1983) Estimating the likelihood of significant coronary artery disease. Am J Med 75: 771–780. 13. Luo Y, Wang C, Chen X, Zhong T, Cai X, et al. (2013) Increased serum and urinary microRNAs in children with idiopathic nephrotic syndrome. Clin Chem 59: 658–666. 30. Wang GK, Zhu JQ, Zhang JT, Li Q, Li Y, et al. (2010) Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J 31: 659–666. 14. Liu R, Chen X, Du Y, Yao W, Shen L, et al. (2012) Serum microRNA expression profile as a biomarker in the diagnosis and prognosis of pancreatic cancer. Clin Chem 58: 610–618. 31. Vacchi-Suzzi C, Bauer Y, Berridge BR, Bongiovanni S, Gerrish K, et al. Supporting Information Table S1 Differentially-expressed miRNAs in ACAD serum samples compared to normal controls deter- mined by TaqMan Low Density Assay. (DOCX) Table S2 ROC curves and the corresponding AUCs of the five selected miRNAs for all the ACAD patients and controls in training set and validation set. (DOCX) Table S2 ROC curves and the corresponding AUCs of the five selected miRNAs for all the ACAD patients and controls in training set and validation set. (DOCX) Table S3 ROC curves and the corresponding AUCs of different serum-miRNA panels for all the ACAD patients and controls in training set and validation set. (DOCX) In summary, we successfully identified five miRNAs that showed different expression levels in ACAD patient serum and controls. Moreover, the five miRNAs had a high diagnostic value in identifying coronary artery disease, particularly with atypical presentation. The bioinformatics prediction indicated all five miRNAs may be involved in the pathogenesis of CAD. As a novel Serum MiRNA Profile of Atypical Coronary Artery Disease Serum MiRNA Profile of Atypical Coronary Artery Disease cardiac remodeling [29] and elevated plasma miR-208 has been identified as one of the most promising biomarkers for myocardial injury [30,31]. Our results showed that levels of miR-208 were significantly elevated in ACAD patients when compared to normal controls. Intriguingly, a recent study reported that levels of miR- 208 were much higher in angina pectoris cases than in acute myocardial ischemia cases [32], implying that miR-208 could be used to monitor the early stages of myocardial injury. Our ROC analysis showed that the sensitivity and specificity of miR-208 for ACAD diagnosing is much higher than that of hsTnI. Based on these results, we concluded that serum miR-208 is more valuable than Troponin I in diagnosing ACAD. For miR-502, which is often expressed in myogenic differentiation [33], has been regarded as a ‘‘myogenic miRNA’’. One group suggested that miR-502 could suppress the autophagy process and play an atheroprotective role by directly targeting RAB1B and AP2B1. While reduced miR-502 could then become the potential section that accounted for the inconspicuous myocardial insult [34,35], the exact mechanism underlying miR-502’s role in ACAD requires further study. invasive biomarker, the established miRNAs and their panel could potentially decrease the number of individuals with under- recognized coronary lesions and help to avoid misdiagnosis and excessive medical treatment. Author Contributions Conceived and designed the experiments: JW SJ JS JG. Performed the experiments: JW YP YZ. Analyzed the data: JW YP. Contributed reagents/materials/analysis tools: YZ. Contributed to the writing of the manuscript: JW JS JG. Discussion At present, more than 40 studies have revealed alterations of miRNAs in serum or plasma in cardiovascular disease. These observations have led to rapid progress in the use of these small molecules as biomarkers for cardiovascular disease. However, those studies are mainly focused on miRNAs that are specifically expressed in skeletal muscle or cardiac tissues, and most of the selected candidate miRNA markers are only for acute myocardial infarction and CAD. This led us to perform a comprehensive analysis of the dynamic changes and diagnostic value of serum miRNAs in ACAD patients. To our knowledge, no previous studies have been conducted on circulating miRNAs in large cohorts of ACAD patients. In the current study, using a genome- wide TaqMan low-density array technology to determine the whole serum miRNAs expression levels of ACAD patients and normal controls followed by RT-qPCR confirmation in individual samples, we successfully identified a novel five miRNAs signature that included miR-487a, miR-502, miR-208, miR-215 and miR- 29b and was markedly altered in the serum of ACAD patients. Further ROC curve analysis indicated that the AUC as well as the sensitivity and specificity of the selected miRNAs were better than the traditional protein marker hsTnI. We therefore conclude that these five circulating miRNAs may serve as independent biomarkers for the diagnosis of ACAD. ACAD is usually referred to as atypical angina pectoris and silent myocardial ischemia, which could lead to a delay in proper evaluation or management. This description suggests that symp- tomatology is a less specific, and possibly less sensitive in defining myocardial injury. In light of the limitation of routine lab techniques [16], innovative and reliable biomarkers for the detection of ACAD are urgently needed. Though extraordinary efforts have been directed towards determining the molecular and pathological characteristics of this disease in order to develop novel diagnostic and therapeutic strategies, none have been validated for use in the clinical setting. Recent studies have observed that miRNAs are stable when present in the circulation, and can be readily quantified by RT- qPCR technology. More importantly, unique serum miRNA expression profiles have been suggested for various diseases including cardiovascular disease and may serve as finger prints September 2014 \| Volume 9 \| Issue 9 \| e107012 PLOS ONE \| www.plosone.org 8 34. Le Guezennec X, Brichkina A, Huang YF, Kostromina E, Han W, et al. (2012) Wip1-dependent regulation of autophagy, obesity, and atherosclerosis. Cell Metab 16: 68–80. 35. Zhai H, Song B, Xu X, Zhu W, Ju J (2013) Inhibition of autophagy and tumor growth in colon cancer by miR-502. Oncogene 32: 1570–1579. Serum MiRNA Profile of Atypical Coronary Artery Disease References (2012) Perturbation of microRNAs in rat heart during chronic doxorubicin treatment. PLoS One 7: e40395. 15. Wang S, Xiang J, Li Z, Lu S, Hu J, et al. (2013) A plasma microRNA panel for early detection of colorectal cancer. Int J Cancer. doi:10.1002/ijc.28136. [Epub ahead of print]. 32. Li C, Fang Z, Jiang T, Zhang Q, Liu C, et al. (2013) Serum microRNAs profile from genome-wide serves as a fingerprint for diagnosis of acute myocardial infarction and angina pectoris. BMC Med Genomics 6: 16. 16. Lobbes MB, Kooi ME, Lutgens E, Ruiters AW, Lima Passos V, et al. (2010) Leukocyte counts, myeloperoxidase, and pregnancy-associated plasma protein a as biomarkers for cardiovascular disease: towards a multi-biomarker approach. Int J Vasc Med 2010: 726207. 33. Dmitriev P, Barat A, Polesskaya A, O’Connell MJ, Robert T, et al. (2013) Simultaneous miRNA and mRNA transcriptome profiling of human myoblasts reveals a novel set of myogenic differentiation-associated miRNAs and their target genes. BMC Genomics 14: 265. 9 PLOS ONE \| www.plosone.org September 2014 \| Volume 9 \| Issue 9 \| e107012 September 2014 \| Volume 9 \| Issue 9 \| e107012 Serum MiRNA Profile of Atypical Coronary Artery Disease PLOS ONE \| www.plosone.org September 2014 \| Volume 9 \| Issue 9 \| e107012 PLOS ONE \| www.plosone.org 10
https://openalex.org/W2029382380	https://europepmc.org/articles/pmc3470392?pdf=render	English	null	12-(4-Methoxybenzoyl)-2-methylbenzo[<i>f</i>]pyrido[1,2-<i>a</i>]indole-6,11-dione	Acta crystallographica. Section E	2,012	cc-by	4,014	J. Josephine Novina,a G. Vasuki,b* Yun Liuc and Jin-Wei Sunc 18096 measured reﬂections 3852 independent reﬂections 2856 reﬂections with I > 2(I) Rint = 0.029 aDepartment of Physics, Idhaya College for Women, Kumbakonam-1, India, bDepartment of Physics, Kunthavai Naachiar Govt. Arts College (W) (Autonomous), Thanjavur-7, India, and cInstitute of Chemistry and Chemical Engineering, Xuzhou Normal University, Xuzhou 221116, Jiangsu, People’s Republic of China Correspondence e-mail: vasuki.arasi@yahoo.com Reﬁnement R[F 2 > 2(F 2)] = 0.043 wR(F 2) = 0.120 S = 1.04 3852 reﬂections 271 parameters H-atom parameters constrained max = 0.26 e A˚ 3 min = 0.20 e A˚ 3 Received 14 August 2012; accepted 24 September 2012 Key indicators: single-crystal X-ray study; T = 293 K; mean (C–C) = 0.002 A˚; R factor = 0.043; wR factor = 0.120; data-to-parameter ratio = 14.2. Data collection: APEX2 (Bruker, 2004); cell reﬁnement: APEX2 and SAINT (Bruker, 2004); data reduction: SAINT and XPREP (Bruker, 2004); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 for Windows (Farrugia, 1997); software used to prepare material for publication: PLATON (Spek, 2009). In the title compound, C25H17NO4, the indolizine fused naphthaquinone unit is approximately planar [r.m.s deviation = 0.0678 A˚ ] and makes a dihedral angle of 57.82 (5) with the benzene ring of the methoxybenzene group. The naphtho- quinone O atoms deviate, in the same sense, from the mean plane of the fused six-membered rings by 0.2001 (14) and 0.0516 (14) A˚ . In the crystal there is – stacking of inversion- related pairs of molecules [interplanar spacing = 3.514 (2) A˚ ]. In the title compound, C25H17NO4, the indolizine fused naphthaquinone unit is approximately planar [r.m.s deviation = 0.0678 A˚ ] and makes a dihedral angle of 57.82 (5) with the benzene ring of the methoxybenzene group. The naphtho- quinone O atoms deviate, in the same sense, from the mean plane of the fused six-membered rings by 0.2001 (14) and 0.0516 (14) A˚ . In the crystal there is – stacking of inversion- related pairs of molecules [interplanar spacing = 3.514 (2) A˚ ]. The authors thank the Sophisticated Analytical Instrument Facility, IIT Madras, Chennai, for the data collection. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: PK2443). Related literature For general background to the applications and biological activity of indolizine derivatives, see: Sˇvorc et al. (2009). For the synthesis of indolizines, see: Babaev et al. (2005), and for their use as intermediates in the synthesis of indolizidines, see: Kloubert et al. (2012). For the crystal structures of similar compounds, see: Liu et al. (2011); Ramesh et al. (2009). For standard bond lengths, see: Allen et al. (1987). 12-(4-Methoxybenzoyl)-2-methyl- benzo[f]pyrido[1,2-a]indole-6,11-dione Data collection Bruker Kappa APEXII CCD diffractometer Absorption correction: multi-scan (SADABS; Bruker, 2004) Tmin = 0.972, Tmax = 0.982 J. Josephine Novina,a G. Vasuki,b* Yun Liuc and Jin-Wei Sunc organic compounds Experimental Crystal data C25H17NO4 Mr = 395.40 Monoclinic, P21=c a = 8.1346 (3) A˚ b = 23.2926 (8) A˚ c = 10.1505 (3) A˚ = 97.304 (2) V = 1907.67 (11) A˚ 3 Z = 4 Mo K radiation = 0.09 mm1 T = 293 K 0.30 0.20 0.20 mm Data collection Bruker Kappa APEXII CCD diffractometer Absorption correction: multi-scan (SADABS; Bruker, 2004) Tmin = 0.972, Tmax = 0.982 18096 measured reﬂections 3852 independent reﬂections 2856 reﬂections with I > 2(I) Rint = 0.029 Reﬁnement R[F 2 > 2(F 2)] = 0.043 wR(F 2) = 0.120 S = 1.04 3852 reﬂections 271 parameters H-atom parameters constrained max = 0.26 e A˚ 3 min = 0.20 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 V = 1907.67 (11) A˚ 3 Z = 4 Mo K radiation = 0.09 mm1 T = 293 K 0.30 0.20 0.20 mm Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: PK2443). Comment Indolizines, the nitrogen containing heterocyclic systems, are widely distributed in nature. In particular, indolizine derivatives are an important class of heterocyclic bioactive compounds with a wide range of applications, such as pharmaceutical drugs, potential central nervous system depressants, calcium entry blockers, cardiovascular agents, spectral sensitizers and novel dyes. Polycyclic indolizine derivatives have been found to have high-efficiency long- wavelength fluorescence quantum yield. Several polyhydroxylated indolizines are interesting as inhibitors of glycosides. They have also been tested as antimycobacterial agents against mycobacterial tuberculosis, for the treatment of angina pectoris, aromatase inhibitory, antiinflammatory, antiviral, analgesic and antitumor activities (Švorc et al., 2009). Moreover, the application of indolizines themselves are as intermediates in the synthesis of indolizidines (Kloubert et al., 2012) and many natural alkaloids contain in their structure a saturated (swainsonine) or aromatic (camptothecin) indolizine moiety (Babaev et al., 2005). The benzo[f]pyrido[1,2-a]indole-6,11-diones are benzo-fused indolizines, and occur in several marine alkaloids (Liu et al., 2011). The synthesis of these compounds has drawn much research interest. In view of their importance, the crystal structure determination of the title compound was carried out and results are presented herein. In the title compound, C25H17NO4, the fused naphthaquione–indolizine ring system (N/C1–C16/O1/O2) is approximately planar with a maximum deviation of 0.1193 (14) Å for atom C11 and -0.2001 (14) Å for atom O1, respectively. The fused ring systems make a dihedral angle of 57.82 (5)° with that of benzene ring of the methoxybenzene group. The torsion angles C11—C18—C19—C20 = -21.0 (2)° and C11—C18—C19—C24 = 161.52 (16)° also indicate that the aromatic ring is at different plane from the plane of the fused ring systems. The sum of bond angles around N [359.99 (43)°] indicates that atom N exhibits sp2 hybridization. The geometric parameters of the title compound (Fig. 1) agree well with a reported similar structure 12-benzoyl-2-methylnaphtho[2,3-b]-indolizine-6,11-dione [Liu et al., 2011]. The O2 atom is essentially coplanar with the ring, deviating by only -0.0516 (14) Å, while O1 deviates by -0.2001 (14) Å from the best- fit plane. The discrepancy in bond length is also observed for C9—C11 [1.400 (2) Å], which is slightly shorter than the average of 1.434 (1) Å calculated for indoles in the Cambridge Structure Database (Allen et al., 1987). The endocyclic angle at C7 is contracted to 114.76 (15)° while those at C8 is expanded to 125.71 (15)°, respectively. References Allen, F. H., Kennard, O., Watson, D. G., Brammer, L., Orpen, A. G. & Taylor, R. (1987). J. Chem. Soc. Perkin Trans. 2, pp. S1–19. Babaev, E. V., Vasilevich, N. I. & Ivushkina, A. S. (2005). Beilstein J. Org. Chem. 1, 1–3. Bruker (2004). SADABS, APEX2, SAINT and XPREP. Bruker AXS Inc., Madison, Wisconsin, USA. , , Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Kloubert, T., Kretschmer, R., Go¨rls, H. & Westerhausen, M. (2012). Acta Cryst E68 o2631–o2632 g , ( ) pp y , Kloubert, T., Kretschmer, R., Go¨rls, H. & Westerhausen, M. (2012). Acta Kloubert, T., Kretschmer, R., Go¨rls, H. & Westerhausen, M. (2012). Acta Cryst. E68, o2631–o2632. Cryst. E68, o2631–o2632. Liu, Y., Wang, S.-H., Shen, S.-R. & Yang, Z.-H. (2011). Acta Cryst. E67, o1550. Ramesh, P., Sundaresan, S. S., Lakshmi, N. V., Perumal, P. T. & Ponnuswamy, M. N. (2009). Acta Cryst. E65, o994. M. N. (2009). Acta Cryst. E65, o994. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122 Spek, A. L. (2009). Acta Cryst. D65, 148–155. ˇ ˇ ˇ Sˇvorc, Lˇ ., Vra´bel, V., Kozˇı´sˇek, J., Marchalı´n, Sˇ. & Sˇafa´rˇ, P. (2009). Acta Cryst. E65, o695–o696. o3040 Novina et al. Acta Cryst. (2012). E68, o3040 doi:10.1107/S1600536812040408 supplementary materials Acta Cryst. (2012). E68, o3040 [doi:10.1107/S1600536812040408] Acta Cryst. (2012). E68, o3040 [doi:10.1107/S1600536812040408] Refinement All H atoms were positioned geometrically and treated as riding on their parent atoms: C—H =0.93 and 0.96 Å for CH and CH3 H atoms, respectively, with Uiso(H) =KUeq (parent C-atom), where K=1.5 for CH3 H atoms and K=1.2 for CH H- atoms. Experimental 4-Methyl pyridine (3.0 mmol), 2-bromo-1-(4-methoxyphenyl)ethanone (1.0 mmol), 1,4-naphthaquionone (1.0 mmol), and hydrated copper chloride (0.1 mmol) were mixed in 15 ml of CH3CN and heated to reflux for 12 h. After completion of the reaction, the reaction mixture was separated by silica gel column chromatography to afford the title compound (yield: 91%). Computing details Data collection: APEX2 (Bruker, 2004); cell refinement: APEX2 and SAINT (Bruker, 2004); data reduction: SAINT and XPREP (Bruker, 2004); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 for Windows (Farrugia, 1997); software used to prepare material for publication: PLATON (Spek, 2009). prepare material for publication: PLATON (Spek, 2009). Figure 1 The molecular structure of the title compound with displacement ellipsoids drawn at the 50% probability level. Comment This would appear to be a real effect caused by the fusion of the indolizine with naphthalene ring resulting an angular distortion as observed in the reported structure 3′-benzyloxy-3-hydroxy-3,3′-bi-1H-indole-2,2′(3H,3′H)-dione monohydrate [Ramesh et al., 2009]. The widening of exocyclic angle O4—C22—C21 [125.05 (16)°] from the normal value of 120°, may be due to steric repulsion between atoms H21 and H25C (H21—H25C = 2.367 Å). In the crystal, there is π-π stacking of inversion-related pairs of molecules [interplanar spacing = 3.514 (2) Å]. Acta Cryst. (2012). E68, o3040 sup-1 supplementary materials Figure 1 Figure 1 The molecular structure of the title compound with displacement ellipsoids drawn at the 50% probability level. Figure 1 The molecular structure of the title compound with displacement ellipsoids drawn at the 50% probability level. The molecular structure of the title compound with displacement ellipsoids drawn at the 50% probability level. sup-2 Acta Cryst. (2012). E68, o3040 supplementary materials Figure 2 C t l ki f th titl d i d i t l d th bi t f th d Figure 2 g Crystal packing of the title compound viewed approximately down the bisector of the a and c axes. 12-(4-Methoxybenzoyl)-2-methylbenzo[f]pyrido[1,2-a]indole- 6,11-dione 12-(4-Methoxybenzoyl)-2-methylbenzo[f]pyrido[1,2-a]indole- 6,11-dione Crystal data C25H17NO4 Mr = 395.40 Monoclinic, P21/c Hall symbol: -P 2ybc a = 8.1346 (3) Å b = 23.2926 (8) Å c = 10.1505 (3) Å β = 97.304 (2)° V = 1907.67 (11) Å3 Z = 4 F(000) = 824 Dx = 1.377 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 5015 reflections θ = 2.2–26.3° µ = 0.09 mm−1 T = 293 K Block, brown 0.30 × 0.20 × 0.20 mm F(000) = 824 Dx = 1.377 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 5015 reflections θ = 2.2–26.3° µ = 0.09 mm−1 T = 293 K Block, brown 0.30 × 0.20 × 0.20 mm sup-3 Acta Cryst. (2012). E68, o3040 supplementary materials Data collection Bruker Kappa APEXII CCD diffractometer Radiation source: fine-focus sealed tube Graphite monochromator ω and φ scan Absorption correction: multi-scan (SADABS; Bruker, 2004) Tmin = 0.972, Tmax = 0.982 18096 measured reflections 3852 independent reflections 2856 reflections with I > 2σ(I) Rint = 0.029 θmax = 26.3°, θmin = 2.2° h = −10→10 k = −28→29 l = −12→10 Refinement Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.043 wR(F2) = 0.120 S = 1.04 3852 reflections 271 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0533P)2 + 0.4967P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.26 e Å−3 Δρmin = −0.20 e Å−3 Secondary atom site location: difference Fourie map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0533P)2 + 0.4967P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.26 e Å−3 Δρmin = −0.20 e Å−3 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0533P)2 + 0.4967P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.26 e Å−3 Δρmin = −0.20 e Å−3 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq N 0.17994 (16) 0.53308 (6) 0.57068 (13) 0.0388 (3) O2 0.34773 (17) 0.42161 (6) 0.56349 (14) 0.0628 (4) C8 0.24197 (19) 0.50723 (7) 0.46488 (15) 0.0376 (4) C9 0.21764 (19) 0.54500 (7) 0.35825 (16) 0.0383 (4) O1 0.2104 (2) 0.55356 (6) 0.12630 (12) 0.0652 (4) C6 0.3785 (2) 0.43608 (7) 0.33619 (18) 0.0435 (4) C18 0.0992 (2) 0.64903 (7) 0.32447 (17) 0.0439 (4) C19 0.2224 (2) 0.67504 (7) 0.24748 (16) 0.0379 (4) C1 0.3460 (2) 0.47115 (7) 0.22307 (18) 0.0443 (4) C21 0.5041 (2) 0.68874 (7) 0.20229 (17) 0.0431 (4) H21 0.6165 0.6806 0.2219 0.052 O3 −0.03169 (17) 0.67344 (7) 0.33481 (16) 0.0714 (5) C16 0.1778 (2) 0.51365 (9) 0.69868 (16) 0.0477 (4) H16 0.2195 0.4776 0.7238 0.057* C10 0.2554 (2) 0.52654 (7) 0.22716 (17) 0.0444 (4) C12 0.11594 (19) 0.58666 (7) 0.53067 (16) 0.0399 (4) C7 0.3231 (2) 0.45262 (8) 0.46517 (17) 0.0426 (4) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-4 Acta Cryst. (2012). Special details E68, o3040 supplementary materials supplementary materials sup 5 A t C t (2012) E68 3040 C14 0.0491 (2) 0.60280 (9) 0.75177 (18) 0.0509 (5) C23 0.2821 (2) 0.74026 (8) 0.0772 (2) 0.0539 (5) H23 0.2453 0.7662 0.0101 0.065 O4 0.54786 (16) 0.75454 (6) 0.02430 (14) 0.0634 (4) C20 0.3900 (2) 0.66254 (7) 0.27206 (16) 0.0409 (4) H20 0.4265 0.6358 0.3373 0.049* C13 0.0496 (2) 0.62101 (8) 0.62437 (18) 0.0464 (4) H13 0.0052 0.6567 0.5991 0.056* C22 0.4494 (2) 0.72723 (7) 0.10299 (17) 0.0443 (4) C2 0.3967 (2) 0.45322 (9) 0.1042 (2) 0.0553 (5) H2 0.3732 0.4758 0.0285 0.066* C11 0.14110 (19) 0.59490 (7) 0.39734 (16) 0.0403 (4) C24 0.1712 (2) 0.71529 (8) 0.14967 (18) 0.0483 (4) H24 0.0598 0.7253 0.1334 0.058* C15 0.1142 (2) 0.54769 (9) 0.78676 (18) 0.0539 (5) H15 0.1129 0.5346 0.8732 0.065* C4 0.5135 (3) 0.36798 (9) 0.2080 (2) 0.0644 (6) H4 0.5702 0.3335 0.2030 0.077* C5 0.4614 (2) 0.38467 (8) 0.3266 (2) 0.0538 (5) H5 0.4824 0.3611 0.4008 0.065* C17 −0.0160 (3) 0.63983 (11) 0.8546 (2) 0.0731 (7) H17A −0.0058 0.6197 0.9379 0.110* H17B −0.1305 0.6486 0.8270 0.110* H17C 0.0467 0.6748 0.8649 0.110* C3 0.4816 (3) 0.40227 (9) 0.0974 (2) 0.0646 (6) H3 0.5174 0.3911 0.0178 0.077* C25 0.7210 (2) 0.74482 (11) 0.0475 (3) 0.0754 (7) H25A 0.7748 0.7666 −0.0149 0.113* H25B 0.7430 0.7047 0.0368 0.113* H25C 0.7624 0.7565 0.1362 0.113* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 N 0.0378 (7) 0.0458 (8) 0.0328 (7) −0.0053 (6) 0.0042 (6) 0.0017 (6) O2 0.0696 (9) 0.0613 (9) 0.0577 (8) 0.0171 (7) 0.0086 (7) 0.0195 (7) C8 0.0367 (8) 0.0409 (9) 0.0352 (9) −0.0036 (7) 0.0045 (7) 0.0012 (7) C9 0.0375 (8) 0.0406 (9) 0.0370 (9) −0.0022 (7) 0.0061 (7) 0.0005 (7) O1 0.1040 (11) 0.0559 (8) 0.0363 (7) 0.0112 (8) 0.0117 (7) 0.0047 (6) C6 0.0366 (9) 0.0411 (10) 0.0529 (11) −0.0042 (7) 0.0058 (8) −0.0034 (8) C18 0.0452 (10) 0.0427 (10) 0.0444 (10) 0.0067 (8) 0.0081 (8) 0.0021 (8) C19 0.0417 (9) 0.0344 (9) 0.0377 (9) 0.0031 (7) 0.0049 (7) −0.0014 (7) C1 0.0436 (9) 0.0431 (10) 0.0479 (10) −0.0067 (7) 0.0123 (8) −0.0052 (8) C21 0.0389 (9) 0.0444 (10) 0.0453 (10) 0.0049 (7) 0.0033 (7) −0.0012 (8) O3 0.0561 (8) 0.0741 (10) 0.0893 (11) 0.0229 (7) 0.0302 (8) 0.0266 (8) C16 0.0476 (10) 0.0604 (12) 0.0346 (9) −0.0068 (9) 0.0031 (8) 0.0086 (9) C10 0.0543 (10) 0.0411 (10) 0.0388 (10) −0.0048 (8) 0.0097 (8) 0.0002 (8) C12 0.0353 (8) 0.0445 (9) 0.0402 (9) −0.0055 (7) 0.0056 (7) −0.0012 (8) C7 0.0374 (9) 0.0446 (10) 0.0447 (10) −0.0034 (7) 0.0014 (7) 0.0053 (8) C14 0.0436 (10) 0.0670 (13) 0.0433 (10) −0.0125 (9) 0.0095 (8) −0.0108 (9) Atomic displacement parameters (Å2) sup-5 Acta Cryst. Special details (2012). Acta Cryst. (2012). E68, o3040 Special details E68, o3040 supplementary materials C23 0.0517 (11) 0.0515 (11) 0.0579 (12) 0.0064 (9) 0.0044 (9) 0.0211 (9) O4 0.0549 (8) 0.0670 (9) 0.0710 (9) −0.0051 (7) 0.0179 (7) 0.0200 (7) C20 0.0460 (9) 0.0394 (9) 0.0365 (9) 0.0073 (7) 0.0017 (7) 0.0036 (7) C13 0.0422 (9) 0.0507 (10) 0.0473 (10) −0.0040 (8) 0.0102 (8) −0.0081 (8) C22 0.0485 (10) 0.0394 (9) 0.0462 (10) −0.0028 (8) 0.0108 (8) −0.0006 (8) C2 0.0608 (12) 0.0542 (11) 0.0543 (11) −0.0064 (9) 0.0202 (9) −0.0091 (9) C11 0.0399 (9) 0.0423 (9) 0.0395 (9) −0.0012 (7) 0.0079 (7) 0.0011 (7) C24 0.0383 (9) 0.0473 (10) 0.0582 (11) 0.0071 (8) 0.0024 (8) 0.0112 (9) C15 0.0534 (11) 0.0751 (14) 0.0335 (9) −0.0127 (10) 0.0073 (8) −0.0006 (9) C4 0.0555 (12) 0.0530 (12) 0.0865 (16) 0.0040 (10) 0.0153 (11) −0.0176 (12) C5 0.0478 (10) 0.0449 (10) 0.0682 (13) 0.0010 (8) 0.0054 (9) −0.0037 (9) C17 0.0779 (15) 0.0912 (17) 0.0537 (12) −0.0094 (13) 0.0219 (11) −0.0256 (12) C3 0.0655 (13) 0.0615 (13) 0.0712 (14) −0.0054 (11) 0.0266 (11) −0.0222 (12) C25 0.0506 (12) 0.0877 (17) 0.0921 (17) −0.0101 (11) 0.0251 (12) 0.0067 (14) Geometric parameters (Å, º) N—C16 1.378 (2) C14—C13 1.361 (3) N—C8 1.381 (2) C14—C15 1.416 (3) N—C12 1.393 (2) C14—C17 1.502 (3) O2—C7 1.228 (2) C23—C24 1.365 (2) C8—C9 1.389 (2) C23—C22 1.386 (3) C8—C7 1.433 (2) C23—H23 0.9300 C9—C11 1.400 (2) O4—C22 1.359 (2) C9—C10 1.467 (2) O4—C25 1.416 (2) O1—C10 1.218 (2) C20—H20 0.9300 C6—C5 1.384 (2) C13—H13 0.9300 C6—C1 1.407 (2) C2—C3 1.379 (3) C6—C7 1.489 (2) C2—H2 0.9300 C18—O3 1.223 (2) C24—H24 0.9300 C18—C19 1.477 (2) C15—H15 0.9300 C18—C11 1.479 (2) C4—C3 1.375 (3) C19—C20 1.385 (2) C4—C5 1.382 (3) C19—C24 1.390 (2) C4—H4 0.9300 C1—C2 1.388 (2) C5—H5 0.9300 C1—C10 1.489 (2) C17—H17A 0.9600 C21—C20 1.379 (2) C17—H17B 0.9600 C21—C22 1.380 (2) C17—H17C 0.9600 C21—H21 0.9300 C3—H3 0.9300 C16—C15 1.347 (3) C25—H25A 0.9600 C16—H16 0.9300 C25—H25B 0.9600 C12—C13 1.402 (2) C25—H25C 0.9600 C12—C11 1.407 (2) C16—N—C8 129.76 (15) C21—C20—C19 121.72 (15) C16—N—C12 121.34 (15) C21—C20—H20 119.1 C8—N—C12 108.89 (13) C19—C20—H20 119.1 N C8 C9 107 44 (14) C14 C13 C12 120 95 (18) C23 0.0517 (11) 0.0515 (11) 0.0579 (12) 0.0064 (9) 0.0044 (9) 0.0211 (9) O4 0.0549 (8) 0.0670 (9) 0.0710 (9) −0.0051 (7) 0.0179 (7) 0.0200 (7) C20 0.0460 (9) 0.0394 (9) 0.0365 (9) 0.0073 (7) 0.0017 (7) 0.0036 (7) C13 0.0422 (9) 0.0507 (10) 0.0473 (10) −0.0040 (8) 0.0102 (8) −0.0081 (8) C22 0.0485 (10) 0.0394 (9) 0.0462 (10) −0.0028 (8) 0.0108 (8) −0.0006 (8) C2 0.0608 (12) 0.0542 (11) 0.0543 (11) −0.0064 (9) 0.0202 (9) −0.0091 (9) C11 0.0399 (9) 0.0423 (9) 0.0395 (9) −0.0012 (7) 0.0079 (7) 0.0011 (7) C24 0.0383 (9) 0.0473 (10) 0.0582 (11) 0.0071 (8) 0.0024 (8) 0.0112 (9) C15 0.0534 (11) 0.0751 (14) 0.0335 (9) −0.0127 (10) 0.0073 (8) −0.0006 (9) C4 0.0555 (12) 0.0530 (12) 0.0865 (16) 0.0040 (10) 0.0153 (11) −0.0176 (12) C5 0.0478 (10) 0.0449 (10) 0.0682 (13) 0.0010 (8) 0.0054 (9) −0.0037 (9) C17 0.0779 (15) 0.0912 (17) 0.0537 (12) −0.0094 (13) 0.0219 (11) −0.0256 (12) C3 0.0655 (13) 0.0615 (13) 0.0712 (14) −0.0054 (11) 0.0266 (11) −0.0222 (12) C25 0.0506 (12) 0.0877 (17) 0.0921 (17) −0.0101 (11) 0.0251 (12) 0.0067 (14) Geometric parameters (Å, º) Acta Cryst. Special details E68, o3040 sup-6 supplementary materials C8—C9—C11 109.24 (14) O4—C22—C21 125.05 (16) C8—C9—C10 119.70 (15) O4—C22—C23 115.09 (16) C11—C9—C10 130.71 (15) C21—C22—C23 119.86 (16) C5—C6—C1 119.22 (17) C3—C2—C1 120.6 (2) C5—C6—C7 119.38 (17) C3—C2—H2 119.7 C1—C6—C7 121.39 (15) C1—C2—H2 119.7 O3—C18—C19 120.77 (16) C9—C11—C12 106.48 (14) O3—C18—C11 120.06 (16) C9—C11—C18 130.43 (15) C19—C18—C11 119.03 (14) C12—C11—C18 122.99 (15) C20—C19—C24 117.97 (15) C23—C24—C19 120.92 (16) C20—C19—C18 122.49 (15) C23—C24—H24 119.5 C24—C19—C18 119.50 (15) C19—C24—H24 119.5 C2—C1—C6 119.25 (17) C16—C15—C14 122.00 (17) C2—C1—C10 119.19 (17) C16—C15—H15 119.0 C6—C1—C10 121.54 (15) C14—C15—H15 119.0 C20—C21—C22 119.14 (16) C3—C4—C5 120.08 (19) C20—C21—H21 120.4 C3—C4—H4 120.0 C22—C21—H21 120.4 C5—C4—H4 120.0 C15—C16—N 119.00 (18) C4—C5—C6 120.7 (2) C15—C16—H16 120.5 C4—C5—H5 119.6 N—C16—H16 120.5 C6—C5—H5 119.6 O1—C10—C9 122.39 (16) C14—C17—H17A 109.5 O1—C10—C1 121.42 (16) C14—C17—H17B 109.5 C9—C10—C1 116.13 (15) H17A—C17—H17B 109.5 N—C12—C13 118.38 (15) C14—C17—H17C 109.5 N—C12—C11 107.93 (14) H17A—C17—H17C 109.5 C13—C12—C11 133.63 (17) H17B—C17—H17C 109.5 O2—C7—C8 123.46 (17) C4—C3—C2 120.1 (2) O2—C7—C6 121.77 (16) C4—C3—H3 119.9 C8—C7—C6 114.76 (15) C2—C3—H3 119.9 C13—C14—C15 118.33 (17) O4—C25—H25A 109.5 C13—C14—C17 121.7 (2) O4—C25—H25B 109.5 C15—C14—C17 119.98 (18) H25A—C25—H25B 109.5 C24—C23—C22 120.34 (17) O4—C25—H25C 109.5 C24—C23—H23 119.8 H25A—C25—H25C 109.5 C22—C23—H23 119.8 H25B—C25—H25C 109.5 C22—O4—C25 118.39 (16) C16—N—C8—C9 −178.53 (15) C22—C21—C20—C19 −1.8 (3) C12—N—C8—C9 0.38 (17) C24—C19—C20—C21 −0.2 (3) C16—N—C8—C7 −1.0 (3) C18—C19—C20—C21 −177.69 (16) C12—N—C8—C7 177.96 (15) C15—C14—C13—C12 −1.4 (3) N—C8—C9—C11 0.40 (18) C17—C14—C13—C12 177.84 (17) C7—C8—C9—C11 −177.21 (15) N—C12—C13—C14 0.7 (2) N—C8—C9—C10 −173.46 (14) C11—C12—C13—C14 −176.11 (17) C7—C8—C9—C10 8.9 (2) C25—O4—C22—C21 −2.3 (3) O3—C18—C19—C20 154.62 (18) C25—O4—C22—C23 178.04 (18) C11—C18—C19—C20 −21.0 (2) C20—C21—C22—O4 −177.84 (17) O3—C18—C19—C24 −22.9 (3) C20—C21—C22—C23 1.8 (3) su 109.24 (14) O4—C22—C21 125.05 (16) 119.70 (15) O4—C22—C23 115.09 (16) 130.71 (15) C21—C22—C23 119.86 (16) 119.22 (17) C3—C2—C1 120.6 (2) 119.38 (17) C3—C2—H2 119.7 121.39 (15) C1—C2—H2 119.7 120.77 (16) C9—C11—C12 106.48 (14) 120.06 (16) C9—C11—C18 130.43 (15) 119.03 (14) C12—C11—C18 122.99 (15) 117.97 (15) C23—C24—C19 120.92 (16) 122.49 (15) C23—C24—H24 119.5 119.50 (15) C19—C24—H24 119.5 119.25 (17) C16—C15—C14 122.00 (17) 119.19 (17) C16—C15—H15 119.0 121.54 (15) C14—C15—H15 119.0 119.14 (16) C3—C4—C5 120.08 (19) 120.4 C3—C4—H4 120.0 120.4 C5—C4—H4 120.0 119.00 (18) C4—C5—C6 120.7 (2) 120.5 C4—C5—H5 119.6 120.5 C6—C5—H5 119.6 122.39 (16) C14—C17—H17A 109.5 121.42 (16) C14—C17—H17B 109.5 116.13 (15) H17A—C17—H17B 109.5 118.38 (15) C14—C17—H17C 109.5 107.93 (14) H17A—C17—H17C 109.5 133.63 (17) H17B—C17—H17C 109.5 123.46 (17) C4—C3—C2 120.1 (2) 121.77 (16) C4—C3—H3 119.9 114.76 (15) C2—C3—H3 119.9 118.33 (17) O4—C25—H25A 109.5 121.7 (2) O4—C25—H25B 109.5 119.98 (18) H25A—C25—H25B 109.5 120.34 (17) O4—C25—H25C 109.5 119.8 H25A—C25—H25C 109.5 119.8 H25B—C25—H25C 109.5 118.39 (16) −178.53 (15) C22—C21—C20—C19 −1.8 (3) 0.38 (17) C24—C19—C20—C21 −0.2 (3) −1.0 (3) C18—C19—C20—C21 −177.69 (16) 177.96 (15) C15—C14—C13—C12 −1.4 (3) 0.40 (18) C17—C14—C13—C12 177.84 (17) −177.21 (15) N—C12—C13—C14 0.7 (2) −173.46 (14) C11—C12—C13—C14 −176.11 (17) 8.9 (2) C25—O4—C22—C21 −2.3 (3) 154.62 (18) C25—O4—C22—C23 178.04 (18) −21.0 (2) C20—C21—C22—O4 −177.84 (17) −22.9 (3) C20—C21—C22—C23 1.8 (3) sup-7 Acta Cryst. Special details (2012). Special details (2012). E68, o3040 supplementary materials supplementary materials C11—C18—C19—C24 161.52 (16) C24—C23—C22—O4 179.80 (17) C5—C6—C1—C2 0.4 (3) C24—C23—C22—C21 0.1 (3) C7—C6—C1—C2 −178.47 (16) C6—C1—C2—C3 −1.5 (3) C5—C6—C1—C10 179.22 (16) C10—C1—C2—C3 179.66 (17) C7—C6—C1—C10 0.3 (2) C8—C9—C11—C12 −1.00 (18) C8—N—C16—C15 177.94 (16) C10—C9—C11—C12 171.95 (17) C12—N—C16—C15 −0.9 (2) C8—C9—C11—C18 175.43 (16) C8—C9—C10—O1 167.00 (17) C10—C9—C11—C18 −11.6 (3) C11—C9—C10—O1 −5.3 (3) N—C12—C11—C9 1.22 (18) C8—C9—C10—C1 −10.4 (2) C13—C12—C11—C9 178.26 (17) C11—C9—C10—C1 177.31 (16) N—C12—C11—C18 −175.55 (14) C2—C1—C10—O1 7.4 (3) C13—C12—C11—C18 1.5 (3) C6—C1—C10—O1 −171.35 (17) O3—C18—C11—C9 140.6 (2) C2—C1—C10—C9 −175.19 (16) C19—C18—C11—C9 −43.8 (3) C6—C1—C10—C9 6.0 (2) O3—C18—C11—C12 −43.5 (3) C16—N—C12—C13 0.5 (2) C19—C18—C11—C12 132.13 (17) C8—N—C12—C13 −178.57 (14) C22—C23—C24—C19 −2.1 (3) C16—N—C12—C11 178.02 (14) C20—C19—C24—C23 2.1 (3) C8—N—C12—C11 −1.00 (17) C18—C19—C24—C23 179.74 (17) N—C8—C7—O2 −0.2 (3) N—C16—C15—C14 0.2 (3) C9—C8—C7—O2 176.99 (17) C13—C14—C15—C16 1.0 (3) N—C8—C7—C6 −179.40 (14) C17—C14—C15—C16 −178.26 (18) C9—C8—C7—C6 −2.2 (2) C3—C4—C5—C6 −0.7 (3) C5—C6—C7—O2 −0.6 (3) C1—C6—C5—C4 0.7 (3) C1—C6—C7—O2 178.29 (16) C7—C6—C5—C4 179.60 (16) C5—C6—C7—C8 178.63 (15) C5—C4—C3—C2 −0.4 (3) C1—C6—C7—C8 −2.5 (2) C1—C2—C3—C4 1.5 (3) sup-8 Acta Cryst. (2012). E68, o3040
https://openalex.org/W4321168218	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0281826&type=printable	English	null	Impacts of medical and non-medical cannabis on the health of older adults: Findings from a scoping review of the literature	PloS one	2,023	cc-by	23,606	PLOS ONE RESEARCH ARTICLE Objective Copyright: © 2023 Wolfe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. To perform a scoping review to map the evidence regarding the health effects of cannabis use for medical and non-medical purposes in older adults. Editor: Francesca Baratta, University of Turin, ITALY Received: December 7, 2021 Accepted: January 31, 2023 Published: February 17, 2023 Abstract Citation: Wolfe D, Corace K, Butler C, Rice D, Skidmore B, Patel Y, et al. (2023) Impacts of medical and non-medical cannabis on the health of older adults: Findings from a scoping review of the literature. PLoS ONE 18(2): e0281826. https://doi. org/10.1371/journal.pone.0281826 Background Cannabis legalization has enabled increased consumption in older adults. Age-related men- tal, physical, and physiological changes may lead to differences in effects of cannabis in older adults compared to younger individuals. Editor: Francesca Baratta, University of Turin, ITALY Editor: Francesca Baratta, University of Turin, ITALY Impacts of medical and non-medical cannabis on the health of older adults: Findings from a scoping review of the literature Dianna WolfeID1, Kim Corace2,3, Claire Butler1, Danielle Rice1, Becky Skidmore1, Yashila Patel1, Premika Thayaparan1, Alan MichaudID1, Candyce Hamel1, Andra Smith4, Gary Garber5,6, Amy Porath7, David Conn8,9, Melanie Willows2,3, Hanan Abramovici10, Kednapa Thavorn1,6, Salmaan Kanji1,5,6, Brian HuttonID1,6* 1 Ottawa Hospital Research Institute, Ottawa, Canada, 2 Institute of Mental Health Research at The Royal, University of Ottawa, Ottawa, Canada, 3 Department of Family Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Canada, 4 School of Psychology, Faculty of Social Sciences, University of Ottawa, Ottawa, Canada, 5 Department of Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Canada, 6 School of Epidemiology and Public Health, University of Ottawa, Ottawa, Canada, 7 Canadian Center for Substance Use and Addiction, Ottawa, Canada, 8 Department of Psychiatry, University of Toronto, Toronto, Canada, 9 Baycrest Health Sciences, Toronto, Canada, 10 Health Canada, Office of Cannabis Science and Surveillance, Ottawa, Canada a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 PLOS ONE PLOS ONE Discussion The effects of medical cannabis are inconsistent within specific patient conditions. For older adults, generally, the available evidence suggests cannabis use may be associated with greater frequencies of mental health issues, substance use, and acute healthcare use, and the benefit-to-risk ratio is unclear. Studies with a balanced assessment of benefits and harms may guide appropriate public health messaging to balance the marketing pressures of cannabis to older adults. Findings From 31,393 citations, 133 publications describing 134 unique studies (26 SRs, 36 RCTs, 72 NRSs) were included. Medical cannabis had inconsistent therapeutic effects in specific patient conditions (e.g., end-stage cancer, dementia), with a number of studies suggesting possible benefits while others found no benefit. For medical cannabis, harmful associations outnumbered beneficial, and RCTs reported more negative effects than NRSs. Cannabis use was associated with greater frequencies of depression, anxiety, cognitive impairment, substance use and problematic substance use, accidents/injuries, and acute healthcare use. Studies often were small, did not consistently assess harms, and did not adjust for confounding. Competing interests: DC is Project Lead for the Canadian Coalition for Seniors’ Mental Health (CCSMH) project on development of Canadian Guidelines on Alcohol, Benzodiazepine, Cannabis and Opioid Use Disorders funded by Health Canada (SUAP) 2017-20; Project Lead for CCSMH Project Cannabis Use and Older Adults: Developing E- Learning Modules and Knowledge Translation Tools for Clinicians and Students, funded by Health Canada (2020-2022). BH has previously received honoraria from Eversana Inc for provision of methodologic advice related to the conduct of systematic reviews. This does not alter the adherence to PLOS Medicine policies on sharing data and materials. All other authors have no conflicts to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Methods Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Electronic databases (MEDLINE, Embase, PsycINFO, Cochrane Library) were searched for systematic reviews (SRs), randomized controlled trials (RCTs) and non-randomized/ observational studies (NRSs) assessing the health effects and associations of cannabis use (medical or non-medical) in adults 50 years of age. Included studies met age-related inclu- sion criteria or involved a priori identified health conditions common among older adults. Records were screened using a liberal accelerated approach and data charting was per- formed independently by two reviewers. Descriptive summaries, structured tables, effect direction plots and bubble plots were used to synthesize study findings. Funding: This work was funded as a catalyst grant in 2019 by the Canadian Institutes of Health Research (https://cihr-irsc.gc.ca/) and the Canadian Centre for Substance Use and Addiction (https://www.ccsa.ca/). The funders had no role in the design of the planned study or preparation of this manuscript. The funders had no role in the PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 1 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults design of the planned study or preparation of this manuscript. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 Introduction Legalization of cannabis has increased access for consumers in a growing number of countries, including Canada and the United States. In the first year following legalization in Canada, the proportion of older adults who reported using cannabis in the previous three months increased significantly over pre-legalisation estimates, from 10.0 to 11.9% in those 45 to 64 years of age, and from 4.1 to 5.9% in those 65 years and older [1]. The proportion of adults 65 years and older who reported daily use of cannabis also rose significantly, from 1.6 to 2.6%, the greatest increase in any age group [1]. More than a half of those over 65 years of age reported using cannabis strictly for medical reasons, while a quarter of those over 65 years of age reported trying cannabis for the first time in the previous three months [2]. Prior to legaliza- tion (2008) in the US, 95% of adults over 50 that had consumed cannabis in the past year had initiated use before the age of 30, suggesting that a substantial number may have used cannabis over the long term rather than initiating at middle age for medical purposes [3]. More recent US surveys (2015–17) support this finding: more than 75% of adults over 50 who consumed cannabis in the past year had used continuously for at least three years, while less than 6% had initiated use for the first time in the past year [4]. Recent data suggest 6–7% of older adults use cannabis, and amongst them approximately 75% use it for medical purposes [5]. Older adults generally suffer from more chronic health conditions than younger adults (e.g., chronic pain, insomnia) [6, 7] and, thus, may be attracted to cannabis for medical purposes [3, 8] by market- ing efforts that exaggerate the medical and “wellness” benefits of cannabis, while minimizing its harms [9], and by word-of-mouth anecdotal “evidence.” Legalization, increased access, and non-evidence-based marketing may plausibly increase the proportion of older adults who con- sume cannabis for both medical and non-medical purposes [10]. Study eligibility criteria Table 1 provides a summary of study eligibility criteria. Review methods A scoping review protocol was developed a priori, guided by established scoping review method- ology [22–25], and was registered with the Open Science Framework (doi 10.17605/OSF.IO/ 5JTAQ), and published [26]. Protocol amendments are described along with rationale in S1 Text. Introduction Past work has suggested med- ical cannabinoids may offer certain benefits from consumption when conventional treatment does not help for conditions that include neuropathic pain, chemotherapy induced nausea and vomiting, spasticity (from multiple sclerosis and spinal cord injury), palliative and end-of-life PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 2 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults pain [11]. However, negative outcomes may also become increasingly prevalent, including increased risks of cannabis use disorder and problematic cannabis use, cognitive impairments, drug interactions due to polypharmacy, adverse reactions due to multiple health conditions, and injuries and visits to emergency departments (EDs). Several age-related mental, physical, physiological, and pharmacokinetic changes may con- tribute to critical differences in the health effects of cannabis between older and younger adults. Declines in cognitive functioning, including executive function, memory, and atten- tion, as well as structural changes to both the grey and white matter of the brain occur with age. Cannabis use has been associated with worsening of these outcomes [12–15]. Polyphar- macy is highly prevalent in older adults and may increase susceptibility to drug interactions with cannabis [16–19], while the physiologic changes of aging may alter cannabis pharmacoki- netics and pharmacodynamics [8, 20, 21], potentially further increasing the risk of adverse drug effects and interactions. Currently, Canadian guidelines support the use of cannabis for a limited number of condi- tions in all age ranges, when standard treatments are ineffective (e.g., neuropathic and pallia- tive pain, chemotherapy-induced nausea and vomiting, spasticity in multiple sclerosis and spinal cord injury) [11]. A greater understanding of the beneficial and harmful health effects of cannabis for both medical and non-medical consumption in older adults is needed, as well as targeted dissemination of evidence-based education and information to physicians, health care providers or practitioners, public health organizations, and the general public, including older adults and their caregivers and family members. We carried out a scoping review of the litera- ture to evaluate the depth of the available evidence regarding impacts and associations of use of cannabis for medical and non-medical purposes on the health of older adults, with the intent of mapping the evidence and identifying priority areas for future research. Key questions The following review question was addressed: What evidence exists regarding the beneficial and harmful effects of cannabis for medical and non-medical purposes in older adults? Findings within different categories of the following subgroups, concepts, and contexts were of interest: older adult age categories (e.g., 50–64 years, 65+ years); sex or gender; race or ethnicity; mental or physical comorbidities; frailty; co-use of prescription or non-prescription drugs, alcohol, or illicit substances; consumption method (e.g., smoking, vaporizing, oils, edi- bles); residential setting (e.g., community, long-term care, hospital); employment status; mari- tal status; accommodation status (e.g., alone, shared, shelter). PLOS ONE Scoping review: Medical and non-medical cannabis in older adults Table 1. Study eligibility criteria. PCC framework domain Element Criteria details Participants Age 80% of the study sample aged 50 years and older. Formulae were used to determine age eligibility when studies included adults of all ages (see the “Additional Details of Scoping Review Methods” section below for details). Disease conditions that affect mainly older adults were used as proxies for age in cases where the age of participants was not explicitly reported: end-stage cancer, Alzheimer’s disease or dementia, and Parkinson’s disease. Chemotherapy for any indication was not considered a proxy for age because patients did not necessarily have end-stage cancer. “Current” cannabis use As defined by the study, but not more than one year in the past. Evaluations of the effects of age of initiation of cannabis use, lifetime/ever cannabis use, or previous cannabis use, where the older adult was no longer using cannabis, were excluded. Other characteristics Individuals of any sex/gender or race were of interest. Healthy individuals as well as those with physical or mental health conditions, whether acute or chronic, were of interest. Concept Interventions Cannabis must have been the intervention or exposure in the study. Types of use: medical purposes (overseen by a physician or other health provider or self-medicated; hereafter “medical cannabis”) or non-medical purposes (hereafter “non-medical cannabis”), of any type, with any mode of consumption (e.g., pills/capsules, smoking, vaporizing, oils, edibles). Cannabis types: whole-plant/loose-leaf cannabis; purified whole-plant extracts (e.g., Nabidiolex1 (purified cannabidiol (CBD)), Tetrabinex1 (purified delta-9-tetrahydrocannabinol (THC)), Sativex1 (purified 1:1 THC:CBD)); cannabinoid derivatives, developed through modification of molecular structure (e.g., nabilone); and other cannabinoids (e.g., dronabinol), whether found in the cannabis plant or elsewhere and that interact with the endocannabinoid system [27]. Comparisons: use vs no use (or placebo), types of use, types of cannabis, modes of consumption, doses, etc. Analyses comparing age categories amongst those who use cannabis were not of interest because cannabis was not the exposure of interest. Outcomes Any physical health, mental health, physical brain structure, pharmacokinetic, and global quality of life outcomes, well as measures related to the use or problematic use of other drugs and alcohol. Examples are provided in the “Additional Details of Scoping Review Methods” section below. PLOS ONE We excluded single-arm studies that only reported prevalence or incidence of cannabis use in older adults and those assessing cannabis use as an outcome (however, cannabis use disorder (CUD) as a mental health outcome was eligible). Context Setting, geography, time period, follow-up duration Current cannabis consumption in all settings, in any geographic area, and including all periods of time and durations of follow-up. Consumption of other illicit drugs or substances, or prescribed pharmaceuticals was allowed. Study design Systematic reviews (including overviews of reviews), randomized controlled trials (RCT), non-randomized/ observational studies (NRS) (note that observational studies are included in the NRS acronym; additional design-specific criteria are provided in the “Additional Details of Scoping Review Methods” section below). Regarding studies included in systematic reviews, we included those that met our a priori eligibility criteria (if not already captured by our search). We excluded qualitative studies, diagnostic test accuracy studies, studies developing or validating diagnostic criteria for CUD or other cannabis-related mental health disorders, editorials, letters, commentaries, abstracts, case reports, case series under 25 patients, and narrative reviews. Language Only English and French publications were considered for reasons of timeliness and cost. https://doi org/10 1371/journal pone 0281826 t001 Table 1. Study eligibility criteria. We excluded qualitative studies, diagnostic test accuracy studies, studies developing or validating diagnostic criteria for CUD or other cannabis-related mental health disorders, editorials, letters, commentaries, abstracts, case reports, case series under 25 patients, and narrative reviews. Only English and French publications were considered for reasons of timeliness and cost. reflect causal effects. Therefore, findings from RCTs and cohort studies for which causation may be inferred have been differentiated from associations reported in cross-sectional and case-control studies. Description of methods Table 2 provides a brief description of the methods, with complete methods described in S2 Text. Of note, where possible, we have summarized findings according to their potential to PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 3 / 37 Extent of literature identified Amongst more than 31,000 citations screened, 133 publications were included: 4 overviews of reviews reported in 5 publications [32–36]; 22 systematic reviews [37–58]; 36 RCTs reported PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 4 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults Table 2. Methods in brief. Review stage Details Literature search Ovid MEDLINE1, including Epub Ahead of Print and In-Process & Other Non- Indexed Citations, Embase Classic+Embase, and PsycINFO, using the Ovid Platform, and the Cochrane Library on Wiley Filters applied for study designs of interest No language or date restrictions Search strategies in S3 Text Peer reviewed using Peer Review of Electronic Search Strategies (PRESS) Checklist [28] Searches run 14 June 2019 and updated to 30 November 2020 Bibliographies of included systematic reviews screened For feasibility, no grey literature searching was performed Study selection Citations collated and duplicates removed in EndNotea, with unique records uploaded to online systematic review management software (DistillerSR1b) Two levels of citation screening: (1) title/abstract and (2) full text. Pilot screening at each level. Liberal accelerated approach for Level 1, using DistillerSR’s1 artificial intelligence (AI) active machine learning to prioritize references Dual independent screening for Level 2, with conflict resolution through discussion Once an estimated recall of 95% of included studies was achieved, the AI reviewer was assigned to exclude the remaining citations A human reviewer screened all citations excluded by the AI reviewer, with conflicts resolved by a second human reviewer Data charting Data charting in DistillerSR1, using standardized piloted forms. Dual independent data extraction, with conflict resolution through discussion Study characteristics, demographic data, and outcome data extracted Results data limited to direction of effect, statistical significance, and type of analysis (uni- or multivariable). Raw data without an analysis were not extracted. a Thomson Reuters. EndNote X7. b DistillerSR. Version 2.35. Evidence Partners; 2021. Accessed May 2019 to November 2021. https://www. evidencepartners.com. https://doi.org/10.1371/journal.pone.0281826.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 https://doi.org/10.1371/journal.pone.0281826.t002 Extent of literature identified Direction of effect: based upon the effect estimate relative to the null value (e.g., for harmful outcomes, any value < 1 was “beneficial” and any value > 1 was “harmful”) Statistical significance: as reported in the study, whether through confidence interval, standard error, p-value, or a statement of significance Risk of bias Not performed for primary studies, as per scoping review methodology Quality of systematic reviews appraised using the AMSTAR-2 tool [29] by two reviewers, independently, with consensus through discussion Data synthesis/ mapping/ reporting Descriptive statistics of the available data and narrative summaries of results, complimented by tables and figures Separate syntheses for each patient condition Study design (RCT, cohort, case-control, cross-sectional) and analysis type (adjusted or unadjusted) in NRSs considered in narrative summaries to reflect level of evidence and potential for causal effects. The words “effects” or “impacts” were used to imply potential causation in RCTs and cohort studies, while “associations” and “correlations” were used to imply non-causal relationships between exposures and outcomes in cross-sectional and case-control studies. Bubble plots and direction of effect figures used to visualize evidence Use of “people-first” language to communicate results in a non-stigmatizing way [30] Reported was structured according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis extension for scoping reviews (PRISMA-ScR) statement [31] (S4 Text). https://doi.org/10.1371/journal.pone.0281826.t002 5 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults Fig 1. Overview of publication selection process. https://doi.org/10.1371/journal.pone.0281826.g001 Fig 1. Overview of publication selection process. https://doi.org/10.1371/journal.pone.0281826.g001 Fig 1. Overview of publication selection process. https://doi.org/10.1371/journal.pone.0281826.g001 in 35 publications [20, 59–92]; and 72 NRSs reported in 71 publications [4, 93–162]. Fig 1 summarizes the study selection process, while Fig 2 provides a visual representation of the evi- dence base; the listing of citations excluded during full text screening is provided in S5 Text. Detailed evidence tables presenting key information regarding each study have been provided in S6 Text due to the considerable length of these data. Three of four overviews of reviews focused on cancer [32–35], while one focused on the use of medical cannabis in any condition, including cancer, Alzheimer’s disease, and Parkinson’s disease [36]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 Extent of literature identified None of the overviews focused specifically on cannabis use in older adults (i.e., they focused on patients of all ages, but provided at least one synthesis for older adults or a rel- evant condition), and none included the most recent systematic reviews. Thus, no further syn- thesis of these publications was undertaken. All but one of the systematic reviews focused on the efficacy and safety of cannabis in the treatment of one or more medical conditions [37–43, 45–58]; the other evaluated associa- tions between cannabis use in older adults and the incidence of cancer [44]. All but one review rated low or critically low on AMSTAR-2 assessment; the final review rated moder- ate [57]. Twenty-two of the 105 included primary study publications (21%) were synthe- sized in at least one of the included systematic reviews, including 17 of 35 included RCTs [60, 62–66, 70–72, 74, 76, 80, 82, 83, 86, 87, 89] (49%) and 5 of 70 included NRSs (7%) [101, 137, 148, 156, 158]. Given the low coverage of primary studies of interest to our scoping review in the included systematic reviews and the relatively low quality of the included reviews, we have prioritized reporting evidence from primary studies over that from sys- tematic reviews in the following sections. 6 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults Fig 2. Visual representation of included evidence by study design and patient condition. An overview of the included number of publications is shown; some publications included more than one study. The size of each bubble is relative to the number of publications it represents. https://doi org/10 1371/journal pone 0281826 g002 Fig 2. Visual representation of included evidence by study design and patient condition. An overview of the included number of publications is shown; some publications included more than one study. The size of each bubble is relative to the number of publications it represents. https://doi.org/10.1371/journal.pone.0281826.g002 https://doi.org/10.1371/journal.pone.0281826.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 Interventions and exposures evaluated in the included primary studies Cannabis interventions were generally well reported in RCTs. Sativex1 (n = 8) [62, 67, 70, 76, 78, 82, 80], nabilone (n = 7) [61, 68, 72, 77, 79, 83, 85], dronabinol (Marinol1) (n = 5) [63, 74, 75, 89, 92], and Namisol1 (n = 5) [20, 60, 86–88] were the primary cannabis products studied, with two studies evaluating unnamed synthetic CBD products [69, 91]. Eight RCTs studied other natural whole plant or extracts [59, 64–66, 71, 73, 84, 90], and one studied an unknown medical-grade CBD powder [66]. Most RCTs evaluated medical cannabis use, overseen by a physician (n = 27); the remaining six studies conducted experiments in laboratory settings, evaluating the immediate effects of cannabis [20, 59, 69, 80, 83, 90]. Nineteen RCTs used a par- allel-group design [61–64, 66, 68, 70, 71, 73–76, 78, 79, 81, 82, 85, 86] and 17 used a cross-over design [20, 59, 60, 65, 67, 69, 72, 77, 80, 83, 84, 87–92]. Cannabis interventions were poorly reported in NRSs, with 46 of 72 studies (64%) not reporting a specific cannabis intervention. Many of these were large population-based cross- sectional studies, using data from national surveys (e.g., National Surveys on Drug Use and Health (NSDUH)). Four other NRSs studied dronabinol [120, 121, 156, 158], three studied nabilone [101, 133, 155], and seventeen studied natural whole plant or extracts [93, 94, 97–99, 102, 119, 129, 131, 137, 147–151, 154, 161]. Many NRSs (n = 28) did not explicitly report the type of cannabis use of interest (i.e., medical or non-medical) [95, 96, 102, 103, 105, 107, 113, 115, 117–119, 122–124, 126–128, 134, 139, 141–143, 145, 146, 157, 159–161]. Fourteen other studies explicitly stated that all cannabis use, whether for medical or non-medical purposes, was evaluated [4, 97, 100, 104, 106, 108–110, 112, 116, 132, 135, 138, 144]. Fifteen NRSs focused strictly on medical use, overseen by a physician [94, 98, 99, 101, 111, 120, 121, 131, PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 7 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults 133, 136, 148, 149, 152, 156, 158]; four focused strictly on non-prescribed medical use (i.e., taken for a perceived or actual medical condition, not for recreational purposes) [150, 151, 154, 155]; and ten focused strictly on non-medical/recreational use [93, 114, 125, 129, 130, 137, 147, 153, 162]. The remaining NRS studied medical use of any kind [140]. Fig 4. Synopsis of findings reported in studies of cannabis use in unique patient conditions and populations. https://doi.org/10.1371/journal.pone.0281826.g004 medical cannabis use as many studies did not clearly report the intent of use or involved a mix- ture of both usage types; findings are thus reported according to population condition to pro- vide an organized synthesis of the literature that will be intuitive to readers. Medical cannabis use can be assumed for sections reporting findings for specific patient medical conditions; for all other sections, we have noted the cannabis use type (medical, non-medical, or mixed), where this information was available. Clinical findings by population In the following sections, we provide detailed descriptions of the included evidence regarding cannabis use in older adults, organized according to the selection criteria of the studies. Studies that selected participants based on specific clinical indications (i.e., end-stage cancer, Alzhei- mer’s disease/dementia, Parkinson’s disease, chronic non-cancer pain, multiple sclerosis, etc.) have been summarized under corresponding headings, while studies that did not select partici- pants based on a clinical indication or that selected only healthy participants have been sum- marized under the heading “Population-based studies and studies of healthy older adults.” We provide summaries of key findings associated with healthy older adults and the older general public in Fig 3, while Fig 4 provides a synopsis of findings pertaining to unique clinical condi- tions and populations. In the main text that follows, where possible, we have prioritized find- ings from studies from which potential causal inferences could be drawn over findings from other designs (i.e., RCTs and cohort studies have been summarized before findings from cross-sectional and case-control studies), in particular within the summary of population- based studies and studies of healthy older adults, where most of the identified evidence was derived from cross-sectional and case-control designs. Additionally, we have prioritized reporting of findings from RCTs and NRSs that adjusted for confounding to focus on the least biased results. To ensure complete mapping of all available evidence, results from unadjusted analyses have also been included; however, they have been summarized more succinctly. As noted above, we were unable to fully separate the reporting of findings for medical versus non- Fig 3. Synopsis of findings reported in population-based studies and studies of healthy older adults. https://doi.org/10.1371/journal.pone.0281826.g003 Fig 3. Synopsis of findings reported in population-based studies and studies of healthy older adults. https://doi.org/10.1371/journal.pone.0281826.g003 Fig 3. Synopsis of findings reported in population-based studies and studies of healthy older adults. 8 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 Fig 4. Synopsis of findings reported in studies of cannabis use in unique patient conditions and populations. PLOS ONE Table 3. Characteristics of population-based studies and studies of healthy older adults. Number of studies Designs Primary studies (n = 28) Type of cannabis use Funding 29 Systematic review: 1 [44] Non-medical: 5 [129, 147, 153, 162] Non-industry: 21 [20, 95, 102, 103, 106, 109, 113, 116, 124, 128, 129, 132, 134, 135, 139, 143, 146, 147, 153, 162] RCTs: 1 [20] Mixed use: 10 [4, 100, 104, 106, 107, 109, 116, 132, 135, 144] Mixed (industry and non-industry): 1 [96] Prospective cohort: 1 [96] Immediate effects in lab: 1 [20] Not funded: 2 [104, 107] Retrospective cohort: 2 [103, 135] Not reported: 11 [95, 102, 103, 105, 113, 124, 128, 134, 139, 143, 146] Not reported: 4 [4, 100, 105, 144] Case-control: 2 [129, 147] Sequential: 11 [4, 102, 104, 105, 116, 124, 128, 134, 139, 144, 146] Cross-sectional: 11 [95, 100, 106, 107, 109, 113, 132, 143, 153, 162] https://doi.org/10.1371/journal.pone.0281826.t003 Table 3. Characteristics of population-based studies and studies of healthy older adults. sequential survey, and case-control designs were restricted to associations between cannabis use and the outcomes assessed and have been reported following the RCT and cohort study findings. Additionally, statistical adjustment for confounding in all NRSs was uncommon, increasing the risk of confounding bias. Consequently, the focus of this summary is on find- ings from the RCT and cohort studies that controlled confounding. Adjusted findings from cross-sectional, sequential survey, and case-control studies have been summarized separately, while salient unadjusted findings from all designs have been summarized more briefly. RCTs and cohort studies. Four primary studies used either a RCT or cohort design in which potential causal effects could be assessed [20, 96, 103, 135]. Harmful effects of cannabis on health outcomes reported in these studies outnumbered beneficial effects (Fig 5, square symbols). A retrospective cohort study found significant adjusted associations between mixed cannabis use and suicide in male Veterans Health Administration (VHA) patients [103]; in female patients, the effect of mixed cannabis use on the risk of suicide became non-significant when adjusted for psychiatric illness [103]. Concerns regarding an increased potential for falls and injuries in older adult consumers led to a small RCT (n = 12) evaluating the immediate effects of Namisol1 (synthetic THC) in the laboratory. Population-based studies and studies of healthy older adults Studies included in this section either sampled only healthy older adults [20, 153, 162] or did not sample participants based on a specific health condition [4, 20, 44, 95, 96, 100, 102–107, 109, 113, 115, 124, 128, 129, 132, 134, 135, 139, 143, 144, 146, 147]. A quarter of all primary studies contributed evidence in this section (n = 28). More than half of these studies evaluated data from large population-based surveys or national/regional databases (n = 15), either as cross-sectional studies at a single point in time [95, 100, 105, 107] or sequential designs that analyzed survey data over multiple years [4, 102, 104, 106, 116, 124, 128, 134, 139, 144, 146]. One systematic review reported on the association between cannabis use in older adults and the incidence of cancer [44]; no systematic reviews explored associations of cannabis use with other outcomes in healthy older adults or the older general public. Generally, cannabis use type could not easily be determined, with 10 primary studies including a mix of medical and non-medical use, and another 11 studies reporting no data related to type of use (Table 3). The majority of primary studies (n = 21) reported non-industry funding. Detailed effect direc- tion plots of evidence from all study designs are provided in S7 Text. A small proportion of primary studies used a RCT or cohort design from which potential causal inferences could be made (n = 4; 14%). Interpretations derived from the cross-sectional, PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 9 / 37 Scoping review: Medical and non-medical cannabis in older adults PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 PLOS ONE Namisol1 was found to cause a statisti- cally significant but not clinically significant increase in body sway compared to both baseline non-use and placebo, and its use was also associated with significantly greater numbers of adverse events (AEs) both compared to placebo and in a dose-dependent manner [20]. A ret- rospective cohort study found in adjusted analyses that 50–64-year-olds who used cannabis daily had a significantly increased hazard of acute care use compared to no cannabis use; how- ever, less frequent use did not significantly increase the hazard of acute care use. No significant effects of cannabis use compared to no use on acute care use were found in those 65+ years of age [135]. In a large prospective cohort study in France, the risk of job loss was significantly higher in adults who used cannabis than in those who did not in an adjusted analysis [96]. This relationship was reported to be dose dependent. The intent of cannabis use (medical ver- sus non-medical) of interest in the study was not reported, and was described in terms of cate- gories of exposure (never used, no consumption in prior 12 months, use less than once a month, use once a month or more); given the study also examined associations with alcohol and tobacco, there is reason to hypothesize the authors’ interests related to non-medical can- nabis use [96]. The pharmacokinetics of oral THC have been evaluated in a lab-based RCT of older adults who smoked fewer than one cannabis cigarette per week [20]. Substantial inter-individual vari- ation in plasma concentrations of THC and its metabolites was identified, a finding that is in 10 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults Fig 5. Bubble plots of findings from population-based studies and studies of healthy older adults. Findings at a glance: Observed associations of cannabis use with physical health outcomes (Panels A, B) and mental health outcomes (Panels C, D), with separate plots for adjusted analyses/RCTs and unadjusted analyses. Some associations were derived from studies with cross-sectional and case-control designs (circles), while others were derived from cohort studies and RCT (squares). Quadrants of plots represent significant and non-significant beneficial and harmfu effects. Each bubble represents an outcome definition for which at least one study reported an effect within a plot quadrant. PLOS ONE Bubble size represents the number of studies that reported that outcome definition in the quadrant. Letters within bubbles denote subgroups or different outcome definitions within an outcome group. https://doi.org/10.1371/journal.pone.0281826.g005 Fig 5. Bubble plots of findings from population-based studies and studies of healthy older adults. Findings at a Fig 5. Bubble plots of findings from population-based studies and studies of healthy older adults. Findings at a glance: Observed associations of cannabis use with physical health outcomes (Panels A, B) and mental health outcomes (Panels C, D), with separate plots for adjusted analyses/RCTs and unadjusted analyses. Some associations were derived from studies with cross-sectional and case-control designs (circles), while others were derived from cohort studies and RCT (squares). Quadrants of plots represent significant and non-significant beneficial and harmful effects. Each bubble represents an outcome definition for which at least one study reported an effect within a plot quadrant. Bubble size represents the number of studies that reported that outcome definition in the quadrant. Letters within bubbles denote subgroups or different outcome definitions within an outcome group. https://doi.org/10.1371/journal.pone.0281826.g005 https://doi.org/10.1371/journal.pone.0281826.g005 line with previous studies of participants of various ages [20]. For some older adults, the time (tmax) when the maximum plasma concentration (Cmax) of THC was reached was delayed beyond 120 minutes (whereas tmax in younger adults in other studies has been reported as 60 minutes or less [163]). However, the value of Cmax was similar to that published for younger adults [163], if reached within 120 minutes [20]. The pharmacodynamic effects of THC first occurred within 20 minutes of dosing, with maximal effects occurring between 55 and 120 minutes [20]. As THC dose increased, there were significant increases in body sway; however, the magnitude of these effects was low and unlikely to increase the risk of falls [20]. No line with previous studies of participants of various ages [20]. For some older adults, the time (tmax) when the maximum plasma concentration (Cmax) of THC was reached was delayed beyond 120 minutes (whereas tmax in younger adults in other studies has been reported as 60 minutes or less [163]). However, the value of Cmax was similar to that published for younger adults [163], if reached within 120 minutes [20]. The pharmacodynamic effects of THC first occurred within 20 minutes of dosing, with maximal effects occurring between 55 and 120 minutes [20]. PLOS ONE As THC dose increased, there were significant increases in body sway; however, the magnitude of these effects was low and unlikely to increase the risk of falls [20]. No PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 11 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults pharmacokinetic studies were identified that evaluated drug interactions with cannabis in this population. Cross-sectional and case-control studies. Cross-sectional, sequential survey, and case- control studies assessed associations of cannabis use with the presence of numerous physical and mental health outcomes (Fig 5A–5D, round symbols). Results reported from adjusted analyses (Fig 5A and 5C, round symbols) have been summarized below. Accidents and injuries were found to be significantly positively associated with cannabis use in adjusted analyses, with outcome definitions varying substantially from study to study. Baseline data from a prospective cohort study of participants 65–79 years of age found a statis- tically significant adjusted association between cannabis use and past-year car accidents and citations [113], while a larger sequential survey of adults 55 years of age found no significant adjusted association between past-year use and past-year vehicle collisions [134]; neither study reported type of cannabis use. Another large cross-sectional study of mixed (medical and non- medical) cannabis use found no significant adjusted associations between either dose (the number of joints smoked in a day) or frequency of use and either (1) past-year emergency department (ED) visits or (2) past-year injuries that caused a person to seek medical help or cut down on usual activities for more than half a day [107]. Regarding other physical health conditions reported from adjusted analyses in cross-sec- tional designs, cannabis use (characterized as any past-year use of marijuana or hashish) was found to be significantly positively associated with past-year diagnoses of HIV/AIDS in those 50–64 years of age (but not those 65 years of age) and diagnoses of sexually transmitted dis- eases in all ages of older adults [146]. Non-alcoholic fatty liver disease was also positively asso- ciated with cannabis use (including current use of Indian hemp, marijuana and other varieties of cannabis and cannabinoids) in individuals with and without cannabis dependence, respec- tively, compared to those who do not use cannabis, although the observed association did not reach statistical significance [95]. PLOS ONE A non-significant negative association of cannabis use with glaucoma was reported in a sequential survey (i.e., fewer people with glaucoma reported using cannabis five or more times a month than people without glaucoma) [124]. Associations of cannabis use with cancer were assessed in two case-control [129, 147] and one cross-sectional [143] studies. Cannabis use was reported to have a significant negative association with two forms of cancer (i.e., cannabis use was lower in those diagnosed with can- cer): head and neck squamous cell carcinoma (non-medical use; case-control study) [129] and prostate cancer in older African American men, diagnosed with prostate-specific antigen levels (use type not reported; cross-sectional study; n = 644) [143]. A second case-control study found non-medical use of hashish/kiff trended toward a positive association with diagnoses of lung cancer (i.e., more incident cases of lung cancer), but the effect did not reach statistical sig- nificance [147]. When use of the tobacco product snuff was included with hashish and kiff, the positive association became statistically significant [147]. Associations of cannabis use with cancer were assessed in two case-control [129, 147] and one cross-sectional [143] studies. Cannabis use was reported to have a significant negative association with two forms of cancer (i.e., cannabis use was lower in those diagnosed with can- cer): head and neck squamous cell carcinoma (non-medical use; case-control study) [129] and prostate cancer in older African American men, diagnosed with prostate-specific antigen levels (use type not reported; cross-sectional study; n = 644) [143]. A second case-control study found non-medical use of hashish/kiff trended toward a positive association with diagnoses of lung cancer (i.e., more incident cases of lung cancer), but the effect did not reach statistical sig- nificance [147]. When use of the tobacco product snuff was included with hashish and kiff, the positive association became statistically significant [147]. A variety of mental health and behavioral outcomes was assessed in the included cross-sec- tional and sequential survey studies in adjusted analyses (Fig 5C). In multiple studies, cannabis use was found to be significantly positively associated with (1) diagnoses of depression (one study of mixed use and two studies did not report type of use) [102, 106, 146] and (2) driving under the influence of alcohol or other substances [113, 146]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 PLOS ONE Significant adjusted positive associations were reported in single survey studies between mixed use of cannabis and impaired cognitive functioning (Executive Function Index [EFI] subscale) [100], executive functioning [100], and attention [100]; and cannabis use of an unreported type and diagnosis of anxiety [146]; increased risk propensity [146]; and illegal high-risk behaviours, such as drug selling, theft, arrest, and attacking others with intention to harm [146]. However, analyses stratified by age suggested that engagement in these illegal high-risk behaviours was only PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 12 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults significantly positively associated with cannabis use in those 50–64 years of age, but not those 65 years [146]. In a survey study that did not report type of use, a non-significant posi- tive association was found between cannabis use and past-year suicidal thoughts [105], and amongst those who used cannabis, a significant positive correlation was found between past- year suicidal thoughts and the frequency of cannabis use [105]. Physical health findings assessed in only unadjusted analyses in cross-sectional and case- control studies (Fig 5B) included significant positive associations with past-year injuries (mixed use) [107], ED visits (mixed use) [107, 116], self-rated health (health-related quality of life [HR-QoL]; mixed use) [107], liver cirrhosis ( 50 years) and hepatitis B or C (mixed use) [116], and chronic obstructive pulmonary disease (COPD; 50 years; mixed use) [116]. Mixed use of cannabis was also found to have significant negative unadjusted associations with “ever” diagnoses of diabetes ( 50 years) [116] and heart conditions ( 65 years) [116]. Mental health findings assessed with only unadjusted analyses (Fig 5D) included significant positive associations with diagnoses of mental health disorders (bipolar, manic, and hypo- manic disorders) [107] and post-traumatic stress disorder (PTSD; mixed use) [107]; driving under the influence (DUI; mixed use) [4]; and impaired cognitive functioning (non-EFI mea- sures; non-medical use) [153, 162]. Many metrics of cognitive function were measured; how- ever, none were significantly associated with cannabis use once univariable analyses were subjected to false discovery rate (FDR) correction [153, 162]. Unadjusted analyses demon- strated non-significant associations between cannabis use and numerous other physical and mental health conditions, and we refer readers to Fig 5B and S3 Dataset for further details. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 PLOS ONE Comparisons of overall brain structure and cortical and sub-cortical grey matter measures between older adults who used non-medical cannabis and those who did not were reported in a small cross-sectional study [153, 162]. After adjustment for age and baseline depression symptoms, there were no differences in total cerebrospinal fluid (CSF), grey matter, or white matter volumes between the two groups; however, some regional volumes (i.e., left putamen, left and right palladium) were found to be significantly greater in those who used cannabis [162]. Only left putamen volume remained significantly different between the two groups, after FDR correction [162]. Preliminary findings also suggested that amongst those who used non-medical cannabis, dose (i.e., past-90-day estimated THC dose), frequency of use (i.e., number of days used in the past 90 days) and the duration of use (i.e., estimated years of use and short-term vs long-term) were significantly negatively associated with regional volumes [153] (see S13 Text). Many cross-sectional and sequential survey studies provided evidence regarding associa- tions of cannabis use with the use and problematic use of other substances, as well as other substance use disorders: (eight studies of mixed use [4, 104, 106, 109, 110, 116, 132, 144], one of non-medical use [162], and four studies did not report type of use [102, 107, 128, 146]). All reported adjusted associations with cannabis use were significant and positive, including asso- ciations with the use of tobacco (use type not reported) [146] and illicit non-cannabis sub- stances (i.e., cocaine, hallucinogens, opioids, etc.; use type not reported) [146], binge alcohol use (use type not reported) [146], misuse of prescription pain relievers (use type not reported) [102] and prescription opioids (mixed use) [144], and opioid dependence (mixed use) [144]. Where reported, age stratified analyses demonstrated similar findings across older adult age categories (use type not reported) [146]. Amongst older adults with past-year mixed cannabis use, both dose (number of joints) and frequency of use (> once a month vs once a month use) were significantly associated with lifetime occurrence of any substance use disorder in adjusted analyses [106]. As well, compared to medical use, non-medical use was positively associated with either cannabis abuse or dependence; however, the association was not statisti- cally significant [4]. PLOS ONE PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 13 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults In unadjusted analyses, significant positive associations were found between cannabis use and the use of several other substances, including alcohol (mixed use) [104], cocaine (mixed use) [116], prescription opioids (use type not reported) [128], any prescription pain reliever (mixed use) [104], non-cannabis prescription psychotherapeutics (e.g., tranquilizers, stimu- lants, sedatives; mixed use) [104], and prescription benzodiazepines (mixed use) [132]. Signifi- cant unadjusted positive associations were also identified with the misuse of the following prescription drugs: any prescription drug (mixed use) [109], sedatives (mixed use) [116], tran- quilizers (mixed use) [116], and non-cannabis prescription psychotherapeutics (mixed use) [104]. Significant unadjusted positive associations were also found with the following use dis- orders and dependences: any substance use disorder (including CUD; mixed use) [104], alco- hol use disorder (DSM-IV and -5 criteria; mixed use) [104, 107, 116], nicotine dependence (mixed use) [104], non-cannabis illicit drug use disorder (mixed use) [104, 107], prescription pain reliever use disorder (mixed use) [104], and non-cannabis prescription psychotherapeutic use disorder (mixed use) [104]. Amongst older adults who used cannabis, in unadjusted analy- ses, frequency of use (mixed use) in the past year (100–365 days vs 1–99 days) was significantly associated with past-year CUD or abuse, while findings for duration of use (continued use for 24 months vs initiation or re-initiation in past year) were not significant [4]. Similarly, overall duration of non-medical use (short-term vs lifetime use) was not associated with timeline fol- low-back measures of alcohol use, Alcohol Use Disorder Identification Test (AUDIT) total score, or the Marijuana Dependence Scale in unadjusted analyses [153]. Regarding types of use, medical cannabis use was significantly positively associated with substitution of cannabis for a prescription drug compared to non-medical use in an unadjusted analysis [109]. End-stage cancer Sixteen systematic reviews [32–36, 39, 41, 45, 46, 50–52, 54–58], eleven RCTs [63, 64, 68, 70, 71, 74–77, 81, 85], and five NRSs [98, 133, 138, 152, 161] provided data regarding the effects of cannabis in end-stage cancer patients. Table 4 reports their key study characteristics. Effect direction plots provided in S8 Text provide a graphical summary of study-specific findings. Across all outcome categories, evidence was sparse and inconsistent. Many primary studies focused on the effect of cannabis on cancer pain, with meta-analyses of RCTs reported in sys- tematic reviews often demonstrating beneficial effects that did not reach statistical significance [39, 41, 46, 51, 57]. These potential improvements in analgesia were offset by several recent meta-analyses that suggested significant increases in AEs [46] and somnolence [39], as well as significantly decreased health-related quality of life (HR-QoL) [50] for patients with cancer using cannabis medications. For all meta-analyses, where reported, the level of evidence for these findings was reported to be very low, indicating that interpretation of the results should Table 4. Characteristics of studies evaluating the impacts of cannabis use in individuals with end-stage cancer. Number of studies Designs Primary studies Cannabis use type Funding 32 Overviews of reviews: 4 (in 5 publications) [32–36] Medical, overseen by physician: 14 [63, 64, 68, 70, 71, 74–77, 81, 85, 98, 133, 152] Non-industry: 5 [63, 64, 68, 74, 75] Systematic review: 12 [39, 41, 45, 46, 50– 52, 54–58] Mixed use: 1 [138] Industry: 4 [70, 76, 133] RCTs: 11 [63, 64, 68, 70, 71, 74–77, 81, 85] Not reported/Unclear: 1 [161] Not funded: 1 [85] Prospective cohort: 3 [98, 133, 161] Not reported: 6 [71, 77, 98, 138, 152, 161] Retrospective cohort: 1 [152] Cross-sectional: 1 [138] https://doi.org/10.1371/journal.pone.0281826.t004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 14 / 37 Table 4. Characteristics of studies evaluating the impacts of cannabis use in individuals with end-stage cancer. End-stage cancer Number of studies Designs Primary studies Cannabis use type Funding 32 Overviews of reviews: 4 (in 5 publications) [32–36] Medical, overseen by physician: 14 [63, 64, 68, 70, 71, 74–77, 81, 85, 98, 133, 152] Non-industry: 5 [63, 64, 68, 74, 75] Systematic review: 12 [39, 41, 45, 46, 50– 52, 54–58] Mixed use: 1 [138] Industry: 4 [70, 76, 133] RCTs: 11 [63, 64, 68, 70, 71, 74–77, 81, 85] Not reported/Unclear: 1 [161] Not funded: 1 [85] Prospective cohort: 3 [98, 133, 161] Not reported: 6 [71, 77, 98, 138, 152, 161] Retrospective cohort: 1 [152] Cross-sectional: 1 [138] https://doi.org/10.1371/journal.pone.0281826.t004 Table 4. Characteristics of studies evaluating the impacts of cannabis use in individuals with end-stage cancer. 14 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults Fig 6. Overall conclusions in systematic reviews regarding the use of cannabis in specific patient conditions, by year of publication. https://doi.org/10.1371/journal.pone.0281826.g006 Fig 6. Overall conclusions in systematic reviews regarding the use of cannabis in specific patient conditions, by year of publication. https://doi.org/10.1371/journal.pone.0281826.g006 be made with caution (see S8 Text for a summary of information from systematic reviews). The six most recent systematic reviews/overviews published from 2019–20 concluded that can- nabis use in end-stage cancer patients results in potentially harmful [39, 50] or no/unclear effects (Fig 6) [36, 41, 45, 46], while older reviews (2014–18) concluded potentially beneficial [56, 57] and unclear/no effects [32–35, 51, 52, 54, 55]. Regarding systematic review quality, one older review [57] was assessed to be of moderate quality based on AMSTAR-2 criteria, while all others were assessed to be of low or critically low quality. Primary studies that evaluated cannabis effects in patients with cancer demonstrated trends within each design: generally, RCTs demonstrated few significant benefits for patients [63, 64, 68, 70, 71, 74–77, 81, 85], while cohort studies reported significant benefits with greater fre- quency [98, 133, 152, 161] (see effect direction plots provided in S8 Text). Differences between RCTs and cohort studies were most apparent regarding mental health outcomes. Many signifi- cant mental health harms were reported in those using cannabis within RCTs that included significantly decreased cognitive functioning, concentration, and memory with either Sati- vex1 or THC extract, respectively [76]; significantly increased psychoactive effects and seda- tion with THC extract [71]; and significantly decreased anorexia-specific quality of life (QoL) with dronabinol [74]. End-stage cancer In addition to these harms, few significant mental health benefits were found: a significantly higher proportion of patients taking dronabinol reported relaxation to be “pleasant” [63] and significantly more patients taking THC extract reported increased social functioning compared to placebo [76]. Conversely, in cohort studies, only significant mental health benefits were identified: nabilone significantly decreased anxiety and significantly increased QoL in adjusted analyses [133] and loose-leaf cannabis significantly decreased anxiety and depression and significantly increased QoL in unadjusted analyses of matched data [161]. Gastrointestinal endpoints were commonly reported in the included RCTs and cohort stud- ies. Five RCTs reported the effects of cannabis on nausea in end-stage cancer patients and found no significant benefits with the use of dronabinol [63], nabilone [68, 77], Sativex1 [76], THC:CBD extract [64], or THC extract [64, 76] compared to placebo. Conversely, two cohort PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 15 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults studies identified significantly improved nausea with nabilone [133] or loose-leaf cannabis [161], when confounding was controlled through matching or multivariable modeling. Simi- larly, the consumption of anti-emetic medication (a proxy for nausea or vomiting) was no dif- ferent in nabilone or placebo recipients in an RCT [68]; however, nabilone was found to significantly reduce the proportion of patients taking anti-emetics in multivariable adjusted models in a prospective cohort study [133]. A recent systematic review conducted meta-analy- ses of nausea and vomiting data, respectively, reported in four RCTs (n = 1,095 patients) and found non-significant harmful effects for both outcomes [39]. Inconsistent findings for overall nausea and vomiting were found within and across the RCTs included in our review: THC sig- nificantly decreased the proportion of patients experiencing any nausea or vomiting on day 1 of administration but not on days 2–4 [71]; after two weeks, THC had no significant effect on a nausea and vomiting score, but THC:CBD significantly worsened it [76]; after six weeks, there were no significant differences between THC extract, THC:CBD extract, or placebo in the frequency of nausea and vomiting [64]. No cohort studies evaluated overall nausea and vomiting as an outcome. No clear and consistent findings were identified for appetite, dietary intake, or weight outcomes. Dronabinol was found to significantly improve measures of appe- tite and protein intake, but had no significant effects on other measures of dietary intake in an RCT [63]. End-stage cancer Similarly, nabilone significantly increased the intake of carbohydrates [85], but had no effect on many other measures of appetite, dietary intake, and weight change in RCTs or adjusted analyses in cohort studies [68, 85, 133]; in another RCT, Sativex1 significantly decreased appetite scores, but had no effect on another measure of appetite loss [76]. As well, Sativa-derived THC and THC:CBD extracts and loose-leaf cannabis had no significant effects on appetite in two RCTs [64, 76] and a prospective cohort study [161]. In meta-analyses reported in two systematic reviews that included the same three RCTs, pooling of various can- nabis products demonstrated non-significant beneficial impacts on appetite [50, 51]. Several other endpoints were reported in primary studies of patients with end-stage cancer, with few statistically significant effects being reported. A variety of sleep measures were evalu- ated in five RCTs [63, 68, 70, 76], with significant benefits found only for sleep disruption scores for Sativex1 [70] and the proportion of patients reporting sleep to be “pleasant” for dro- nabinol [63]; non-significant beneficial or harmful effects were reported for all other sleep out- comes. Incoordination was significantly increased in patients taking THC extract compared to placebo in one trial [71], but no significant effect was found in patients taking synthetic THC compared to placebo in another trial [74]. A recent systematic review presented a meta-analy- sis of four studies reporting dizziness data and found a non-significant trend toward increased dizziness (I2 = 0%) [39]. Two of the RCTs were not included in our review due to a failure to meet all eligibility criteria [164, 165], and the other two RCTs were included in our review but did not have data extracted for the dizziness outcome because only raw data were reported [70, 76]. We included a different RCT reporting an analysis of frequency of vertigo that found also no significant difference between groups [64]. PLOS ONE Number of studies Designs Primary studies Cannabis use type Funding 18 Systematic review: 9 [32, 36–38, 47, 49, 51, 53, 58] Medical, overseen by physician: 9 [60, 72, 86–89, 148, 156, 158] Non-industry: 5 [60, 72, 86–88] RCTs: 6 [60, 72, 86–89] Mixed (industry and non-industry): 1 [89] Non-randomized trial: 2 [148, 156] Not reported: 3 [148, 156, 158] Retrospective cohort: 1 [158] https //doi o g/10 1371/jo nal pone 0281826 t005 Most primary studies evaluated dronabinol [89, 156, 158], nabilone [72], or Namisol1 [60, 86–88] in pill form, while one study evaluated a generic THC extract in oil form [148]. Evi- dence for Alzheimer’s disease/dementia was sparse and inconsistent: few significant clinical benefits were reported in RCTs and many more in the non-randomized trials and cohort study, and several significant harms were reported in RCTs but none in the non-randomized trials and cohort study. Two recent systematic reviews published in 2019 and 2020 concluded, based on the totality of evidence reviewed, that cannabis use may be beneficial in Alzheimer’s disease/dementia patients [34, 47]; however, a third review (2020) suggested that there were no clear benefits or harms [37], and another from 2019 concluded that cannabis use may be harmful [49] (Fig 6). Multiple related RCTs from the Netherlands [60, 86–88] evaluated Namisol1 use in patients with Alzheimer’s disease/dementia and observed no significant physical or mental health benefits. Instead, the studies identified several significant harms, mainly related to wors- ening measures of balance [60, 87, 88] and psychedelic effects [60], despite no difference from placebo in the total number of AEs reported [60, 86, 88]. Although not evaluated as extensively in the literature, dronabinol [89, 156, 158], nabilone [72], and a THC extract [148] were all associated with significantly improved agitation and aggression in individual NRSs, although the analysis of THC extract recipients was unadjusted. When seven studies [86, 87, 89, 156, 166, 167] in a systematic review [49] were pooled in a meta-analysis of cannabis effects on agi- tation, no significant effect was found; however, the meta-analysis had very high heterogeneity (I2 = 86%). PLOS ONE Similarly, in individual studies, dronabinol (NRS, unadjusted analysis) [156], nabilone (RCT) [72], and THC extract (NRS, unadjusted analysis) [148] were observed to have potentially significant benefits on neuropsychiatric symptoms, but not Namisol1 (RCTs)1 [86, 87]. A meta-analysis of five studies [86, 87, 156, 166, 167] in a systematic review [49] found no significant effects on neuropsychiatric symptoms, possibly due to relatively heavier weighting of the two Namisol1 RCTs [86, 87] with non-significant effects. Although there was no statistical heterogeneity in this meta-analysis, there were concerns regarding imprecision. Aberrant motor behaviour and motor activity may also significantly improve with administration of dronabinol [156] or THC extract [148] (both NRSs, unadjusted analy- ses), while dronabinol (one RCT [89]; and unadjusted analyses in one NRS [156] and one cohort study [158]) and nabilone (RCT) [72] may also improve some measures of nutrition. Table 5. Characteristics of studies evaluating the impacts of cannabis use in individuals with Alzheimer’s disease or dementia. Number of studies Designs Primary studies Cannabis use type Funding 18 Systematic review: 9 [32, 36–38, 47, 49, 51, 53, 58] Medical, overseen by physician: 9 [60, 72, 86–89, 148, 156, 158] Non-industry: 5 [60, 72, 86–88] RCTs: 6 [60, 72, 86–89] Mixed (industry and non-industry): 1 [89] Non-randomized trial: 2 [148, 156] Not reported: 3 [148, 156, 158] Retrospective cohort: 1 [158] https://doi.org/10.1371/journal.pone.0281826.t005 Table 5. Characteristics of studies evaluating the impacts of cannabis use in individuals with Alzheimer’s disease or dementia. Number of studies Designs Primary studies Cannabis use type Funding 18 Systematic review: 9 [32, 36–38, 47, 49, 51, 53, 58] Medical, overseen by physician: 9 [60, 72, 86–89, 148, 156, 158] Non-industry: 5 [60, 72, 86–88] RCTs: 6 [60, 72, 86–89] Mixed (industry and non-industry): 1 [89] Non-randomized trial: 2 [148, 156] Not reported: 3 [148, 156, 158] Retrospective cohort: 1 [158] https://doi.org/10.1371/journal.pone.0281826.t005 Table 5. Characteristics of studies evaluating the impacts of cannabis use in individuals with Alzheimer’s disease or dementia. PLOS ONE PLOS ONE Scoping review: Medical and non-medical cannabis in older adults Most primary studies evaluated dronabinol [89, 156, 158], nabilone [72], or Namisol1 [60, 86–88] in pill form, while one study evaluated a generic THC extract in oil form [148]. Evi- dence for Alzheimer’s disease/dementia was sparse and inconsistent: few significant clinical benefits were reported in RCTs and many more in the non-randomized trials and cohort study, and several significant harms were reported in RCTs but none in the non-randomized trials and cohort study. Two recent systematic reviews published in 2019 and 2020 concluded, based on the totality of evidence reviewed, that cannabis use may be beneficial in Alzheimer’s disease/dementia patients [34, 47]; however, a third review (2020) suggested that there were no clear benefits or harms [37], and another from 2019 concluded that cannabis use may be harmful [49] (Fig 6). Multiple related RCTs from the Netherlands [60, 86–88] evaluated Namisol1 use in patients with Alzheimer’s disease/dementia and observed no significant physical or mental health benefits. Instead, the studies identified several significant harms, mainly related to wors- ening measures of balance [60, 87, 88] and psychedelic effects [60], despite no difference from placebo in the total number of AEs reported [60, 86, 88]. Although not evaluated as extensively in the literature, dronabinol [89, 156, 158], nabilone [72], and a THC extract [148] were all associated with significantly improved agitation and aggression in individual NRSs, although the analysis of THC extract recipients was unadjusted. When seven studies [86, 87, 89, 156, 166, 167] in a systematic review [49] were pooled in a meta-analysis of cannabis effects on agi- tation, no significant effect was found; however, the meta-analysis had very high heterogeneity (I2 = 86%). When the meta-analysis was stratified by cannabis product, pooling of three studies [86, 87] that evaluated Namisol1 demonstrated no significant effects (I2 = 0%), while pooling of four studies [89, 156, 166, 167]—two of which did not meet inclusion criteria for our review [166, 167]—that evaluated synthetic cannabinoids (dronabinol or nabilone) trended toward a favourable but non-significant response. However, the latter meta-analysis had significant het- erogeneity (I2 = 90%) and imprecision, suggesting that the studies were too different in some aspects to be pooled. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 Alzheimer’s disease/dementia Six RCTs [60, 72, 86–89] and three NRSs [148, 156, 158] evaluated the effects of medical can- nabis use in patients with Alzheimer’s disease/dementia, in addition to nine systematic reviews that reported at least one synthesis of Alzheimer’s disease/dementia studies (i.e., some of these systematic reviews included multiple patient conditions, not just Alzheimer’s disease/demen- tia) [32, 36–38, 47, 49, 51, 53, 58]. Key study characteristics are reported in Table 5, while detailed effect direction tables summarizing their findings have been provided in S9 Text. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 16 / 37 PLOS ONE When the meta-analysis was stratified by cannabis product, pooling of three studies [86, 87] that evaluated Namisol1 demonstrated no significant effects (I2 = 0%), while pooling of four studies [89, 156, 166, 167]—two of which did not meet inclusion criteria for our review [166, 167]—that evaluated synthetic cannabinoids (dronabinol or nabilone) trended toward a favourable but non-significant response. However, the latter meta-analysis had significant het- erogeneity (I2 = 90%) and imprecision, suggesting that the studies were too different in some aspects to be pooled. Similarly, in individual studies, dronabinol (NRS, unadjusted analysis) [156], nabilone (RCT) [72], and THC extract (NRS, unadjusted analysis) [148] were observed to have potentially significant benefits on neuropsychiatric symptoms, but not Namisol1 (RCTs)1 [86, 87]. A meta-analysis of five studies [86, 87, 156, 166, 167] in a systematic review [49] found no significant effects on neuropsychiatric symptoms, possibly due to relatively heavier weighting of the two Namisol1 RCTs [86, 87] with non-significant effects. Although there was no statistical heterogeneity in this meta-analysis, there were concerns regarding imprecision. Aberrant motor behaviour and motor activity may also significantly improve with administration of dronabinol [156] or THC extract [148] (both NRSs, unadjusted analy- ses), while dronabinol (one RCT [89]; and unadjusted analyses in one NRS [156] and one cohort study [158]) and nabilone (RCT) [72] may also improve some measures of nutrition. However, a meta-analysis of three studies [87, 89, 166] (one of which did not meet our PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 17 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults inclusion criteria [166]) in a systematic review [49] did not find a significant effect on body mass index (BMI), possibly due to heterogeneity (I2 = 62%). When stratified by baseline BMI (high and low), no significant effect of cannabis was found in those with a high baseline BMI (two studies [86, 166]); however, cannabis significantly increased BMI in those with a low baseline BMI (one study [89]). Regarding potential harms, a meta-analysis in a systematic review [49] identified significantly greater risk of sedation or somnolence when four studies [86, 87, 89, 166] evaluating dronabinol, nabilone, and Namisol1 were pooled. As well, in one RCT, a significantly higher number of patients receiving nabilone experienced treatment- emergent AEs, but cognition scores were significantly better in those receiving nabilone com- pared to placebo [72]. PLOS ONE No included studies compared AEs in patients receiving either dronabi- nol or THC extract, respectively, to a placebo group, meaning that the harmful effects of dronabinol and THC extract in this vulnerable patient group may not have been sufficiently evaluated. Parkinson’s disease Five RCTs [65, 66, 69, 79, 83] and five NRSs (two prospective cohort [131, 149] and three cross-sectional studies [97, 154, 160]) evaluated the impacts of cannabis use in individuals with Parkinson’s disease, while five systematic reviews reported at least one synthesis [36, 42, 48, 53, 58]. Key study characteristics are reported in Table 6, and S10 Text presents tables of detailed effect direction plots. Two RCTs randomized subjects to nabilone or placebo in pill form [79, 83], two to oral CBD (synthetic [69] and an unclear formulation [66] as pills), and one to a natural THC:CBD extract (2:1, Cannador, Berlin) [65]. Other natural products of unclear formulation that were smoked, vaped, or used as oil were evaluated in cohort and cross-sectional studies [97, 131, 149, 154]. One cross-sectional study that compared cannabis to other analgesics did not report details of the cannabis products used [160]. Another survey compared the effects of whole plant comparisons (i.e., fresh vs dried, flowers vs leaves) and frequency of use amongst those who used cannabis [154]. Adverse events were reported in RCTs but not in NRSs. Almost all effects and associations reported in NRSs were beneficial; however, given that none of the NRSs controlled for confounders and that the resulting potential risk of bias in the reported size and significance of effects would be high, our main summary focuses on the findings of the five RCTs, with findings from NRSs presented more briefly below. The majority of findings reported in the five RCTs were non-significant for all outcome types (see effect direction plots provided in S10 Text). Similarly, most systematic reviews that summarized the impacts of cannabis use in Parkinson’s disease failed to make clear conclu- sions [36, 42, 48, 58], including two published in 2019 [48] and 2020 [42], although one SR from 2017 suggested possible benefits of use [53] (Fig 6). When significance was reached in RCTs, the effect of cannabis was typically beneficial. However, for all outcomes, significant Table 6. Characteristics of studies evaluating the impacts of cannabis use in individuals with Parkinson’s disease. Parkinson’s disease Number of studies Designs Primary studies Use type Funding 15 Systematic review: 5 [36, 42, 48, 53, 58] Medical, overseen by physician: 5 [65, 66, 79, 131, 149] Non-industry: 3 [69, 83, 154] RCTs: 5 [65, 66, 69, 79, 83] Medical, not prescribed: 1 [154] Mixed (industry and non-industry): 1 [79] Prospective cohort: 2 [131, 149] Mixed use: 1 [97] Not funded: 2 [66, 149] Cross-sectional: 3 [97, 154, 160] Immediate effects in lab: 2 [69, 83] Not reported: 4 [65, 97, 131, 160] Not reported/Unclear: 1 [160] https://doi.org/10.1371/journal.pone.0281826.t006 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 18 / 37 Table 6. Characteristics of studies evaluating the impacts of cannabis use in individuals with Parkinson’s disease. Number of studies Designs Primary studies Use type Funding 15 Systematic review: 5 [36, 42, 48, 53, 58] Medical, overseen by physician: 5 [65, 66, 79, 131, 149] Non-industry: 3 [69, 83, 154] RCTs: 5 [65, 66, 69, 79, 83] Medical, not prescribed: 1 [154] Mixed (industry and non-industry): 1 [79] Prospective cohort: 2 [131, 149] Mixed use: 1 [97] Not funded: 2 [66, 149] Cross-sectional: 3 [97, 154, 160] Immediate effects in lab: 2 [69, 83] Not reported: 4 [65, 97, 131, 160] Not reported/Unclear: 1 [160] Table 6. Characteristics of studies evaluating the impacts of cannabis use in individuals with Parkinson’s disease. Number of studies Designs Primary studies Use type Funding 15 Systematic review: 5 [36, 42, 48, 53, 58] Medical, overseen by physician: 5 [65, 66, 79, 131, 149] Non-industry: 3 [69, 83, 154] RCTs: 5 [65, 66, 69, 79, 83] Medical, not prescribed: 1 [154] Mixed (industry and non-industry): 1 [79] Prospective cohort: 2 [131, 149] Mixed use: 1 [97] Not funded: 2 [66, 149] Cross-sectional: 3 [97, 154, 160] Immediate effects in lab: 2 [69, 83] Not reported: 4 [65, 97, 131, 160] Not reported/Unclear: 1 [160] Characteristics of studies evaluating the impacts of cannabis use in individuals with Parkinson’s disease. https://doi.org/10.1371/journal.pone.0281826.t006 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 18 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults beneficial effects were offset by one or more non-significant effects either in the same study for differing outcome definitions or in other studies. Thus, equivocal evidence was found for all reported outcomes due to lack of consensus within and across RCTs. Parkinson’s disease Significant beneficial impacts of cannabis were found for dyskinesia (one [83] of three RCTs [65, 69, 83] evaluating the outcome), tremors (one RCT reporting both significant and non-significant effects [69]), non-motor symptoms (one RCT reporting both significant and non-significant effects [79]), apparent efficacy (one [79] of three RCTs [65, 66, 79]), activities of daily living (ADLs; one [66] of two RCTs [65, 66]), sleep (one [79] of two RCTs [65, 79]), and anxiety (two RCTs reporting both significant and non-significant effects within the study [69, 79]). Quality of life was significantly reduced in one [66] of three RCTs [65, 66, 79] that assessed this outcome. Many outcomes demonstrated no significant effect of cannabis in the included RCTs: motor symptoms [66, 79, 83]; mobility [65, 66]; pain/discomfort [65, 66, 69, 79]; mental and physical sedation [69, 79]; heart rate/blood pressure/EKG [69, 79]; blood tests [66, 79]; cognitive func- tioning [66, 69, 79]; emotional functioning [66]; depression [79]; social functioning [66]; sui- cidal behaviour/ideation [79]; inappropriate behaviour [79]; mentation, behaviour, and mood [66]; stigma [66]; overall AEs [79]; serious AEs (SAEs) [79]; complication of therapy [66]; response to levodopa treatment [83]; and neuronal viability [66]. Variability in outcome definitions does not fully explain the differences in effects found within and across RCTs. For example, there was high diversity in dyskinesia outcome mea- sures, including a tapping test [69]; the Rush Dyskinesia scale [65, 83]; the Unified Parkinson’s Disease Rating Scale (UPDRS), questions 32–34 [65]; the Bain scale [65]; and numerous mea- sures of duration of dyskinesia with respect to “on” and “off” periods [65, 83]. All were found to be non-significant, except for the Rush Dyskinesia scale in one [83] of the two studies [65, 83] reporting it. In another example, there was no variability in the QoL outcome defini- tion across three studies, and yet one reported a significant harmful effect (n = 21; CBD pow- der) [66], while the other two reported non-significant beneficial effects [n = 19 (Cannador extract) [65] and 38 (nabilone) [79], respectively]. Inconsistencies in effects and significance were present despite the same outcome definition and similar sample sizes, indicating other heterogeneity, such as cannabis products, may have influenced the effects. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 Other indications This review also identified evidence regarding effects of cannabis use in older adults with a variety of other conditions and indications beyond those specified a priori. In the sections below, we provide brief summaries of this evidence, grouped by indication. Direction of effect plots for these indications can be found in S11 Text. Chronic non-cancer pain. One non-randomized trial [99], two prospective cohort studies [101, 111], and three cross-sectional studies [140, 150, 151] assessed cannabis use for general chronic non-cancer pain [99, 101, 111, 140, 150, 151]; one additional cross-sectional study included older adults who used medical cannabis of whom 61% had a pain-related condition (chronic non-cancer or cancer-related) [110], and one RCT included patients with rheumatoid arthritis [62]. All significant physical health, mental health, and AE-related effects and associa- tions were beneficial; however, almost all were reported from unadjusted analyses in NRSs. Evidence from the single RCT suggested significant improvements in several pain measures [62] for rheumatoid arthritis patients taking Sativex1, while a single adjusted analysis in a cross-sectional study demonstrated a significant negative association between use of whole- plant cannabis and waking at night in those with chronic non-cancer pain, generally, although no significant associations were found for other indicators of insomnia such as sleep initiation and early awakening [150]. A single cross-sectional study reported drug- and alcohol-related outcomes in patients with chronic non-cancer pain and found significant associations between cannabis use and misuse of prescription opioids, hazardous alcohol use, and nicotine use in unadjusted analyses; however, only an increased risk of misuse of prescription opioids remained significant after adjustment for confounding [140]. Unadjusted analyses found significant improvements in apparent efficacy and pain out- comes (one non-randomized trial and two cohort studies) [99, 101, 111], sleep (one RCT and one cohort study) [62, 101], vitality (cohort study) [101], global QoL (non-randomized trial) [99], anxiety (cohort study) [101], and overall AEs (cross-sectional study) [140]. Non-signifi- cant effects were also reported for these outcomes, potentially due to differences in patient characteristics or outcome definitions. A cross-sectional study found non-significant positive associations in unadjusted analyses between mixed cannabis use (compared to medical use) and past-year opioid use and benzodiazepine use, and non-significant negative associations with measures of global QoL and physical and mental health [110]. Parkinson’s disease Briefly, in the five included NRSs [97, 131, 149, 154, 160], none of which adjusted for con- founding, whole-plant cannabis use was found to have significant beneficial effects and associ- ations for almost all reported outcomes, with little conflicting evidence across studies. Briefly, in the five included NRSs [97, 131, 149, 154, 160], none of which adjusted for con- founding, whole-plant cannabis use was found to have significant beneficial effects and associ- ations for almost all reported outcomes, with little conflicting evidence across studies. Significant beneficial effects and associations were reported for dyskinesia (cohort study [131] and cross-sectional study [97]), muscle spasticity (cohort study [131] and cross-sectional study [97]), tremors (cohort study [131] and cross-sectional study [97]), pain (two cohort studies [131, 149] and a cross-sectional study [97]), apparent efficacy (cohort study [149] and cross- sectional study [97]), motor symptoms (cohort study) [131], and physical health symptoms (cohort) [131]. In a telephone survey, significant positive associations were reported between whole-plant cannabis use and mobility [97], sleep [97], appetite or food intake [97], libido [97], and attention [97], and significant negative associations were reported with nausea [97], constipation [97], depression [97], and balance or falls [97]. Only non-significant associations were found between whole-plant cannabis use and posture [131], urination [97], and memory [97]. In those who used cannabis, improvement in dyskinesia was not associated with fresh vs dried product or use of flowers vs leaves; however, frequency of use once a day was associ- ated with dyskinesia improvement over < once a day use [154]. One small prospective cohort study that evaluated pain with both short- and long-term follow-up times found significant reductions in pain measured by either the Pain Rating Index (PRI) or a visual analog scale (VAS) at 30 minutes post-treatment compared to pre-treatment, but non-significant reduc- tions in the same measures after 10 weeks of continued cannabis use [149]. Two other PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 19 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults studies used different outcome measures and found significant reductions in pain at 30 min- utes post-use (prospective cohort study) [131] and after 3–84 months of treatment (telephone survey) [97]. Another survey found no association between type of analgesic used (e.g., canna- bis, paracetamol, NSAIDs, pregabalin) and self-reported response to analgesia in PD patients with non-low-back pain [160]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 Other indications Many other non-signifi- cant effects were reported from unadjusted analyses of physical health, mental health, AE- related, and drug-related outcomes across studies of chronic non-cancer pain (S11 Text). Joint replacement. Patients receiving joint replacements were included in three retro- spective cohort studies that evaluated the effects of cannabis use [112, 120, 122]. Patients who self-reported using cannabis [122] or who were administered 10 mg dronabinol [120] had no significant differences in measures of analgesic use in hospital, respectively, compared to patients who did not use cannabis or dronabinol in unadjusted analyses; however, compared to non-use, dronabinol significantly reduced hospital length of stay [120], while self-reported cannabis use did not [122]. Patients who screened positively for urine THC pre-operatively had significantly poorer pre-anaesthetic health scores and were significantly more likely to screen positively for opioids pre-operatively than patients who screened negatively for THC in PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 20 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults unadjusted analyses [112]. However, adjusted analyses demonstrated no significant difference between patients who screened positive or negative for urine THC in post-operative complica- tions, 90-day readmissions, reoperations, or deaths up to 90 days post-op [112]. Many other outcomes with non-significant effects were reported in the three studies. Neuropathy. Three RCTs evaluated the impacts of cannabis use on peripheral neuropathy [91] and diabetic neuropathy [82, 90]. One trial of diabetic neuropathy patients found signifi- cant improvements in pain scores with high-dose THC extracts (28 mg) but not with lower doses (4 or 16 mg) compared to placebo, although other measures of pain (e.g., proportion of patients achieving 30% pain reduction) were not significantly affected, and the benefits were offset by significant increases in somnolence and psychoactive effects [90]. Another trial of patients with peripheral neuropathy found significant improvements in measures of some non-pain sensations but not others with CBD topical cream [91]. A third trial found no signifi- cant differences in any measured physical health or mental health outcomes in diabetic neu- ropathy patients randomized to either Sativex1 or placebo [82]. Chronic obstructive pulmonary disease. The effects of cannabis in patients with COPD were evaluated in two RCTs [59, 80] and one retrospective cohort study [155]. No significant impacts were identified on measures of spirometry when Sativex1 was compared to placebo in a RCT [80]. Other indications Comparisons of high-dose vs low-dose THC, with or without CBD, also sug- gested no effects on spirometry [80], but identified significantly negative psychoactive effects [59] and “bad drug effects [59],” as well as significantly higher risks of hospitalization for COPD or pneumonia [155] and all-cause mortality [155] in adjusted analyses. One RCT iden- tified a significant benefit of high-dose compared to low-dose THC for “good drug effects” and one self-reported measure of anxiety, but no significant difference between groups for another self-reported measure of anxiety/relaxation [59]. Multiple sclerosis. Two RCTs randomized patients with multiple sclerosis to Sativex1 and placebo [67, 78]. Significant benefits in apparent efficacy [78] and some nerve conduction measures [67] were reported; however, no significant effects were found for pain scores [67], muscle spasticity [78], strength [78], gait velocity [78], or sleep [78]. Trauma. Trauma patients who used cannabis were compared to those who did not in two retrospective cohort studies [114, 127]. In adjusted analyses, cannabis use in older adults did not impact mortality [127] or the need for intubation [127]; however, it was significantly asso- ciated with ICU admission [127] and need for an operation due to the trauma [127]. Unad- justed analyses suggested significant associations of non-medical cannabis use with lower blood pressure and higher heart rate (potentially early signs of shock) [114], and non-signifi- cant associations with consciousness/coma [114], injury severity [114], and length of stay in the hospital or ICU [114]. Cardiac conditions. Two retrospective cohort studies evaluated the impact of cannabis use [123] and misuse [125] in patients with cardiac conditions. Adjusted analyses suggested that older adults who used cannabis who suffered an acute myocardial infarction have signifi- cantly reduced risks of shock (ages 50–69 years) and mortality (ages 50–59 only), respectively, compared to those who did not use cannabis [123]. No significant effects were found at any age >50 years on the risks of mechanical ventilation [123], ventricular tachycardia/fibrillation/ cardiac arrest [123], or a composite of death, mechanical ventilation, cardiac arrest, placement of an intra-aortic balloon pump, or shock [123]. In unadjusted analyses, patients undergoing percutaneous coronary interventions who misused cannabis were at significantly greater risk of post-intervention bleeding complications, if they were between the ages of 66 and 75 years, but not in younger or older age categories [125]. No other post-intervention AEs were associ- ated with cannabis use, including vascular complications, stroke/transient ischemic attack, or death [125]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 Other indications 21 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults Homelessness. Past-six-month use of non-medical cannabis in people without housing was evaluated in two cohort studies that used the same sample of participants [93, 142]. A sig- nificantly higher risk of moderate-to-high physical symptomatology was found with moder- ate-risk cannabis use (i.e., Alcohol, Smoking and Substance Involvement Screening Test (ASSIST) score of 4) compared to no moderate-risk cannabis use after adjustment for con- founders [142]. The risk of falling was significantly increased in people without housing who consumed cannabis non-medically in an unadjusted analysis [93]. Alcohol use. One case-control [157] and two cross-sectional studies [115, 130] assessed impacts of cannabis use in participants who consumed alcohol. All reported adjusted analysis findings. Co-use of cannabis and alcohol was associated with increased prevalences of past- year prescription drug misuse (non-medical cannabis use) [130] and past-month binge drink- ing [115], and decreased odds of alcohol-related liver cirrhosis in those 50–59 years of age, but not over 60 years [157]. Additional stand-alone conditions and indications. Six RCTs [20, 61, 73, 81, 84, 92], five cohort studies [121, 136, 137, 145, 159], and one cross-sectional study [141] evaluated the effects of cannabis use on a variety of other patient conditions and indications and reported a mix of findings. A small cross-over study randomizing healthy older adults who did not use cannabis to three doses of Namisol1 and placebo found significantly greater overall AEs for all doses com- pared to placebo and for high-dose (6.5 mg) compared to either lower dose (3 or 5 mg) [20]. No significant effects were found on balance or concentration, and substantial variability was found between participants in pharmacokinetic parameters (see section on “Cannabis use in healthy older adults and the older general public”). In other studies, significantly beneficial effects were found in • Ever and former tobacco use (spirometry outcomes; non-medical use only; cohort study, adjusted findings) [137], • Amyotrophic lateral sclerosis (apparent efficacy, pain score, and muscle spasticity; RCT) [81], • Diabetes (apparent efficacy and various diabetic blood tests; RCT) [73], • Failed back surgery syndrome (pain scores, sleep, mobility, physical health symptoms, QoL, social functioning, and mood; cohort study, unadjusted findings) [136] and • Older adults with sedentary lifestyles (BMI; cohort study, adjusted findings) [159]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 Clinical subgroup findings As per our a priori objectives, data for subpopulations of interest were captured; for brevity, we refer readers to S12 Text, where we provide both descriptive text and tables of findings per- taining to all subgroups. Additionally, in earlier sections, we have reported subgroup findings where they provided additional context of the data being described. Overall, data related to the subgroups of sex, older adult age group (i.e., 50–64 years, 65+ years, etc.), residential setting (e.g., ambulatory versus inpatient care), and illicit drug use were identified. As well, some stud- ies focused entirely on some subpopulations of interest that have been summarized as “patient conditions” in the main text, including existing physical and mental health conditions (i.e., end-stage cancer, Alzheimer’s disease, Parkinson’s disease, etc.), accommodation status (i.e., homelessness [93, 142]), and use of other substances (i.e., alcohol [115, 130, 157], tobacco [137], prescription opioids [117], and heroin [118]). We were unable to locate information related to the following key subpopulations of interest: race/ethnic groups, frailty, employment status, marital status, or other accommodation statuses (e.g., alone, shared). Cannabis comparisons amongst individuals who use cannabis Similar to subgroup data, we planned a priori to gather data on comparisons of cannabis con- sumption other than use vs no use/placebo (i.e., comparisons of use amongst those who used cannabis). For brevity, we direct readers to S13 Text, where we provide a descriptive synthesis of findings with corresponding tables. These data pertain to comparisons of use type (i.e., med- ical vs non-medical), dose (e.g., grams per month; number of joints per month), THC and CBD concentrations, frequency of use, duration of use, cannabis plant characteristics (e.g., flowers vs leaves, fresh vs dried), and use of other substances. Data were identified for a variety of patient conditions. We were unable to locate comparisons of consumption methods (e.g., smoking, vaporising, edibles), which was an a priori comparison of interest. Other indications Significant harmful effects were found for the following indications: • Diabetes: a high-dose cannabis preparation (10 mg Tetrahydrocannabivarin + 200 mg CBD) was found to significantly increase the risk of depression in an RCT [73] • Intraocular hypertension: 40 mg CBD significantly increased both blood pressure (at 60 and 90 minutes) and intraocular pressure, respectively, while low-dose THC (5 mg) significantly increased heart rate at 90 minutes but had no effect on intraocular pressure in an RCT [84]. • Surgery: pain scores were significantly increased for those taking high-dose Nabilone (2 mg) compared to those taking 1 mg Nabilone, ketoprofen, or placebo, respectively in an RCT [61]. • Non-traumatic aneurysmal subarachnoid hemorrhage: significantly higher risk of hospital readmission was found if patients had current CUD compared to those who did not in an adjusted analysis in a cohort study [145]. 22 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults • People who are HIV-positive and taking HIV medication: cannabis use had no significant association with response to HIV therapy in unadjusted analyses, despite being significantly associated with reduced HIV drug adherence in a cross-sectional study [141]. Only non-significant effects were found for the following conditions: Only non-significant effects were found for the following conditions: • Cervical dystonia: dronabinol had no significant effects on pain scores, apparent efficacy, torticollis severity or activities of daily life scores in an RCT [92]. • Reduced appetite due to chronic disease: dronabinol had no significant effects on appetite, food intake, weight, or blood albumin levels in unadjusted analyses in a cohort study [121]. Many other non-significant effects were reported in the above studies. Discussion While we identified studies that reported benefits of medical cannabis, such as alleviation of cancer pain and improved QoL in cancer patients, the extent of evidence for specific patient conditions was generally sparse. Additionally, while benefits were occasionally observed in RCTs, these were often accompanied by an observed increased risk of one or more harms associated with cannabis use. Benefits were more often noted in NRSs; however, few of these studies also included evaluations of harms. Where significant associations were identified, often there was conflicting evidence within and/or across studies. Conflicting findings may reflect heterogene- ity of patient demographics, sample sizes, cannabis interventions, outcome definitions, follow- up times, residual confounding, or other factors. While there may be potential for medical can- nabis to offer benefits to some patients with conditions such as end-stage cancer, Alzheimer’s disease, and Parkinson’s disease, the quantity and quality of evidence is limited at this time. A recent Canadian study found > 50 reasons for authorization of medical cannabis across all ages, with the majority not supported by evidence of long-term efficacy [10]. While we identified studies that reported benefits of medical cannabis, such as alleviation of cancer pain and improved QoL in cancer patients, the extent of evidence for specific patient conditions was generally sparse. Additionally, while benefits were occasionally observed in RCTs, these were often accompanied by an observed increased risk of one or more harms associated with cannabis use. Benefits were more often noted in NRSs; however, few of these studies also included evaluations of harms. Where significant associations were identified, often there was conflicting evidence within and/or across studies. Conflicting findings may reflect heterogene- ity of patient demographics, sample sizes, cannabis interventions, outcome definitions, follow- up times, residual confounding, or other factors. While there may be potential for medical can- nabis to offer benefits to some patients with conditions such as end-stage cancer, Alzheimer’s disease, and Parkinson’s disease, the quantity and quality of evidence is limited at this time. Similar conclusions were observed in recent systematic reviews reporting findings for cannabis use for end-stage cancer [39, 41, 46, 50, 51], Alzheimer’s disease/dementia [37, 38, 47, 49, 51], and Parkinson’s disease [48]. None of these systematic reviews could recommend cannabis use for these conditions. It should be noted that meta-analyses reported in the included systematic reviews often pooled data from differing cannabis products. Discussion Legalization of cannabis in many jurisdictions around the world has the potential to lead to greater availability and accessibility of cannabis products for all age groups [10]. As North American baby boomers born in the 1950s and 1960s age, attitudes toward cannabis use in the older adult cohort have changed, leading to higher rates of use in this demographic [2, 116, 168, 169]. However, the effects of cannabis use in older adults have not been well defined, beyond cannabis use for specific health conditions common to older adults, such as cancer pain, chronic non-cancer pain, and palliative medicine [32, 33, 46, 51, 55]. Previously only one published systematic review of cannabis effects focused specifically on older adults; however, it was limited to medical cannabis use [58]. In the current review, more than 130 studies were PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 23 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults found that contribute to the current evidence regarding positive and negative health effects and associations with cannabis use for both medical and non-medical purposes in older adults. While a large number of studies were mapped in our review, we caution readers not to over-interpret the available data. Many findings were supported by single studies only, were derived from unadjusted analyses in non-randomized designs, were reported only in cross- sectional or case-control studies that are minimally informative for causal inferences, or may have limited biological plausibility. Furthermore, although we prioritized the reporting of find- ings from adjusted analyses in NRSs, it should be noted that we did not assess the level of adjustment made for each finding (e.g., adjustment for all critical confounders), and conse- quently, even adjusted results may still be biased by residual confounding. As well, there was limited assessment of harms in the included NRSs. For these reasons, the benefit-to-risk ratio of cannabis use is unclear. Therefore, we recommend that readers consider the nature and potential limitations of the primary research sources underlying the benefits, harms, and asso- ciations mapped in this scoping review, prior to making decisions about prescribing or con- suming cannabis. A recent Canadian study found > 50 reasons for authorization of medical cannabis across all ages, with the majority not supported by evidence of long-term efficacy [10]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 Discussion Different cannabis products may have differing effects; therefore, pooling data from multiple products in meta-analyses may result in high heterogeneity and reduced likelihood of identifying significant effects. Similarly, as cannabis includes many different cannabinoids wherein the type and route of administra- tion impact side effects, it is also challenging to concisely summarize side effect data. While there exists some evidence of benefit of cannabis for certain conditions in the general adult population > 18 years of age (e.g., anxiety, PTSD), there was limited evidence mapped in our review for these and many other conditions, indicating that focused research in older adults is needed. A recent narrative review suggested that medical cannabis may be considered as a “last-resort” therapy or adjunct/replacement treatment, when all else fails, in cases of Parkin- son’s disease or dementia for some symptoms, although the clinical data are still incomplete [170]. Of note, none of the publications included in our review explicitly reported harms related to drug interactions, medical cannabis misuse, or withdrawal. Additional rigorous research of the effects of cannabis in these and other conditions common to older adults remains vital, and physicians, health providers, patients, caregivers, and other stakeholders may wish to proceed cautiously in using cannabis for medical purposes at this time. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 24 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults Development of tailored dissemination strategies to inform and educate clinicians, healthcare professionals, pharmacists, caregivers, and patients may be important to help reduce the risks associated with the use of cannabis for medical purposes in older adults. Development of tailored dissemination strategies to inform and educate clinicians, healthcare professionals, pharmacists, caregivers, and patients may be important to help reduce the risks associated with the use of cannabis for medical purposes in older adults. Cannabis use for non-medical purposes, specifically, was studied infrequently in the mapped studies. Several observational studies explored the effects of non-medical (sometimes referred to as “recreational”) cannabis use. In these studies, although non-medical cannabis use was found not to compromise lung function in individuals who used tobacco [137], detri- mental effects of non-medical use were identified, albeit in unadjusted analyses: older people without housing that consumed cannabis were more likely to fall [93] and older trauma patients were more likely to demonstrate early signs of shock, if they consumed cannabis [114]. Discussion Of greater prevalence in the literature were observational studies that either evaluated associations with any cannabis use (i.e., medical or non-medical; n = 14 studies) or that didn’t report the type of use (n = 28 studies), with more than half of these studies analyzing large national survey data (i.e., the older general public). In studies of the older general public, no significant associations with beneficial physical or mental health outcomes were reported from adjusted analyses, except possibly reduced future incidence of head/neck [129] and prostate cancers [143]. As illustrated in our bubble plots, associations with harmful physical and mental health outcomes greatly outnumbered beneficial outcomes in RCTs and in adjusted analyses reported in cohort studies. It should be emphasized that associations from cross-sectional and sequential studies do not denote causality, and that cannabis use may simply be more prevalent in older adults with pre-existing physical and mental health conditions. However, the available evidence suggests that any cannabis use may be detrimental to the physical and mental well- being of older adults, although the clinical significance of these findings is unknown and many potentially beneficial outcomes (e.g., pain reduction) were not reported in studies of the older general public. Our scoping review found very few studies that specifically examined cannabis use in older adults, relative to the volume of research available across all age groups. It also identified sev- eral important gaps in the evidence. First, we identified limited findings regarding our a priori subgroups of interest within the population of older adults. We found occasional studies that differentiated some effects of cannabis by sex [61, 64, 130] and age [116, 125, 135, 146, 157]; two related studies that explored whether the effects of Namisol1 in Alzheimer’s patients var- ied by their residential setting [86, 87]; a single study reporting cannabis effects on spirometry in individuals who formerly used tobacco amongst those who ever used tobacco [137]; and no studies reporting cannabis effects in different categories of accommodation status, marital sta- tus, gender, or frailty. We also identified few studies that made comparisons other than canna- bis use vs no use/placebo, such as comparing alternative means of cannabis consumption in older adults (e.g., vaporizing, oils, edibles), doses, types of cannabinoids, cannabinoid ratios, frequencies of use, etc. Discussion These comparisons may be especially salient to the older adult popula- tion, where variations in potency and use patterns may influence health risks and benefits. We also were met with a severe lack of data for several key outcomes, including physical brain out- comes, measures of pharmacokinetics, and drug interactions. Given the plausible effects of cannabis on measures of this nature, additional research is needed to address these gaps. Limitations Certain limitations of the review should be noted. First, given the extremely large volume of citations identified in our initial searches, we used study design filters to reduce search yield by only retrieving publications pertaining to the study designs of interest. These filters use com- prehensive controlled vocabulary and free text to identify pertinent study designs, but it is 25 / 37 25 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults possible that some records of potential interest may not have used the relevant vocabulary at the title, abstract, keyword and indexing level, in which case they could have been overlooked. Second, in determining eligibility of systematic reviews, our criteria defining “systematic review” were very strict, and relevant reviews may have been excluded due to poor reporting of their study methods. Regarding primary studies, our eligibility criteria were somewhat restricted in that we did not include studies reporting raw data only, with no analyses (e.g., studies that reported the numbers of patients with AEs but that did not analyze these data). As a result, single-arm studies were excluded, except if they reported pharmacokinetic data (of which we found none), and raw AE data in included studies were not charted. As well, it should be noted that scoping reviews differ from systematic reviews in several key areas. First, the objective of a scoping review is to map the available evidence on a topic not to meta-ana- lyze or fully synthesize the evidence. As such, we provide a high-level overview of all peer- reviewed evidence, without syntheses. Second, risk of bias assessments are not conducted in scoping reviews, and so the quality of the studies underlying our findings is not known. Regarding limitations of the available literature, many reviews and primary studies did not report age data in sufficient detail to allow determination of eligibility. As well, many primary studies of all ages of adults evaluated age-related differences, using “age” as a covariate in mul- tivariable models; however, these were excluded because the cannabis effect for the older adult age group(s) could not be determined. It should be noted that many RCTs and cohort studies were of small sample size, which may limit generalizability and confidence in their findings. As well, adjustment for confounding was not conducted in many NRSs, further reducing con- fidence in their findings. Limitations The cross-sectional design of many studies provided minimal infor- mation regarding causality, and these designs were especially prevalent in studies of populations without medical conditions, limiting the ability to infer the impacts of cannabis use in the general older adult population. Criteria to define ‘current cannabis use’ also varied between studies, thereby complicating the ability to compare reported effects between studies. As can be seen in the effect direction plots provided in the supplements to this review, consid- erable variability exists in the measures used to capture clinical outcomes of interest. A certain degree of divergence in findings between RCTs and NRSs was noted in some cases, and pat- terns in outcome assessment that varied across study designs also complicates interpretations (i.e., a common omission of harms endpoints in NRSs). In general, there was a lack of overlap- ping treatment/exposure comparisons across multiple studies, likely due in large part to a lack of standardized cannabis interventions for specific conditions and the broad range of non- medical/recreational products available (and variations in route of administration and other factors). Taking these challenges into consideration, while there is a desire for strong interpre- tations of benefits with cannabis use, the general public, patients and clinicians must be aware that the evidence remains weak at this time. While lack of or weak evidence does not necessar- ily correlate to a lack of benefits, at this time further research in older adults that addresses these limitations is urgently needed. We are hopeful that research funders may find this map- ping of evidence helpful in guiding future funding opportunities. Conclusions The current scoping review mapped more than 130 studies that provide evidence regarding the benefits, harms, and associations with health outcomes of cannabis consumption in older adults. The nature of these studies was diverse in terms of populations studied, reasons for consumption, and health effects measured. Within the general population of older adults, the limited evidence base suggests that the harms of cannabis use may outweigh the benefits. Sup- port regarding the benefits of cannabis use in older adults for medical reasons such as end- PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 26 / 37 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults stage cancer, Alzheimer’s disease/dementia, Parkinson’s disease and other indications appears limited at this time: evidence is often inconsistent across studies within specific patient condi- tions. The health effects of cannabis consumption in older adults require further study, with a balanced assessment of both benefits and harms, to guide appropriate public health messaging to balance the marketing pressures of cannabis to the older adults. Supporting information Supporting information S1 Dataset. Final data extracted from systematic reviews. (XLSX) S2 Dataset. Final data extracted from RCTs. (XLSX) S3 Dataset. Final data extracted from observational and non-randomized stu (XLSX) S1 Text. Protocol amendments with rationale. (DOCX) S2 Text. Complete description of the methods. (DOCX) S3 Text. Literature search strategies. (DOCX) S4 Text. PRISMA ScR checklist. (DOCX) S5 Text. Studies excluded at full-text screening. (DOCX) S6 Text. Study characteristics–Evidence tables by study design. (DOCX) S7 Text. Effect direction plots–Healthy adults and general population. (DOCX) S8 Text. Effect direction plots–End stage cancer. (DOCX) S9 Text. Effect direction plots–Alzheimer’s disease and dementia. (DOCX) S10 Text. Effect direction plots–Parkinson’s disease. (DOCX) S11 Text. Effect direction plots, other patient conditions. (DOCX) S12 Text. Summary of available subgroup data. (DOCX) S13 Text. Summary table of cannabis comparisons for older adults who used (DOCX) S3 Dataset. Final data extracted from observational and non-randomized studies. (XLSX) S2 Text. Complete description of the methods. (DOCX) S3 Text. Literature search strategies. (DOCX) S4 Text. PRISMA ScR checklist. (DOCX) S5 Text. Studies excluded at full-text screening. (DOCX) S6 Text. Study characteristics–Evidence tables by study design. (DOCX) S7 Text. Effect direction plots–Healthy adults and general population. (DOCX) S8 Text. Effect direction plots–End stage cancer. (DOCX) S9 Text. Effect direction plots–Alzheimer’s disease and dementia. (DOCX) S10 Text. Effect direction plots–Parkinson’s disease. (DOCX) S11 Text. Effect direction plots, other patient conditions. (DOCX) S12 Text. Summary of available subgroup data. (DOCX) S12 Text. Summary of available subgroup data. (DOCX) S13 Text. Summary table of cannabis comparisons for older adults who used cannabis. (DOCX) 27 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 PLOS ONE Scoping review: Medical and non-medical cannabis in older adults Acknowledgments We would like to thank Raymond Daniel (Assistant Information Specialist) for his role in pro- viding support in database management and document procurement. Author Contributions Conceptualization: Kim Corace, Danielle Rice, Melanie Willows, Brian Hutton. Conceptualization: Kim Corace, Danielle Rice, Melanie Willows, Brian Hutton. Data curation: Dianna Wolfe, Claire Butler, Danielle Rice, Becky Skidmore, Yashila Patel, Premika Thayaparan, Alan Michaud, Candyce Hamel, Brian Hutton. Data curation: Dianna Wolfe, Claire Butler, Danielle Rice, Becky Skidmore, Yashila Patel, Premika Thayaparan, Alan Michaud, Candyce Hamel, Brian Hutton. Formal analysis: Dianna Wolfe, Brian Hutton. Formal analysis: Dianna Wolfe, Brian Hutton. Funding acquisition: Kim Corace, Danielle Rice, Candyce Hamel, Andra Smith, Gary Garber, David Conn, Melanie Willows, Hanan Abramovici, Kednapa Thavorn, Salmaan Kanji, Brian Hutton. Investigation: Dianna Wolfe, Kim Corace, Danielle Rice, Candyce Hamel, Andra Smith, Gary Garber, Amy Porath, David Conn, Melanie Willows, Hanan Abramovici, Kednapa Thavorn, Salmaan Kanji, Brian Hutton. Methodology: Dianna Wolfe, Kim Corace, Becky Skidmore, Candyce Hamel, Brian Hutton. Project administration: Dianna Wolfe, Brian Hutton. Resources: Brian Hutton. Resources: Brian Hutton. Supervision: Dianna Wolfe, Kim Corace, Brian Hutton. Validation: Dianna Wolfe. Validation: Dianna Wolfe. Visualization: Dianna Wolfe. Visualization: Dianna Wolfe. Writing – original draft: Dianna Wolfe, Becky Skidmore, Brian Hutton. Writing – review & editing: Dianna Wolfe, Kim Corace, Claire Butler, Danielle Rice, Becky Skidmore, Yashila Patel, Premika Thayaparan, Alan Michaud, Candyce Hamel, Andra Smith, Gary Garber, Amy Porath, David Conn, Melanie Willows, Hanan Abramovici, Kednapa Thavorn, Salmaan Kanji, Brian Hutton. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281826 February 17, 2023 References Improving Quality of Life: Substance Use and Aging. 2018. Available: http://www.deslibris.ca/ID/10096147 13. Kelleher LM, Stough C, Sergejew AA, Rolfe T. 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https://openalex.org/W2520194699	https://www.econstor.eu/bitstream/10419/169030/1/aej-v18-i43-p707.pdf	English	null	Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area	DOAJ (DOAJ: Directory of Open Access Journals)	2,016	cc-by	8,522	Provided in Cooperation with: The Bucharest University of Economic Studies Suggested Citation: Țarțavulea (Dieaconescu), Ramona Iulia; Belu, Mihaela Gabriela; Paraschiv, Dorel Mihai; Popa, Ioan (2016) : Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area, Amfiteatru Economic Journal, ISSN 2247-9104, The Bucharest University of Economic Studies, Bucharest, Vol. 18, Iss. 43, pp. 707-725 Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. Documents in EconStor may be saved and copied for your personal and scholarly purposes. 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Sofern die Verfasser die Dokumente unter Open-Content-Lizenzen (insbesondere CC-Lizenzen) zur Verfügung gestellt haben sollten, gelten abweichend von diesen Nutzungsbedingungen die in der dort genannten Lizenz gewährten Nutzungsrechte. http://creativecommons.org/licenses/by/4.0/ http://creativecommon Amfiteatru Economic Journal AE Amfiteatru Economic recommends Abstract The process of globalization has stimulated the demand for logistics services at a level of speed and increased efficiency, which involves using of techniques, tools, technologies and modern models in supply chain management. The aim of this research paper is to present a model that can be used in order to achieve an optimized supply chain, associated with minimum transportation costs. The utilization of spatial modeling for determining the optimal locations for logistics centers in a predefined economic area is proposd in this paper. The principal methods used to design the model are mathematic optimization and linear programming. The output data of the model are the precise placement of one up to ten logistics centers, in terms of minimum operational costs for delivery from the optimum locations to consumer points. The results of the research indicate that by using the proposed model, an efficient supply chain that is consistent with optimization of transport can be designed, in order to streamline the delivery process and thus reduce operational costs. Keywords: logistic model, decision-making process, supply chain, optimal location, mathematical modeling, linear programming. JEL Classification: C21, C23, R31 JEL Classification: C21, C23, R31 Please cite this article as: Please cite this article as: Țarțavulea (Dieaconescu), R.I., Belu, M.G., Paraschiv, D.M. and Popa, I., 2016. Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area. Amfiteatru Economic, 18(43), pp. 707-725 SPATIAL MODEL FOR DETERMINING THE OPTIMUM PLACEMENT OF LOGISTICS CENTERS IN A PREDEFINED ECONOMIC AREA Ramona Iulia Țarțavulea (Dieaconescu)1, Mihaela Gabriela Belu2 , Dorel Mihai Paraschiv3 and Ioan Popa4 1) 2) 3)4) Bucharest University of Economic Studies, Romania Article History Received: 17 March 2016 Revised: 13 May 2016 Accepted: 8 June 2016 Corresponding author, Ramona Iulia Țarțavulea (Dieaconescu) ‒ ramona.tartavulea@yahoo.com 1. Literature review The literature review is focused on a few categories of models that apply in logistics, highlighting the hypothesis, algorithms, area of applicability and results. The most relevant models that have served as a point of reference in building the research methodology for this paper are presented and analyzed. Introduction International logistics plays an important role in the process of economic globalization and linking the national markets into a global network, providing methods, tools and systems by which goods and services can be delivered to international customers from all over the world. Corresponding author, Ramona Iulia Țarțavulea (Dieaconescu) ‒ ramona.tartavulea@yahoo.com Vol. 18 • No. 43 • August 2016 707 Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area AE This paper is in the area of academic research with practical application. The main purpose of this paper is to design a model that can be used as an instrument in the decision-making process for the domain of logistics strategies. We aim at presenting a model that can be used by managers/shareholders/investors in the decision-making process regarding the placement of logistic facilities that would ensure the minimum transport cost to the consumption points. By using this model to design a supply chain, we ensure the premises for minimum transportation costs. The applicability of the model refers to predefined economic area, consisting of all the consumption points which are targeted to be supplied by a company. The model uses a series of mathematical methods and classical algorithms, but the element of originality consists of defining a new conceptual framework for identifying the optimal location of logistics centers, so transportation can be more efficient. The first version of this model was presented in the thesis “International logistics strategies. Modeling of logistics decision-making process„ (Ţarţavulea, 2011). Amfiteatru Economic 1.1. The route optimization models If L(y) < ∞ then S = S ∪ {y} For z ϵ M(y) L (z) = min {L (z), L(y) +l (z, y)} repeated until S=X OR L(y) = ∞ The iteration stops when finally set S will contain all points (from 1-7) and L (x) will be equal to the minimum path from s to point x. The solution is the optimum path that consists of arcs chosen in our case with the condition of a minimum sequence of points in the route: 1, 2, 3, 6, 7. 1.1. The route optimization models The decisions regarding the choice of routes are taken in terms of cost minimization, considering the frequency and quantity of delivery as input data (Di Gangi, Montella and Russo, 1994). Further development consists in including the quantity and frequency of delivery as variables to be determined as results of the model. This type of models has been developed at Massachusetts Institute of Technology (MIT). Nuzzolo and Russo have designed a model used in the process of deciding the mode of transport, taking into consideration delivery units that are characteristic for different types of transportation (Nuzzolo and Russo, 1998). The problem of choosing the shortest route between multiple points is solved by the classic algorithm of Dijkstra (Trandafir, 2004), that allows the calculation of the shortest paths from one point s to all vertices x of a connected graph, the solution representing the optimum route for transportation. A simple example of how this algorithm functions is presented in figure no. 1 that illustrates a connected graph formed by 7 points linked by routes which have the necessary transit time mentioned above each one. Figure no. 1: A connected graph with 7 points and the time necessary to travel between them Amfiteatru Economic Figure no. 1: A connected graph with 7 points and the time necessary to travel between them Figure no. 1: A connected graph with 7 points and the time necessary to travel between them Amfiteatru Economic 708 AE Amfiteatru Economic recommends If we note the departing point with s, taking values from 1 to 7, we can determine the optimum route between s and any other point noted with x, where x ϵ X and X = {the lot of all points (1-7) excluding s}. We use the notations: We use the notations: L(x) = l(s, x) = the length of the route from x to s M(x) = set of nodes incident to node x We initially consider L(x) =0 and the lot S = {s}, that is only the departure point. Step 1: For any x ϵ X-S if x ϵ M(x) then L(x) = l(s, x), if not M(x) = +∞ We determine y ϵ X – S so that L(y) = min l (z), where z doesn’t belong to the lot S. 1.3. Dynamic models The component that incites our interest from this type of models is that of space representation. In order to be able to use the space dynamically, you need to divide it into small pieces that give the possibility of easily using it in a model. The potential of space celularisation method in mathematic modeling was first brought to light by Tobler (1979) and further developed by Phipps (1989) and Constanza, Sklar and White (1990). The literature review also revealed that a broad base of methodologies was developed for modeling logistic processes (static scenarios comparison, dynamic simulation, mathematical optimization). • Static Scenario Comparison - include making calculations based on data related to supply-delivery chain, without taking into account their variability over time. For this reason, data collection is relatively easy. Models of this type can be implemented through applications based on worksheets (spreadsheets). They allow static analysis, but rapid alternative scenarios (Chwif, Barretto and Saliby, 2002) • Dynamic Simulation - this type of models capture the evolution of the system over time and thus the planning horizon can be quite large, taking into account the influences of characteristic different periods (e.g. economic cycles, seasonal variations). Stochastic effects are presented so that the degree of realism increases. The effort to collect data on flows of goods and materials, and information is greater than in the first case (Seidel, 2005). • Mathematical Optimization - It is centered on the analysis and modeling of the logistics system. Models can be applied to a single period (single-period) or more (multi- period). Mathematical optimization is based on expressing system restrictions using equations, followed by applying specific algorithms for solving them and finding the optimum solution (Seidel, 2005). 1.2. Spatial models The simplest model in this category is based on the method of determination for the gravitational center. A gravitational model uses a map containing the points of consumption (defined by latitude and longitude). The solution of the model identifies the optimal location for placing a central warehouse that implies minimum costs for transportation to all the consumer points. We can complicate this model by adding more than one logistic center to the expected result of the model. The gravitational model can no longer be used after determining the first optimal location. Latest research performs a combination of a cost minimizing model and a gravity model with dual restriction. This type of model allows setting a certain level of customer service as additional restriction for establishing the optimal locations (Wilson, 2005). The interesting point made in this paper is the fact that the average delivery time can be calculated and used as a measure for the level of customer service a company aims to offer. A gravitational dual restriction model uses a matrix that includes logistic facilities and centers of consumption. On this basis we can design a linear programming model that approximates optimal flows between these locations (Țarțavulea, Belu and Dieaconescu, 2011). Previous research on this subject (Țarțavulea, Belu and Dieaconescu, 2011; Țarțavulea, 2015) used a gravitational model in order to identify one optimum location for placing a central logistic facility, taking into consideration the points of consumption. The aim was to minimize the global transport costs for a company. This article further develops the methodology aiming at solving the complex problem of identifying several optimal locations for placing logistics centers, taking into consideration the ones previously determined, with the purpose of obtaining the lowest cost of delivery to all the point of consumption taken into consideration. Vol. 18 • No. 43 • August 2016 709 Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area AE 2. Research methodology After gathering information about logistics modeling from the literature review, it was decided to design a model that can be used in the decision-making process regarding the logistic activity and strategy of the company. As it is defined, the model can be included in the following categories: a) by the input data and output data: spatial, gravitational; b) by the methodology used for modeling the logistic processes: mathematical optimization, dynamic simulation. The hypotheses of research are: 1. Using modeling on the logistics decision-making processes leads to lower operational costs and improvement in efficiency for the enterprise; 2. Optimal locations for storage / production for a market defined by the points of consumption can be determined using mathematical modeling and linear programming; 3. Optimal location of storage sites in the space defined as the consumer market, determines the minimum cost of transport; 3. Optimal location of storage sites in the space defined as the consumer market, determines the minimum cost of transport; 4. The average time of delivery will decrease with the addition of more optimal locations. Note: throughout the paper the logistics centers will also be referred to as warehouses, or storage locations. The functions that they can undertake can be storage of goods, production activities, packing, promotional improvements or labeling and any kind of logistics activities. 2.1.1. Input data The input data consists of the geographic coordinates for 100 points of consumption, which delimitate an economic area; the demand in each consumer point was estimated. In order to simplify the process, it was decided to use the population as a measure of demand in each city (a city = a point of consumption). The data regarding the geographic coordinates and population of cities were collected from public databases (Wikipedia). Amfiteatru Economic 710 AE Amfiteatru Economic recommends Starting from the research up to date in the field of logistic modeling, modern methods were used for designing the model that can determine the optimum locations for logistics centers based on the demand from the consumption points, with the condition of minimum transportation costs. Some of the methods taken into consideration were: celularisation of space, mathematic algorithms (such as Dijkstra’s algorithm), geometric methods (such as the determination of the gravitational center) and linear programming. A unique combination of these methods leads to the construction of a new logistic model, tested on the economic territory of Romania. 2.1.2. Hypotheses of research The hypotheses of research are: 2.1.4. Assumptions of the model The assumptions of the model are: • Any product can be delivered through a single transport; • The cost of transportation depends on the distance between the nearest warehouse and the point of consumption (the curvature of the Earth is not taken into account); • An average speed is taken into account, generally valid for any route; • A consumer center will be served only by one deposit; • The first location identified, corresponding to the center of gravity, will be considered the central warehouse, which will supply the other locations (identified by repeated simulations); • The average delivery time will decrease with the addition of more optimal locations; • The model does not consider the supply of raw materials to the central warehouse; • Decision behavior is considered to be economically rational. 2.1.3. The structure of the model The general structure of the model is presented in table no. 1, and it shows the main and secondary variables, as well as the input and output data, with technical specifications. Table no. 1: The variables of the model Main variables Secondary variables Technical specification Input variables A geographic region n consumption points (c1, c2 ...cn) Geographic coordinates Longitude Latitude Demand on each point May be approximated by population Output variables A central warehouse DC1 Geographic coordinates Number of served cities Served population The average delivery time m secondary/local warehouses (D2, D3, ... Dm) Geographic coordinates For the practical application m ϵ {2;10} For further development the number of local warehouses can be increased Number of served cities Served population The average delivery time Source: Adapted after Ţarţavulea R.I., 2015 Vol. 18 • No. 43 • August 2016 711 711 Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area AE The input data refers to geographic coordinates of points (cities, consumption centers – which define the area of application for the model) and population afferent to these. 2.2. Design of the model The model is structured into stages, which are presented subsequently. Please note that the first stage of the model undertakes the method used in previous papers (Țarțavulea, Belu and Dieaconescu, 2011; Țarțavulea, 2015), and the next stages represent a further original development of the model. Stage I: Defining the geographic and economic area selected for the application of the model Choosing the geographic region for which we want to determine the optimum location/s for placing logistic centers. Choosing the geographic region for which we want to determine the optimum location/s for placing logistic centers. Selecting a number of points of consumption to be taken into account for products delivery (n=100 is proposed for the practical application). Each point shall be defined by its geographic coordinates (latitude and longitude) and by the estimated demand (it can be approximated by market studies or in order to simplify, we can use the population number). Stage II: Identifying the optimum location for one central warehouse, assuring the lowest total cost of delivery to all points of consumption. First, we need to transform the geographic coordinates into decimal numbers, in order to be able to utilize them in calculations. This is carried out by using a Microsoft Excel formula (Țarțavulea, Belu and Dieaconescu, 2011). We calculate the geographical coordinates for the gravitational center of the consumer points we defined, by using specific formulas (Țarțavulea, 2015). We obtain the optimum position for a central repository in the defined region, which is determined by the latitude and longitude calculated in Step 2, resulting in 712 Amfiteatru Economic AE Amfiteatru Economic recommends point DC1 (the gravitational center of the defined consumer points). We calculate the distances from the central warehouse (DC1) to each point of consumption, which are required for Stage V. To determine the distances between the optimal location and all other cities have used a mathematical algorithm (Țarțavulea, Belu and Dieaconescu, 2011). For each point of consumption we determine the average delivery time, calculated using the formula: 𝑇𝑖𝑑𝑖𝑣 (1) 𝑇𝑇𝑖𝑖= 𝑑𝑑𝑖𝑖∗𝑣𝑣 where: 𝑇𝑇𝑖𝑖= 𝑑𝑑𝑖𝑖∗𝑣𝑣 where: Ti - estimated delivery time from the warehouse to every point of consumption; di - the distance from the central warehouse to the point of consumption i; v - the average speed applicable in selected geographic region. Stage III: Determining the second optimal location for placing an additional secondary logistic center. The geographical space is divided into z cells. The smaller the cells are (meaning higher number of cells) the more exact the approximation algorithm will be and the estimated solutions for the secondary storage will approach optimum placement. where: d1c - the distance from cell 1 to the central warehouse 𝑐𝑖𝑝 c’1 = ∑𝑐𝑐𝑖𝑖 𝑝𝑝 1 (6) where: (6) c’1 = ∑𝑐𝑐𝑖𝑖 𝑝𝑝 1 where: ci ϵ {1, 2... p}, the set of consumer points that can be served by cell 1, that is they fit the condition: dmin1i = d1i ; 𝑐𝑛 ci ϵ {1, 2... p}, the set of consumer points that can be served by cell 1, that is they fit the condition: dmin1i = d1i ; 𝑐𝑛 cT - total demand for consumption in all consumption points, calculated as: 𝑐𝑖𝑛 cT = ∑𝑐𝑐𝑖𝑖 𝑛𝑛 1 (7) cT = ∑𝑐𝑐𝑖𝑖 𝑛𝑛 1 (7) (7) cT = ∑𝑐𝑐𝑖𝑖 𝑛𝑛 1 Next, for each cell of between 2 and z the sum of distances from the cell to each point of consumption is calculated using the formula: 𝑑𝑑𝑑𝑑𝑐𝑖𝑛𝑑𝑐𝑗 Next, for each cell of between 2 and z the sum of distances from the cell to each point of consumption is calculated using the formula: 𝑑𝑑𝑑𝑑𝑐𝑖𝑛𝑑𝑐𝑗 sj = ∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑗𝑗𝑗𝑗∗ 𝑐𝑐𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 1 + 𝑑𝑑𝑗𝑗𝑗𝑗∗(1 + 𝑐𝑐′𝑗𝑗 𝑐𝑐𝑇𝑇) (8) (8) where: where: j ϵ{1, 2... z}, the set of cells in which we divided the geographic space in Stage 1 of the model; j ϵ{1, 2... z}, the set of cells in which we divided the geographic space in Stage 1 of the model; dji - distance from cell j to consumer point i, i ϵ {1, 2... 100}, i ϵ N; dci - distance from the central warehouse to the consumer point i, i ϵ {1,2,.,100}; dminji = min (dji , dci); j j dminji - the minimum between the distance from cell j to the point of consumption i and the distance from the central warehouse DC1 to the point of consumption i; 𝑐 p p djc - the distance from cell j to the central warehouse DC1. Choosing the geographic region for which we want to determine the optimum location/s for placing logistic centers. For the first cell the sum of the distances from this cell to each point of consumption is calculated according to the formula: 𝑑𝑑𝑑𝑑𝑖𝑛 s1 =∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑1𝑖𝑖 𝑛𝑛 1 (2) where: (2) s1 =∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑1𝑖𝑖 𝑛𝑛 1 where: s1 =∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑1𝑖𝑖 𝑛𝑛 1 where: s1 =∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑1𝑖𝑖 𝑛𝑛 1 where: s1 - the sum of distances from the first cell to each consumer point; s1 - the sum of distances from the first cell to each consumer point; d1i - distance from cell 1 to the consumer point i, i ϵ {1, 2... 100}, i ϵ N; dci - distance from the central warehouse to the consumer point i, i ϵ {1, 2... 100}; dmin1i - min (d1i, dci) that is the minimum between the distance from cell 1 to the consumer point i and the distance from the central warehouse to the consumer point i. dmin1i - min (d1i, dci) that is the minimum between the distance from cell 1 to the consumer point i and the distance from the central warehouse to the consumer point i. If considered the fact that for the consumer point with a higher demand, more than one transport is necessary, then the distance should be weighted with the level of demand, modifying the formula: 𝑑𝑑𝑑𝑑𝑖𝑐𝑖𝑛 s1 = ∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑1𝑖𝑖∗ 𝑐𝑐𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 1 (3) where: s1 = ∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑1𝑖𝑖∗ 𝑐𝑐𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 1 (3) h s1 = ∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑1𝑖𝑖∗ 𝑐𝑐𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 1 (3) where: where: ci - the level of demand in the consumer point i; 𝑐 p cM - the average demand, calculated as: 𝑐𝑖𝑛𝑛 p cM - the average demand, calculated as: 𝑐𝑖𝑛𝑛 cM - the average demand, calculated as: 𝑐𝑖𝑛𝑛 cM = ∑𝑐𝑐𝑖𝑖 𝑛𝑛 1 𝑛𝑛 (4) cM = ∑𝑐𝑐𝑖𝑖 𝑛𝑛 1 𝑛𝑛 (4) Since the secondary warehouse will be supplied from the central repository, the length of the road between the two locations must be added, weighted by the volume of demand which the secondary deposit serves. The formula is amended as follows: 𝑑𝑑𝑑𝑑𝑐𝑖𝑛𝑑𝑐 s1 = ∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑1𝑖𝑖∗ 𝑐𝑐𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 1 + 𝑑𝑑1𝑐𝑐∗(1 + 𝑐𝑐′1 𝑐𝑐𝑇𝑇) (5) s1 = ∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑1𝑖𝑖∗ 𝑐𝑐𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 1 + 𝑑𝑑1𝑐𝑐∗(1 + 𝑐𝑐′1 𝑐𝑐𝑇𝑇) (5) (5) (5) Vol. 18 • No. 43 • August 2016 713 Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area AE AE where: where: The general algorithm is: S = min {s1, s2......sz} Amfiteatru Economic 714 AE Amfiteatru Economic recommends (11) sj = ∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑗𝑗𝑗𝑗∗ 𝑐𝑐𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 1 + 𝑑𝑑𝑗𝑗𝑗𝑗∗(1 + 𝑐𝑐′𝑗𝑗 𝑐𝑐𝑇𝑇) where: j ϵ{1,2......,z}, the set of cells in which we divided the geographic space in Stage 1 of the model; dji - distance from cell j to consumer point i, i ϵ {1,2......,100}, i ϵ N; j dci - distance from the central warehouse to the consumer point i, i ϵ {1,2,.,100}; dci - distance from the central warehouse to the consumer point i, i ϵ {1,2,.,100} dki - min (dD1i , dD2i , ...., dDri ), where dDri = distance from the warehouse r to the consumer point i, i ϵ {1,2,.,100}, k is the number of deposits that have been determined so far; dki - min (dD1i , dD2i , ...., dDri ), where dDri = distance from the warehouse r to the consumer point i, i ϵ {1,2,.,100}, k is the number of deposits that have been determined so far; dki - min (dD1i , dD2i , ...., dDri ), where dDri = distance from the warehouse r to the consumer point i, i ϵ {1,2,.,100}, k is the number of deposits that have been determined so far; ; dminji - min (dji , dci, dki) - the minimum between the distance from cell j to the point of consumption i, the distance from the central warehouse DC1 to the point of consumption i and distances from all previously determined secondary storage and consumer point i; djc - the distance from cell j to the central warehouse; c’1, cT, ci, cM – see Stage III for definition. c’1, cT, ci, cM – see Stage III for definition. The secondary deposit Dk, k ϵ {1,2.., m} will have the coordinates for the cell that corresponds to the minimum sum (sj = S), j ϵ {1,2.....,z}. The secondary deposit Dk, k ϵ {1,2.., m} will have the coordinates for the cell that corresponds to the minimum sum (sj = S), j ϵ {1,2.....,z}. For each point of consumption we will determine the optimal warehouse from which the delivery will be made, that is the nearest optimal location to the point of consumption (operation which is repeated for each addition of a warehouse, changing the distribution scheme). where: 𝑐𝑖𝑝 c’j = ∑𝑐𝑐𝑖𝑖 𝑝𝑝 1 (9) c’j = ∑𝑐𝑐𝑖𝑖 𝑝𝑝 1 (9) where: c’j = ∑𝑐𝑐𝑖𝑖 𝑝𝑝 1 (9) where: c’j = ∑𝑐𝑐𝑖𝑖 𝑝𝑝 1 where: c’j = ∑𝑐𝑐𝑖𝑖 𝑝𝑝 1 (9) where: ci ϵ {1,2,....,p}, the set of consumer points which may be served by cell 1, that is they fulfill the condition: dminji = dji ; 𝑐 j j cT - the total demand requested in all consumer points, calculated by the formula: 𝑐𝑖𝑛 cT = ∑𝑐𝑐𝑖𝑖 𝑛𝑛 1 (10) (10) cT = ∑𝑐𝑐𝑖𝑖 𝑛𝑛 1 Stage IV: Determining up to m optimum points for placing local logistic centers. Stage IV: Determining up to m optimum points for placing local logistic centers. Making use of the space division in cells from the previous stage, the iterative steps described in stage III are repeted. Making use of the space division in cells from the previous stage, the iterative steps described in stage III are repeted. The optimal location for a third warehouse is identified and this helps reduce the average delivery time to any client from the points of consumption. Next the locations for deposits from 4 to m are determined. where: For each point of consumption, we will calculate the distance from the warehouse which is nearest (it was determined in Stage IV) and the delivery time, calculated using the formula: 𝑇𝑖𝑑𝑖𝑣 𝑇𝑇𝑖𝑖= 𝑑𝑑𝑖𝑖∗𝑣𝑣 (12) where: (12) 𝑇𝑇𝑖𝑖= 𝑑𝑑𝑖𝑖∗𝑣𝑣 where: where: Ti - estimated delivery time from the warehouse to every point of consumption; Ti - estimated delivery time from the warehouse to every point of consumption; di - the distance from the nearest warehouse to the point of consumption i; v - the average speed in the selected geographic region. Stage V: Calculating the average time necessary for delivery from the central warehouse at any point of consumption First, the sum of the length of roads which must be covered for delivery is calculated from the central warehouse to all the selected cities plus the distance to the central repository, using the formula: 𝑑𝑑𝑝𝑖𝑛 S = ∑ max (𝑑𝑑𝑖𝑖, 𝑑𝑑𝑖𝑖∗ 2∗𝑝𝑝𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 𝑖𝑖=1 ) (13) where: S = ∑ max (𝑑𝑑𝑖𝑖, 𝑑𝑑𝑖𝑖∗ 2∗𝑝𝑝𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 𝑖𝑖=1 ) (13) where: (13) where: where: di - is the length of the road from the central warehouse to the point of consumption i; di - is the length of the road from the central warehouse to the point of consumption i; ci - the demand for the consumption point I; di - is the length of the road from the central warehouse to the point of consumption i; ci - the demand for the consumption point I; p p cM - the average consumer demand. cM - the average consumer demand. cM - the average consumer demand. Vol. 18 • No. 43 • August 2016 715 Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area AE Basically, the sum of distances will be composed of the lengths of roads traveled from the central warehouse to the points of consumption, taking into account at least one transport (if the consumer demand is twice smaller than the average consumer demand) or the road weighted by consumer demand (if it is higher than half the average consumer demand). It is considered that the average consumer demand requires two shipments. where: The calculated amount is divided by the number of consumption points and the result is multiplied by the average speed the defined geographical region and legal average time of delivery is obtained by the formula: 𝑇𝑆𝑣 𝑇𝑇𝑚𝑚= 𝑆𝑆 𝑛𝑛∗𝑣𝑣 (14) where: 𝑇𝑇𝑚𝑚= 𝑆𝑆 𝑛𝑛∗𝑣𝑣 (14) where: where: Tm - the average delivery time; Tm - the average delivery time; g y n - number of points of consumption; S - sum of distances from a logistics center to any point of consumer; v - the legal average speed in the specified geographical region. v - the legal average speed in the specified geographical region. Stage VI: Calculation of the average time of delivery from secondary logistic centers to any point of consumption. For each number of optimal locations (from 2 to m) we calculate the total amount of roads covered to ensure delivery to all points of consumption, depending on the size of demand for each of them. We must take into consideration that at each addition of a further location the scheme of distribution to the points of consumption changes (each point of consumption will be served by the nearest logistic center). We apply steps 1 and 2 from the previous stage, calculating the average time of delivery characteristic for each number of optimal locations (from 2 to m logistic center) We calculate the difference between the average delivery time for m locations and m-1, in order to reduce the average time of delivery to each addition of a location. The formula is: (15) ∆ t = Tm-Tm-1 ∆ t = Tm-Tm-1 Notations: Notations: DC1: the first optimal location identified for the entire region, which is the central logistic center (determined by the method of the center of gravity for consumption points); D2 – D10: the optimal secondary locations identified consecutively by applying the algorithm of the model; D2 – D10: the optimal secondary locations identified consecutively by applying th algorithm of the model; c1 – cn: the centers of consumption from 1 to n. c1 – cn: the centers of consumption from 1 to n. 2.3. The graphical representation of the model For a better understanding of the way the system of delivery operates within the supply chain that can be built using the proposed model, we created figure no. 2, which is a graphical representation of a hypothetical geographic area defined by a series of consumption points, that must be characterized by their geographical coordinates and the exact size of consumer demand (for goods / services for which you want to identify optimal placement locations of logistics centers for storage facilities and / or production). The consumption points c1, c2 ... cn represent the model input data, which are the first set on the map, and the central warehouse location DC1 and secondary deposits D2, D3, ... Amfiteatru Economic 716 AE Amfiteatru Economic recommends D10 are output variables, namely the model results. Any point of consumption but will be served from the nearest warehouse, be it at primary (DC1) or secondary one (D2-D10). D10 are output variables, namely the model results. Any point of consumption but will be served from the nearest warehouse, be it at primary (DC1) or secondary one (D2-D10). Figure no. 2: The delivery configuration for the supply chain proposed by the model in a hypothetical geographic area Figure no. 2: The delivery configuration for the supply chain proposed by the model in a hypothetical geographic area The contour of the hypothetical figure delimits the geographical area chosen for the model application. The contour of the hypothetical figure delimits the geographical area chosen for the model application. 2.4. Case study In order to validate the model, the geographic area of Romania was chosen, placing 100 points of consumption that define the space of application. The consumer points represent actual cities, defined by their exact geographic coordinates and their demand which is approximated by their population number (97% of them have over 10.000 inhabitants). For Vol. 18 • No. 43 • August 2016 717 Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area AE the application of the model on the case study of Romania, we considered m=10, so a number of ten optimal locations for placing logistics centers will be identified. the application of the model on the case study of Romania, we considered m=10, so a number of ten optimal locations for placing logistics centers will be identified. The case study follows precisely the stages that have been defined in the theoretical structure of the model. First the latitude and longitude of the cities were processed in order to be able to use them in calculations made in an excel sheet. The formula used to transform the geographic coordinates in decimal numbers is: (16) Next, the decimal numbers were synthetized in a new table (a selection can be consulted in Appendix A), based on which a chart was created to show all selected cities in form of their spatial distribution in two dimensions - latitude and longitude (figure no. 3). We mention that in order to simplify, the curvature of the geographical space, namely altitude was not taken into account. Figure no. 3: Spatial distribution of the 100 selected cities in Romania Figure no. 3: Spatial distribution of the 100 selected cities in Romania Figure no. 3: Spatial distribution of the 100 selected cities in Romania It is considered necessary to add two hypotheses to the model, which are specific to the application of the model in the form of the case study: It is considered necessary to add two hypotheses to the model, which are specific to the application of the model in the form of the case study: • The consumption points are considered to be cities; • The demand manifested in the points of consumption is approximated by the city's population. 2.4. Case study Amfiteatru Economic 718 AE Amfiteatru Economic recommends For identifying the optimal location for a central repository that ensures a minimum total cost of delivery to all points of consumption, the calculations start with Stage I, defined in Section 2.2. The first step in identifying the optimal location for a central warehouse is to determine the geographical coordinates of the center of gravity. 3. Results and discussion As a result of calculations, the point corresponding to the computed coordinates (45.46 N, 25.63 E) was identified as the optimal location for placement of a single central repository (DC1), which ensures minimal costs for distribution of products in the 100 selected cities in Romania. Next, the distances between DC1 and all the consumer points were calculated using the algorithm described in step 4, Stage II. In order to identify the optimal location for the second logistic center, the first step is to divide the given geographic area in z cells as shown in the figure no. 4. Each cell is 0.01 degrees latitude and 0.01 degrees longitude, which means a width and a length equal to 0.01 111.1385 (number of km / degree) = 1.1113 kilometers. Hence, the surface of a cell would be approximately 1234 km2, thus the maximum error in the application model is 1 km. Since the map of Romania starts at a longitude of 21,5o minimum to maximum longitude 28o, and a minimum latitude 44,1o to maximum latitude of 47,4o, the total number of cells z is 214 500 according to the formula: [(28 - 21,5) / 0.01] * [(47,4 - 44,1)/0,01] = 650 * 330 = 214 500 (17) (17) For cell 1 (the order is not important, it can start from any part of the map) the sum of distances from the cell to each points of consumption defined in the first stage is calculated according to the formula: 𝑑𝑑𝑑𝑑𝑐𝑖𝑛𝑑𝑐 s1 = ∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑1𝑖𝑖∗ 𝑐𝑐𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 1 + 𝑑𝑑1𝑐𝑐∗(1 + 𝑐𝑐′1 𝑐𝑐𝑇𝑇) (18) s1 = ∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑1𝑖𝑖∗ 𝑐𝑐𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 1 + 𝑑𝑑1𝑐𝑐∗(1 + 𝑐𝑐′1 𝑐𝑐𝑇𝑇) (18) where: dmin1i - min (d1i, dci) the minimum between the distance from cell 1 to the point of consumption i and the distance from the central warehouse DC1 to the point of consumption i; 𝑐𝑝 p ; d1c - distance from cell 1 to the central warehouse; 𝑐𝑖𝑝 p ; d1c - distance from cell 1 to the central warehouse; 𝑐𝑖𝑝 c’1 = ∑𝑐𝑐𝑖𝑖 𝑝𝑝 1 , where: ci ϵ {1,2....,p}, set of the points of consumption that can be served by cell 1, and satisfy the condition: dmin1i = d1i. c’1 = ∑𝑐𝑐𝑖𝑖 𝑝𝑝 1 , where: ci ϵ {1,2....,p}, set of the points of consumption that can be served by cell 1, and satisfy the condition: dmin1i = d1i. Vol. 18 • No. 3. Results and discussion 43 • August 2016 719 719 Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area AE AE Figure no. 4: Dividing the geographic space of Romania into cells Figure no. 4: Dividing the geographic space of Romania into cells The other variables are defined in Stage III, step 2 from the theoretical model. The other variables are defined in Stage III, step 2 from the theoretical model. Next, for each cell from 2 to n the sum of distances from the cell at each point of consumption is calculated. For each amount calculated the weighted distance from cell to the central repository identified in stage II is added (the secondary deposit must be supplied from the central warehouse). The general formula is: 𝑑𝑑𝑑𝑑𝑐𝑖𝑛𝑑𝑐𝑗 sj = ∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑗𝑗𝑗𝑗∗ 𝑐𝑐𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 1 + 𝑑𝑑𝑗𝑗𝑗𝑗∗(1 + 𝑐𝑐′𝑗𝑗 𝑐𝑐𝑇𝑇) (19) sj = ∑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑗𝑗𝑗𝑗∗ 𝑐𝑐𝑖𝑖 𝑐𝑐𝑀𝑀 𝑛𝑛 1 + 𝑑𝑑𝑗𝑗𝑗𝑗∗(1 + 𝑐𝑐′𝑗𝑗 𝑐𝑐𝑇𝑇) (19) S = min {s1, s2......sz}; the minimum sum calculated with the previous formula was chosen and therefore was identified the cell where we must place the second point which is an optimal location for placing a secondary deposit, given the already identified center of gravity = central warehouse. For each point of consumption, we calculate the distance from it to D2 storage and the delivery time, calculated using the formula: 𝑇𝑖𝑑𝑖𝑣 𝑇𝑇𝑖𝑖= 𝑑𝑑𝑖𝑖∗𝑣𝑣 (20) (20) 𝑇𝑇𝑖𝑖= 𝑑𝑑𝑖𝑖∗𝑣𝑣 By applying this algorithm in the computer programming language C + +, the result obtained was: Optimum location 2 (D2): lat = 46.6467, long = 23.4633 um location 2 (D2): lat = 46.6467, long = 23.4633 Making use of the cellularisation of space method applied previously, the iterative steps in stage IV were followed. This way the optimal location for a third logistic center is identified, and so on until ten optimal locations are found consequently. Each number of Amfiteatru Economic 720 AE Amfiteatru Economic recommends logistic centers is placed in order to insure the minimum average delivery time to any customer at one of the points of consumption. As an additional logistic center is placed, the number of served cities for each of the optimal locations changes, as well as the total distance that needs to be covered and the average delivery time. The results for 3 logistic centers are presented in table no. 2. Table no. 3. Results and discussion 2: The results for 3 consequently determined logistic centers Optimal location Latitude Longitude Number of served cities Population served DC1 45.4659 25.6257 37 2 728 347 D2 46.6467 23.4633 33 2 183 393 D3 44.4467 26.1733 30 4 069 014 Table no. 2: The results for 3 consequently determined logistic centers The total distance that should be traveled in order to reach all the consumption points is: 15525.5274 km, and the weighted total distance that will be covered in order to deliver the products to the cities, taking into consideration the population number for each, is: 29115.3158 km (for bigger cities there might be more than one transport necessary, the condition for doing more than one transport is ci > cM 2 , as explained in Stage IV, Section 2.2). If the objective is to to place four logistic centers in Romania, according to the placement of consumer points, the cities will be served by the nearest warehouse. The map will be divided into four zones of influence, depending on the logistic center that will serve them. Figure no. 5 illustrates how the consumer points are ascribed to the 4 logistic centers determined so far. Figure no. 5: The division of cities depending of the supplying source (warehouse) The results for each number of optimal locations between 4 and 10 are presented in Appendix B. The last iteration of the model reveals the results for ten optimal locations, placed as presented in figure no. 6. Figure no. 5: The division of cities depending of the supplying source (warehouse) The results for each number of optimal locations between 4 and 10 are presented in Appendix B. The last iteration of the model reveals the results for ten optimal locations, placed as presented in figure no. 6. The results for each number of optimal locations between 4 and 10 are presented in Appendix B. The last iteration of the model reveals the results for ten optimal locations, placed as presented in figure no. 6. Vol. 18 • No. 43 • August 2016 721 721 721 Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area AE AE Figure no. 6: The optimal placement for ten logistics centers in Romania Figure no. 3. Results and discussion 0.0000 2.0000 4.0000 6.0000 8.0000 1 2 3 4 5 6 7 8 9 10 The average delivery time (in hours) Number of logistic centers Figure no. 7: The decrease of average delivery time on adding a supplementary location 0.0000 2.0000 4.0000 6.0000 8.0000 1 2 3 4 5 6 7 8 9 10 The average delivery time (in hours) Number of logistic centers Figure no. 7: The decrease of average delivery time on adding a supplementary location Investors which are interested in building logistic centers in our country can rely on this model to decide what number of warehouses is appropriate to have (depending on the level of customer service that they want to offer – based on the average delivery time) and where it is best to position them, in order to have a minimum cost of delivery. Investors which are interested in building logistic centers in our country can rely on this model to decide what number of warehouses is appropriate to have (depending on the level of customer service that they want to offer – based on the average delivery time) and where it is best to position them, in order to have a minimum cost of delivery. 3. Results and discussion 6: The optimal placement for ten logistics centers in Romania Next, we proceed to Stage V, in which we calculate the average delivery time necessary for transportation from the central logistic center to all the points of consumption. After calculating the sum of all roads that need to be covered, the formula for Tm is used and the result is 6 h 55 min. We calculate the average delivery time for each number of logistic centers (from 2 to 10) using the algorithm described in Stage VI from the theoretical model presented in Section 2.2. Also of interest is the amount of time that is saved by adding a supplementary optimum location, noted with ∆ t. The results are synthesized in table no. 3. Table no. 3: The average delivery time for 1 to 10 consecutive d t i d ti l l ti Table no. 3: The average delivery time for 1 to 10 consecutive determined optimal locations Number of Logistic centers Total weighted distance (km) Average delivery time: Tm (in hours and minutes) ∆ t (in hours and minutes) 1 48373.6462 6.9105 (6h 55m) 2 37998.0405 5.4283 (5h 26m) 1.4822 (1h 29m) 3 29115.3158 4.1593 (4h 10m) 1.2690 (1h 16m) 4 22565.2016 3.2236 (3h 13m) 0.9357 (0h 56m) 5 19213.6671 2.7448 (2h 45m) 0.4788 (0h 29m) 6 15897.3821 2.2711 (2h 16m) 0.4738 (0h 28m) 7 14199.1577 2.0285 (2h 02m) 0.2426 (0h 15m) 8 12697.1597 1.8139 (1h 49m) 0.2146 (0h 13m) 9 11965.6024 1.7094 (1h 43m) 0.1045 (0h 06m) 10 11090.5295 1.5844 (1h 35m) 0.1250 (0h 08m) In order to observe better the trend of decrease in the average delivery time. Figure no. 7 was built and it may be of help in the procedure of establishing the level of customer service desired. Amfiteatru Economic 722 AE Amfiteatru Economic recommends Figure no. 7: The decrease of average delivery time on adding a supplementary location Investors which are interested in building logistic centers in our country can rely on this model to decide what number of warehouses is appropriate to have (depending on the level of customer service that they want to offer – based on the average delivery time) and where it is best to position them, in order to have a minimum cost of delivery. Conclusions The enunciated hypotheses have been verified by the empirical application of the model, which has been validated and proven to be functional. The model can be used as an instrument by managers or investors, in order to design the most convenient supply chain, making use of information regarding the demand and the localization of consumer points. It is also an instrument for cost minimization (for new investments or new market penetration) or expenses reduction (in case of relocations imposed by decrease of demand in certain areas). Both circumstances are characteristic to the economic situation that has been generated after the appearance of the financial crisis and that continues to cause effects on multinational companies and not only. Also, this model can be used by companies that have a green logistics strategy and want to locate their facilities in order to optimize the transportation cost, along with minimization of fuel consumption and CO2 emissions. Facility location decision is the critical part in strategic logistics planning. Now-a-days the location of the facilities i.e. warehouses, logistics hubs/centers etc. is the main concern of the companies related to this business (Sheikh, 2013). The model is considered to be clearly defined by its input and output variables, as well as the stages described in more steps, so it is easy to be applied on other geographic areas, may they be economic regions, economic unions or continents. It can be also particularized for certain products, or companies, given that the specific demand can be estimated correctly. Please note that the number of consumer points that we have chosen (of 100), can also be modified without any changes in the model, as well as the maximum number of logistic centers (which was set at 10 for this case study). Vol. 18 • No. 43 • August 2016 723 Spatial Model for Determining the Optimum Placement of Logistics Centers in a Predefined Economic Area AE As a future direction of research, reverse flows can be included in the model that we proposed. Recycling old products or parts is the next step in approaching a green logistics strategy and it is possible for reverse flows to be taken into account in the model. Wilson, D., 2005. Location Modeling in Logistics, A decision Maker Defined Approach. In Advances and Applications for Management and Decision Making. In: Modelling and Simulation Society, International Congress on Modelling and Simulation MODSIM. Melbourne, Australia, 13 December 2005. Melbourne: Modelling and Simulation Society of Australia and New Zealand Inc. References Melbourne: Modelling and Simulation Society of Australia and New Zealand Inc. References Chwif, L., Barretto, M. and Saliby, E., 2002. Supply Chain Analysis: Spreadsheet or Simulation?. In: INFORMS Computing Society, Winter Simulation Conference. San Diego, U.S.A., 08-11 December 2002. San Diego: WSC. Constanza, R., Sklar, F. and White, M., 1990. Modeling Coastal Landscape Dynamics. Bioscience, [e-journal] 40(2). Available through: JSTOR website <http://www.jstor.org/stable/1311342> [Accessed 11 April 2016]. Di Gangi, M., Montella, B. and Russo, F., 1994. Mode choice models for freight transportation: the Italian market. IATBR: Valle Nevado, Santiago. Nuzzolo, A. and Russo F., 1998. A logistic approach for freight modal choice model. In: PTRC (Planning and Transport Research and Computation), European Transportation Forum. London, U.K, 14-18 September 1998. London: PTRC. Phipps, M., 1989. Dynamical Behavior of Cellular Automata under the Constraint of Neighborhood Coherence. Geographical Analysis, 21(3), pp. 197-215. Seidel, T., 2005. BTO Network Design Methodologies and Tools. [internal research paper] Berlin: Project ILIPT – Intelligent Logistics for Innovative Product Technologies. Sheikh, A.A., 2013. Evaluation of the potential locations for logistics hubs: A case study for a logistics company. DEd. KTH Royal Institute of Technology. Available at: <http://docplayer.net/13998244-Evaluation-of-the-potential-locations-for-logistics- hubs-a-case-study-for-a-logistics-company.html > [Accessed 22.03.2016]. Tobler, W.R., 1979. Cellular Geography. In: S. Gale, G. Olsson, ed. 1979. Philosophy in Geography. Dortrecht: Riedel. pp. 279-386. Trandafir, R., 2004. Modele și Algoritmi de Optimizare, Seria Matematica. București: AGIR. Ţarţavulea R.I., 2011, Strategii logistice internaționale. Modelarea proceselor decizionale logistice. Ph. D. Bucharest University of Economic Studies. Ţarţavulea R.I., 2015. Model for Determining the Optimum Location for Performance Improvement in Supply-Chain Strategies. European Journal of Interdisciplinary Studies, 7(1), pp. 39-54. Ţarţavulea R.I., Belu M.G. and Dieaconescu V.C., 2011. Spatial modeling in logistics decision-making processes. Identifying the optimal location for a single central warehouse. Annals of The University of Oradea, Economic Sciences Series, 20(1) pp. 137-143. Wikipedia, 2016. Lista orașelor din România. [online] Available at: <https://ro.wikipedia.org/wiki/Lista_ora%C8%99elor_din_Rom%C3%A2nia> [Accessed 05.03.2016]. 724 Amfiteatru Economic AE Amfiteatru Economic recommends Wilson, D., 2005. Location Modeling in Logistics, A decision Maker Defined Approach. In Advances and Applications for Management and Decision Making. In: Modelling and Simulation Society, International Congress on Modelling and Simulation MODSIM. Melbourne, Australia, 13 December 2005. Melbourne: Modelling and Simulation Society of Australia and New Zealand Inc. Wilson, D., 2005. Location Modeling in Logistics, A decision Maker Defined Approach. In Advances and Applications for Management and Decision Making. In: Modelling and Simulation Society, International Congress on Modelling and Simulation MODSIM. Melbourne, Australia, 13 December 2005. Appendix A Table A1: The input variables of the model for the case study on Romania (selection of first 5 cities from a total of 100) City Longitude Latitude Population Abrud 23.0833 46.3167 6803 Aiud 23.7333 46.3167 28909 Alba-Iulia 23.6500 46.1333 66369 Alexandria 25.4000 43.9500 50591 Arad 21.3333 46.1667 172827 Table A1: The input variables of the model for the case study on Romania (selection of first 5 cities from a total of 100) Appendix B Table B1: The results for placing ten logistic centers in Romania Optimal Location Latitude Longitude Number of served cities Number of served population DC1 45.4659 25.6257 6 444 873 D2 46.6467 23.4633 16 1 116 966 D3 44.4467 26.1733 11 2 731 976 D4 45.4467 27.9833 10 888 670 D5 46.8667 26.9733 15 1 198 212 D6 44.3567 23.8033 12 676 892 D7 46.2967 21.8033 6 559 115 D8 44.2367 28.6833 6 470 905 D9 44.8667 24.9033 8 470 046 D10 46.5167 24.6333 10 423 099 Vol. 18 • No. 43 • August 2016 725
https://openalex.org/W3195878018	https://europepmc.org/articles/pmc8561025?pdf=render	English	null	Corrigendum: Machine Learning-Based Analysis of Magnetic Resonance Radiomics for the Classification of Gliosarcoma and Glioblastoma	Frontiers in oncology	2,021	cc-by	737	CORRECTION published: 18 October 2021 doi: 10.3389/fonc.2021.774369 Keywords: gliosarcoma, glioblastoma, machine learning, radiomics, differentiation Approved by: Frontiers Editorial Ofﬁce, Frontiers Media SA, Switzerland Correspondence: Xuzhu Chen radiology888@aliyun.com †These authors share ﬁrst authorship Approved by: Frontiers Editorial Ofﬁce, Frontiers Media SA, Switzerland Corrigendum: Machine Learning- Based Analysis of Magnetic Resonance Radiomics for the Classiﬁcation of Gliosarcoma and Glioblastoma Zenghui Qian 1†, Lingling Zhang 2†, Jie Hu 1, Shuguang Chen 3, Hongyan Chen 2, Huicong Shen 2, Fei Zheng 2, Yuying Zang 2 and Xuzhu Chen 2 Zenghui Qian 1†, Lingling Zhang 2†, Jie Hu 1, Shuguang Chen 3, Hongyan Chen 2, Huicong Shen 2, Fei Zheng 2, Yuying Zang 2 and Xuzhu Chen 2* Zenghui Qian 1†, Lingling Zhang 2†, Jie Hu 1, Shuguang Chen 3, Hongyan Chen 2, Huicong Shen 2, Fei Zheng 2, Yuying Zang 2 and Xuzhu Chen 2* 1 Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, China, 2 Department of Radiology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China, 3 School of Mathematical Sciences, Nankai University, Tianjin, China A Corrigendum on Machine Learning-Based Analysis of Magnetic Resonance Radiomics for the Classiﬁcation of Gliosarcoma and Glioblastoma By Qian Z, Zhang L, Hu J, Chen S, Chen H, Shen H, Zheng F, Zang Y and Chen X (2021). Front. Oncol. 11:699789. doi: 10.3389/fonc.2021.699789 Specialty section: This article was submitted to Cancer Imaging and Image-directed Interventions, a section of the journal Frontiers in Oncology Received: 11 September 2021 Accepted: 04 October 2021 Published: 18 October 2021 Citation: Qian Z, Zhang L, Hu J, Chen S, Chen H, Shen H, Zheng F, Zang Y and Chen X (2021) Corrigendum: Machine Learning-Based Analysis of Magnetic Resonance Radiomics for the Classiﬁcation of Gliosarcoma and Glioblastoma. Front. Oncol. 11:774369. doi: 10.3389/fonc.2021.774369 Specialty section: This article was submitted to Cancer Imaging and Image-directed Interventions, a section of the journal Frontiers in Oncology Specialty section: This article was submitted to Cancer Imaging and Image-directed Interventions, a section of the journal Frontiers in Oncology In the published article, there was a mistake in the order of authors. The co-ﬁrst author was incorrectly written as the second author. The correct author list with the correct author order appears below. In the published article, there was a mistake in the order of authors. The co-ﬁrst author was incorrectly written as the second author. The correct author list with the correct author order appears below. “Zenghui Qian1†, Lingling Zhang2†, Jie Hu1, Shuguang Chen3, Hongyan Chen2, Huicong Shen2, Fei Zheng2, Yuying Zang2, Xuzhu Chen2*” Received: 11 September 2021 Accepted: 04 October 2021 Published: 18 October 2021 The authors apologize for this error and state that this does not change the scientiﬁc conclusions of the article in any way. The original article has been updated. Copyright © 2021 Qian, Zhang, Hu, Chen, Chen, Shen, Zheng, Zang and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. CORRECTION published: 18 October 2021 doi: 10.3389/fonc.2021.774369 Citation: Qian Z, Zhang L, Hu J, Chen S, Chen H, Shen H, Zheng F, Zang Y and Chen X (2021) Corrigendum: Machine Learning-Based Analysis of Magnetic Resonance Radiomics for the Classiﬁcation of Gliosarcoma and Glioblastoma. Front. Oncol. 11:774369. doi: 10.3389/fonc.2021.774369 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2021 Qian, Zhang, Hu, Chen, Chen, Shen, Zheng, Zang and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. October 2021 \| Volume 11 \| Article 774369 Frontiers in Oncology \| www.frontiersin.org
https://openalex.org/W4212885379	https://www.nature.com/articles/s41598-022-06870-9.pdf	English	null	Heteroscedastic sparse Gaussian process regression-based stochastic material model for plastic structural analysis	Scientific reports	2,022	cc-by	11,475	Heteroscedastic sparse Gaussian process regression‑based stochastic material model for plastic structural analysis OPEN Baixi Chen, Luming Shen* & Hao Zhang Describing the material flow stress and the associated uncertainty is essential for the plastic stochastic structural analysis. In this context, a data-driven approach-heteroscedastic sparse Gaussian process regression (HSGPR) with enhanced efficiency is introduced to model the material flow stress. Different from other machine learning approaches, e.g. artificial neural network (ANN), which only estimate the deterministic flow stress, the HSGPR model can capture the flow stress and its uncertainty simultaneously from the dataset. For validating the proposed model, the experimental data of the Al 6061 alloy is used here. Without setting a priori assumption on the mathematical expression, the proposed HSGPR-based flow stress model can produce a better prediction of the experimental stress data than the ANN model, the conventional GPR model, and Johnson Cook model at elevated temperatures. After the HSGPR-based flow stress model is implemented into finite element analysis, two numerical examples with synthetic material properties are performed to demonstrate the model’s capability in stochastic plastic structural analysis. The results have shown that with sufficient data, the distribution of the structural load carrying capacity at elevated temperatures and the variation of load–displacement curves during the loading and unloading processes can be accurately predicted by the HSGPR-based flow stress model. The flow stress behavior takes an important role in plastic structural analysis. Many constitutive models have been proposed to describe the flow stress of material and they can mainly be divided into two types, i.e., phenomeno- logical constitutive models and physics-based constitutive models. Phenomenological constitutive models include the Johnson Cook model1,2, Hockett-Sherby model3 and Arrhenius model4, etc. For physical-based constitu- tive modelling, there are Zerilli–Armstrong model5 and Rusinek–Klepaczko model6 etc. The above-mentioned conventional flow stress models could achieve good description in many situations, but the applications are limited by their mathematical expression. When there is a new alloy or a complex non-linear flow behavior, the original expression will become unreliable, and a new mathematical expression often needs to be developed. For example, Samantaray et.al7 modified the expression of the Zerilli–Armstrong model to make it suitable for the modified 9Cr-1Mo steel. However, the development of a new mathematical expression is non-trivial and can be time-consuming8. g To overcome the abovementioned limitations of the conventional constitutive approaches, machine learn- ing has been introduced to model the flow stress behavior by many researchers8–14. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ the same material will show different behavior under the same conditions. For example, Aakash et.al16 reported that the flow stress-plastic strain curve of Al6061 aluminum alloys had large variation even under the same temperature. Without considering the material uncertainty, the reliability of the constitutive model cannot be guaranteed, and the simulation given by this constitutive model can be unreliable. the same material will show different behavior under the same conditions. For example, Aakash et.al16 reported that the flow stress-plastic strain curve of Al6061 aluminum alloys had large variation even under the same temperature. Without considering the material uncertainty, the reliability of the constitutive model cannot be guaranteed, and the simulation given by this constitutive model can be unreliable. g g y By keeping the advantage of machine learning and taking the material uncertainty into account, a Bayesian based machine learning algorithm, Gaussian process regression (GPR), has been adopted to model material behavior17. Different from other machine learning methods only providing the deterministic estimation, the GPR model can capture both the underlying relation and corresponding uncertainty of the data simultaneously via the Bayesian approach18. In recent years, GPR model has achieved wide applications in several areas, includ- ing structural reliability analysis19, material strength estimation20, and deformation forecasting21. However, the time complexity of the conventional GPR model is the cube of the data points ( O n3 ). When more factors are included and the dataset become large, it will be hard to train and use the conventional GPR model. Hence, in order to improve the model efficiency, an enhanced GPR model, i.e. heteroscedastic sparse Gaussian process regression (HSGPR)22, is introduced to capture the flow stress behavior in the present work. Compared with the conventional GPR model, the HSGPR model uses the sparse technology to achieve higher efficiency and reduce the time complexity from O n3 to O nmd + nm222,23, where n is the number of samples, d is the dimension of the input and m is the number of the basis functions with d < < n and m < < n. Besides, different from the con- ventional GPR model, which usually uses the constant uncertainty model, the HSGPR model encodes a more advanced input-dependent uncertainty model in its structure. HSGPR‑based flow stress model l Basic theory of HSGPR. To achieve the sparse Gaussian process regression, the basis functions approach is used here22,23. As shown in the equations below, the output y is regarded as a linear combination of m nonlinear basis functions φ(x) of the inputs x: (1) y = φ(x)w+ ∈ (2) φ(x) = φ1(x), . . . , φj(x), . . . , φm(x) (3) φj(x) = exp −1 2 x −pj T ŴT j Ŵj x −pj (1) y = φ(x)w+ ∈ y = φ(x)w+ ∈ (1) (2) φ(x) = φ1(x), . . . , φj(x), . . . , φm(x) (2) (3) φj(x) = exp −1 2 x −pj T ŴT j Ŵj x −pj (3) where x ∈R1×d is the input vector and d is the dimension of the input, y ∈R1 is the output, φj(x) is the basis function. In this paper, the radial basis function kernel is chosen as the basis function for the HSGPR model and its expression is presented in Eq. (3). m is the number of the basis functions,w is the weight of basis functions and has the prior distribution N(0, A) where A is an m × m constant diagonal matrix and represents the covari- ance matrix of the prior distribution, pj ∈R1×d and Ŵj ∈Rd×d are the parameters of the basis function and Ŵj is a diagonal matrix. ∈ is the uncertainty of the output and is assumed to follow the normal distribution N 0, √β2 and β reflects the intensity of the uncertainty. To consider the input dependent uncertainty and enhancing the uncertainty estimation, the HSGPR model regards β as the linear combination of the nonlinear basis functions as given below. (4) β(x) = exp(φ(x)u + b) β(x) = exp(φ(x)u + b) (4) where φ(x) is same as the basis functions in Eq. (2), u is the weight of the basis functions and has the prior distribution N(0, N) where N is an m × m diagonal matrix, and b is the constant part of Eq. (4) and is used to consider the situation of constant uncertainty intensity. The exponential function used here is to make β positive. In terms of training dataset X∗, y∗ , X∗= x∗ i n i=1 ∈Rn×d is the n × d matrix storing n training inputs vectors and y∗= y∗ i n i=1 ∈Rn×1 is the vector storing n training outputs. www.nature.com/scientificreports/ The advanced uncertainty model enables the HSGPR model to produce more accurate uncertainty estimation than the conventional GPR model22.l p y In this paper, the HSGPR model which takes the temperature and plastic strain as input variable and flow stress as output variable is used to identify the stochastic temperature-dependent elasto-plastic behavior. Without loss of generality, the experimental data of the Al6061 aluminum alloy at elevated temperatures are used in the present work to verify the HSGPR-based flow stress model. The obtained HSGPR-based flow stress model is then compared with the Johnson Cook model, which was often used to describe the Al6061 aluminum alloy in the previous studies24–27, ANN model and the conventional GPR model. For the purpose of completeness, the methodology of numerically implementing the HSGPR model-based flow stress model into finite element analy- sis and stochastic plastic structural analysis is introduced in this work as well. Finally, two numerical examples with the synthetic flow stress data are used to demonstrate the capability of the HSGPR-based flow stress model in stochastic plastic structural analysis. Heteroscedastic sparse Gaussian process regression‑based stochastic material model for plastic structural analysis OPEN Quan et al.8 used one of the popular machine learning algorithms, the artificial neural network (ANN), to evaluate the flow stress of Al7050 aluminum alloy and the obtained ANN-based flow stress model showed higher accuracy than the Arrhenius model. Besides the ANN model, the support vector regression was also used by Tang et al. to predict the flow stress of Al2519 aluminum alloy15. The high agreement between the experimental data and the machine learning- based flow stress model was observed in the above-mentioned studies. Unlike the phenomenological constitutive models and physical-based constitutive models, machine learning-based flow stress models do not require any pre-postulated mathematical model and can predict the flow behavior more efficiently and more conveniently, as long as sufficient amount of experimental data are available8.l gfi p Although the machine learning based flow stress models showed good performance in the previous stud- ies, most of the existing machine learning based models are deterministic and do not consider the material uncertainty. Due to the manufacture error of the material and the variation of the chemical compositions, even \| https://doi.org/10.1038/s41598-022-06870-9 Scientific Reports \| (2022) 12:3017 www.nature.com/scientificreports/ HSGPR‑based flow stress model The log marginal likelihood lnp y∗ of the training data can be expressed as https://doi.org/10.1038/s41598-022-06870-9 Scientific Reports \| (2022) 12:3017 \| www.nature.com/scientificreports/ (5) ln p y∗ = −1 2 w −y∗TB w −y∗ + 1 2 ln \|B\| −n 2 ln 2π −1 2wTAw + 1 2 ln \|A\| −1 2 ln TB + A −1 2uTNu + +1 2 ln \|N\| −m 2 ln 2π (5) where = φ x∗ i n i=1 ∈Rn×m is the n × m matrix storing the basis functions for each training inputs, B is the n × n diagonal matrix with diagonal elements B(i.i) = β x∗ i and represents the uncertainty intensity at the training inputs X∗ , and w = TB + A −1TBy∗.hh where = φ x∗ i n i=1 ∈Rn×m is the n × m matrix storing the basis functions for each training inputs, B is the n × n diagonal matrix with diagonal elements B(i.i) = β x∗ i and represents the uncertainty intensity at the training inputs X∗ , and w = TB + A −1TBy∗.hh The parameters mentioned above can be determined by the training process. The training process is con- ducted by maximizing the log marginal likelihood of the training data. The optimization is achieved by using the quasi-Newton approach. After training the model, the parameters and weights in the basis functions can be updated to the optimal values. The output y at the unseen input x is normally distributed as N µ, δ2 with (6) µ = φ(x)w (7) δ2 = v + β (8) v = φ(x)−1φ(x)T (9) β = exp φ(x)u + b (6) (7) (9) where φ(x) is the updated basis functions; w , u and b are the updated parameters and = TB + A is a m × m matrix. µ and δ are the expectation and standard deviation of the prediction of the unseen input x. HSGPR‑based flow stress model. When the HSGPR model is used to model the material flow stress σp at elevated temperatures, the plastic strain εp and the temperature T (K) are used as the input variables and the flow stress σp (MPa) is used as the output variable. The number of basis functions can be determined by trial and error. A larger number of basis functions can give higher accuracy but will increase the model complexity. Based on Eq. HSGPR‑based flow stress model (6) to Eq. (9) and the Gaussian prior distribution assumed before, the obtained HSGPR-based stochastic flow stress model σp εp, T follows the Gaussian distribution with the mean of φ εp, T w and the variance of δ2 εp, T , as expressed below (10) σp εp, T = φ εp, T w + δ εp, T · R (10) (11) δ2 εp, T = φ εp, T φ εp, T T + exp φ εp, T u + b (11) where R is a standard normal distribution of N 0, 12 . The possible curve of the HSGPR based stochastic flow stress model can be sampled by sampling standard normal random variable R first and then substituting the sampled random variable into Eq. (10). The procedure of the HSGPR-based flow stress modelling is illustrated as a flow chart in Fig. 1. Model evaluation. To evaluate the accuracy of the obtained flow stress models, the average absolute relative error (AARE) is used here. The model with lower AARE has higher accuracy.h h g y The expression of AARE is presented below: (12) AARE = 1 N N i=1 σ exp pi −σ pre pi σ exp pi (12) where σ exp pi and σ pre pi are the ith experimental flow stress and the ith predicted mean flow stress of the model respectively, and N is the number of the data.l To evaluate the uncertainty of the flow stress predicted by the models, the mean log likelihood (MLL) of the experimental data is introduced here. The MLL reflects the average likelihood of obtaining the experimental data under the given model. The model with a higher MLL means that it is more likely to reproduce the data under the given model and the material uncertainty predicted by this model has higher accuracy. Th f b l g y p y g y The expression of MLL is given below: g y p y g y The expression of MLL is given below: g y p y g The expression of MLL is given below: g The expression of MLL is given below: (13) MLL = 1 N N i=1   − σ exp pi −σ pre pi 2 2δ2 i −1 2 ln δ2 i   −1 2 ln (2π) (13) where δi is the standard deviation of the ith flow stress predicted by the model. where δi is the standard deviation of the ith flow stress predicted by the model. Application for material modelling pp g Material data. Without loss of generality, the experimental flow stress-plastic strain data of Al6061 alu- minum alloy from the literature28 are applied in the present work. There were six temperatures (20, 100, 150, 200, 250, 300 °C) considered in the experiments. The experiment data obtained by the uniaxial tension test con- ducted on a standard “dog bone” type coupon by MTS 810 (10-kip capacity). Design of the uniaxial tension test followed the guidelines in ASTM E8. A total of 98 stress–strain curves (totally 24,011 data points) are used here. The detailed breakdown of the obtained curves is illustrated in Table 1 and all data curves are presented in Fig. 2.lff h p g Kolmogorov–Smirnov hypothesis test is conducted on the flow stress data at different temperature and differ- ent plastic strain to examine if the flow stress follows the Gaussian distribution. The p-values of the hypothesis test on the flow stress are presented in Table 2. As demonstrated in Table 2, the test does not reject the hypothesis, indicating that the flow stress obeys the Gaussian distribution, at the 5% significance level. Hence, it is reasonable to use the normal distribution for the uncertainty ∈ in the HSGPR model and Eq. (10) to describe the stochastic behavior of the flow stress. l In addition, the experimental data at temperature of 250 °C are used as the testing dataset for the HSGPR model, while the rest of data (21,523 data points) are used as the training dataset for the ANN model and HSGPR model and conventional GPR model. HSGPR‑based flow stress model where δi is the standard deviation of the ith flow stress predicted by the model. where δi is the standard deviation of the ith flow stress predicted by the model. https://doi.org/10.1038/s41598-022-06870-9 https://doi.org/10.1038/s41598-022-06870-9 Scientific Reports \| (2022) 12:3017 \| www.nature.com/scientificreports/ ature.com/scientificreports/ Figure 1. The flow chart of establishing HSGPR-based stochastic flow stress model. p Figure 1. The flow chart of establishing HSGPR-based stochastic flow stress model. igure 1. The flow chart of establishing HSGPR-based stochastic flow stress model. Figure 1. The flow chart of establishing HSGPR-based stochastic flow stress model. Table 1. Number of the stress–strain curves for each lot and each temperature. Temperature (°C) 20 100 150 200 250 300 Number of curves 19 18 12 18 12 19 Number of data points 4513 5194 3910 4168 2488 3738 Table 1. Number of the stress–strain curves for each lot and each temperature. Results and discussion Four models, namely, the Johnson Cook (JC) model29, the ANN model, the HSGPR model, and the conventio GPR model are used to model the flow stress of the Al6061 aluminum alloys at elevated temperatures.hll l y p The Johnson–Cook flow stress model was used by many researchers to model the flow behavior of Al6 aluminum24–27. The formulation of JC model is expressed in Eq. (14). Scientific Reports \| (2022) 12:3017 \| https://doi.org/10.1038/s41598-022-06870-9 www.nature.com/scientificreports/ Figure 2. The experimental flow stress-plastic strain data of Al6061 aluminum alloys at the temperature of (a) 20 °C, (b) 100 °C, (c) 150 °C, (d) 200 °C, (e) 250 °C and (f) 300°C28. Figure 2. The experimental flow stress-plastic strain data of Al6061 aluminum alloys at the temperature of ( 20 °C, (b) 100 °C, (c) 150 °C, (d) 200 °C, (e) 250 °C and (f) 300°C28. Table 2. The P-value of the Kolmogorov–Smirnov hypothesis test on the flow stress data at various temperatures and plastic strains. P-value Temperature (°C) 20 100 150 200 250 300 Plastic strain εp 0.00 0.61 0.92 0.84 0.57 0.99 0.79 0.01 0.51 0.52 0.95 0.78 0.94 0.85 0.02 0.65 0.76 0.93 0.99 0.98 0.92 0.03 0.62 0.89 0.94 0.98 0.98 0.95 0.04 0.58 0.98 0.60 0.96 0.99 0.79 0.05 0.54 0.98 0.52 0.62 0.96 0.71 Table 2. The P-value of the Kolmogorov–Smirnov hypothesis test on the flow stress data at various temperatures and plastic strains. P-value Temperature (°C) 20 100 150 200 250 300 Plastic strain εp 0.00 0.61 0.92 0.84 0.57 0.99 0.79 0.01 0.51 0.52 0.95 0.78 0.94 0.85 0.02 0.65 0.76 0.93 0.99 0.98 0.92 0.03 0.62 0.89 0.94 0.98 0.98 0.95 0.04 0.58 0.98 0.60 0.96 0.99 0.79 0.05 0.54 0.98 0.52 0.62 0.96 0.71 Table 2. The P-value of the Kolmogorov–Smirnov hypothesis test on the flow stress data at various temperatures and plastic strains. Table 2. The P-value of the Kolmogorov–Smirnov hypothesis test on the flow stress data at various temperatures and plastic strains. https://doi.org/10.1038/s41598-022-06870-9 Scientific Reports \| (2022) 12:3017 \| www.nature.com/scientificreports/ ntificreports/ Figure 3. The relationship between the AARE on the testing dataset and the number of basis functions m. Figure 3. The relationship between the AARE on the testing dataset and the number of basis functions m. Results and discussion (14) σp = c1 + c2εc3 p 1 − T −Tr Tm −Tr c4 (14) where σp (MPa) is the flow stress, εp is the plastic strain, T (K) is the temperature of material, c1 , c2 , c3 and c4 are the model parameters, Tr is the room temperature of 293.15 K (i.e. 20 °C) and Tm is the melt temperature of 925 K for the Al6061 aluminum alloy.h The parameters c1 , c2 , c3 of JC model are calibrated by the stress–strain curves at room temperature (i.e. 20 °C) first. The Pearson correlation coefficients between c1 and c2 , and between c1 and c3 are -0.002 and -0.021 respectively, which means that the correlation is negligible and c1 is independent of c2 and c330. As the Pear- son correlation coefficients between c2 and c3 is 0.96, c2 and c3 are assumed to be linearly correlated with each other here. By conducting Kolmogorov–Smirnov test, the parameters c1,c2 and c3 follow normal distribution N 260.72, 17.322 MPa , N 504.26, 106.992 MPa and N 0.82, 0.142 respectively at the 5% significance level. The parameter c4 is calibrated by the data of zero plastic strain. c4 obeys the normal distribution N 1.277, 0.0692 at the 5% significance level.hl gi The HSGPR-based flow stress model for the Al6061 aluminum alloys is established by the approach described in “Section HSGPR-based flow stress model”. In order to determine the suitable number of basis functions m, the relationship between the AARE on the testing dataset and m is drawn in Fig. 3. It can be observed from Fig. 3 that the improvement of the model accuracy is negligible when m is larger than 10. Hence, 10 basis functions are sufficient to produce a good estimate and more basis functions will only increase the model complexity without obvious contribution to the model accuracy. For the sake of the model efficiency and accuracy, m = 10 is adopted in the rest of the paper. With m = 10, the optimal parameters of the HSGPR model can be obtained by the training process and presented in “Appendix A” of Supplementary Information.h p p pp pp y The conventional GPR model and the ANN model are established by using the MATLAB toolbox as well for comparison. Results and discussion For the GPR model, the squared exponential kernel function is used as the kernel function, and the subset of the data points approximation is set as the fitting method to address the efficiency issue. The quasi-Newton method is used to optimize the parameters in the GPR model and the default value in MATLAB is used for all other settings of the GPR model. The optimal parameters of the GPR model (i.e., the signal stand- ard deviation, the characteristic length scales and the noise standard deviation) are 228.8175, 244.4820 and 17.8537 respectively. The ANN structure is adopted from literature9 directly. As demonstrated in the reference9, the network with one hidden layer consisting of 20 nodes is the suitable setting for estimating the flow stress.ll l Since the flow stress model needs to be called to predict the flow stress at each integration point and each increment step in the finite element analysis (FEA), the prediction time of the data-driven flow stress model will significantly influence the efficiency of the model in the FEA. The prediction time of the ANN model, HSGPR model and GPR model for 10,000 input points in the MATLAB platform are 0.019 s, 0.011 s and 26.78 s, respectively. Compared with the conventional GPR model, the HSGPR model and ANN model have lower time consumption during the prediction, which means they have higher efficiency in the FEA.h p g p y gfi y The AARE and MLL of the three machine learning models (namely, the HSGPR model, GPR model and ANN model) on the training and testing dataset are presented in Table 3. Since only deterministic prediction is given by the ANN model, only the AARE of the ANN model is calculated. The ANN model, HSGPR model and GPR model have similar AARE on the training stage, while the performance of ANN model on the testing dataset is the worst among the three models with the AARE of 12.4%. In addition, with the advanced uncertainty model, the HSGPR model presents obviously larger MLL and smaller AARE than the GPR model on the testing dataset. The larger MLL means the testing dataset is more likely to be reproduced by the HSGPR model than the GPR model. g g y p y In order to further evaluate the generalization ability of the three machine learning approaches, the cross- validation procedure is performed here as well. Results and discussion The data points at one selected temperature are excluded for validation, while the rest of the data are used for training. Hence, there are totally 6 cross-validation tests. The Scientific Reports \| (2022) 12:3017 \| https://doi.org/10.1038/s41598-022-06870-9 www.nature.com/scientificreports/ Table 3. AARE and MLL of ANN model, HSGPR-based flow stress model and GPR-based flow stress model on the training and testing dataset. Dataset Model AARE (%) MLL Training HSGPR 5.2 − 4.04 GPR 5.7 − 4.23 ANN 5.2 – Testing HSGPR 6.5 − 3.74 GPR 8.7 − 7.08 ANN 12.4 – Table 3. AARE and MLL of ANN model, HSGPR-based flow stress model and GPR-based flow stress model on the training and testing dataset. Table 3. AARE and MLL of ANN model, HSGPR-based flow stress model and GPR-based flow stress mod on the training and testing dataset. Table 3. AARE and MLL of ANN model, HSGPR-based flow stress model and GPR-based flow stress model on the training and testing dataset. Table 4. AARE of ANN-based flow stress model, HSGPR-based flow stress model, GPR-based flow stress model on cross validation set. Validation No Excluded temperature for validation (°C) AARE (%) HSGPR GPR ANN 1 20 9.9 30.0 17.2 2 100 8.6 8.7 8.5 3 150 4.2 4.4 7.2 4 200 4.5 6.6 4.5 5 250 6.5 8.7 12.4 6 300 8.1 33.1 23.6 Table 4. AARE of ANN-based flow stress model, HSGPR-based flow stress model, GPR-based flow stress model on cross validation set. Table 5. AARE and MLL of the ANN model, HSGPR-based flow stress model, GPR-based flow stress model and JC model on the whole dataset. Model AARE (%) MLL HSGPR 5.3 − 4.01 GPR 6.0 − 4.21 ANN 6.0 – JC 31.1 − 10.18 Model AARE (%) MLL HSGPR 5.3 − 4.01 GPR 6.0 − 4.21 ANN 6.0 – JC 31.1 − 10.18 Table 5. AARE and MLL of the ANN model, HSGPR-based flow stress model, GPR-based flow stress model and JC model on the whole dataset. Table 5. AARE and MLL of the ANN model, HSGPR-based flow stress model, GPR-based flow stress model and JC model on the whole dataset. AARE of the three models on the excluded validation set is demonstrated in Table 4. In the validation test 1 and 6, the validation temperature is not covered by the training dataset, so the model extrapolation ability can be tested. Results and discussion Notably, the HSGPR model have superior accuracy over the GPR model and ANN model on extrapola- tion. Besides, the performance of the HSGPR model on the interpolation validation set (No. 2–5) is also better than the other two models. It can be concluded that the HSGPR model has better generalization ability than the conventional GPR model and ANN model on flow stress prediction. l As a conventional constitutive model, the JC model is directly evaluated using the whole dataset (including all the experimental data). For comparison, the three machine learning approaches are also evaluated on the whole dataset and the results are listed on Table 5. Due to the limitation of the mathematical expression, the performance of the JC model is worse than the machine learning approach. The HSGPR model has the smallest AARE and the largest MLL among all the models, which means that the HSGPR model shows better performance than the other models.i As for the material uncertainty predicted by the models, it can be visualized by the 95% confidence region (i.e. mean ± 2 × standard deviation ) and the coefficient of variation (COV, i.e. standard deviation/mean ) of the flow stress model and is plotted in Figs. 4, 5, 6, 7. Since the ANN cannot provide the material uncertainty, only the HSGPR-based flow stress model, GPR-based flow stress model and JC model are plotted and discussed in the next.i At the low temperatures (20 °C to 150 °C), it can be observed from Figs. 4, 5, 6, 7 that the 95% confidence region given by the three models could cover most of the data at low temperatures. Besides, as shown in Fig. 4, the COVs given by the three models are similar and are close to the COV given by the experimental data at the temperatures of 20 °C to 150 °C. At the high temperatures (200 °C to 300 °C), the mean curves and material uncertainty estimated by the HSGPR model are consistent with the experimental data, while the material uncertainty is overestimated by the GPR model and the COV given by GPR model is much larger than the experimental value. It is notable to mention that the advanced uncertainty model within HSGPR model results in the better uncertainty estimation Scientific Reports \| (2022) 12:3017 \| https://doi.org/10.1038/s41598-022-06870-9 www.nature.com/scientificreports/ Figure 4. Results and discussion The obtained HSGPR-based flow stress model, GPR-based flow stress model and JC model with respect to the temperatures at the plastic strains of 0.02 and 0.04. (a) HSGPR model at the plastic strains of 0.02, (b) HSGPR model at the plastic strains of 0.04, (c) GPR model at the plastic strains of 0.02, (d) GPR model at the plastic strains of 0.04, (e) JC model at the plastic strains of 0.02, (f) JC model at the plastic strains of 0.04. Figure 4. The obtained HSGPR-based flow stress model, GPR-based flow stress model and JC model with respect to the temperatures at the plastic strains of 0.02 and 0.04. (a) HSGPR model at the plastic strains of 0.02, (b) HSGPR model at the plastic strains of 0.04, (c) GPR model at the plastic strains of 0.02, (d) GPR model at the plastic strains of 0.04, (e) JC model at the plastic strains of 0.02, (f) JC model at the plastic strains of 0.04. than the GPR model. In addition, as limited by its mathematic expression, the JC model cannot describe the severe temperature softening of Al6061 alloys at high temperatures. As shown in Fig. 7, the estimated flow stress given by the JC model deviates from the experiment data at high temperature, which causes the high AARE of the JC model in Table 5. The poor performance of the JC model for the Al6061 alloy at high temperatures is consistent with the results in the work of Fan et.al.25. Although it is possible to modify the JC model to improve the accuracy, those modifications are material-dependent and need to postulate the mathematical expression as well31. Establishing a new mathematic expression of the modified JC model for each new material is always a time-consuming work and sometimes a suitable modification is hard to find. Different from the JC model, the HSGPR-based flow stress model is a material-independent data-driven flow stress model. Without postulating the specific mathematical form, the HSGPR framework can be used to establish a flow stress model directly from the material data with high accuracy. g y In addition, it should be admitted that the current HSGPR based model use the uncertainty model ∈ with the normal distribution to approximate the uncertainty of material data. If the data deviated from the normal distribution, the uncertainty ∈ with other distribution needs to be used. Results and discussion The new distribution of ∈ can be selected by trial and error, and the distribution, which can produce the maximum MLL, is regarded as the suitable one. https://doi.org/10.1038/s41598-022-06870-9 Scientific Reports \| (2022) 12:3017 \| www.nature.com/scientificreports/ / p / Figure 5. The flow stress obtained using the HSGPR-based model with respect to the plastic strain at the temperature of (a) 20 °C, (b) 100 °C, (c) 150 °C, (d) 200 °C, (e) 250 °C and (f) 300 °C. Figure 5. The flow stress obtained using the HSGPR-based model with respect to the plastic strain at the temperature of (a) 20 °C, (b) 100 °C, (c) 150 °C, (d) 200 °C, (e) 250 °C and (f) 300 °C. Application in plastic stochastic structural analysis S h SG b d h fl d l d d Application in plastic stochastic structural analysis Since the HSGPR-based stochastic flow stress model is introduced for the first time in the current work and has not been implemented in the finite element analysis (FEA) for plastic stochastic structural analysis previ- ously, the numerical implementation of the HSGPR-based flow stress model into the FEA for plastic stochastic structural analysis is proposed in this section. And the model is then validated by two numerical examples with the synthetic material data. Numerical implementation in FEA. The obtained HSGPR-based stochastic flow stress model can be combined with the von Mises yield criteria to describe the plastic behavior of the material in the FEA. The yield function with the HSGPR-based flow stress model can be expressed as Scientific Reports \| (2022) 12:3017 \| https://doi.org/10.1038/s41598-022-06870-9 www.nature.com/scientificreports/ (15) f σ, εp, T = σ −σp εp, T (16) σ = (σ11 −σ22)2 + (σ33 −σ22)2 + (σ11 −σ33)2 + 6 σ 2 12 + σ 2 13 + σ 2 23 Figure 6. The flow stress obtained using GPR-based model with respect to the plastic strain at the temperature of (a) 20 °C, (b) 100 °C, (c) 150 °C, (d) 200 °C, (e) 250 °C and (f) 300 °C. Figure 6. The flow stress obtained using GPR-based model with respect to the plastic strain at the temperature of (a) 20 °C, (b) 100 °C, (c) 150 °C, (d) 200 °C, (e) 250 °C and (f) 300 °C. (15) f σ, εp, T = σ −σp εp, T (15) f σ, εp, T = σ −σp εp, T (16) + (σ33 −σ22)2 + (σ11 −σ33)2 + 6 σ 2 12 + σ 2 13 + σ 2 23 2 (15) f σ, εp, T = σ −σp εp, T (16) σ = (σ11 −σ22)2 + (σ33 −σ22)2 + (σ11 −σ33)2 + 6 σ 2 12 + σ 2 13 + σ 2 23 2 (15) (16) σ = (σ11 −σ22)2 + (σ33 −σ22)2 + (σ11 −σ33)2 + 6 σ 2 12 + σ 2 13 + σ 2 23 2 (16) where σ is the equivalent von Mises stress, σp is the HSGPR-based flow stress, εp is the von Mises equivalent strain, T is temperature, and [σ11, σ22, σ33, σ12, σ13.σ23] is the Cauchy stress vector. https://doi.org/10.1038/s41598-022-06870-9 Scientific Reports \| (2022) 12:3017 \| www.nature.com/scientificreports/ om/scientificreports/ Figure 7. Application in plastic stochastic structural analysis S h SG b d h fl d l d d The flow stress obtained using the JC model with respect to the plastic strain at the temperature of (a) 20 °C, (b) 100 °C, (c) 150 °C, (d) 200 °C, (e) 250 °C and (f) 300 °C. Figure 7. The flow stress obtained using the JC model with respect to the plastic strain at the temperature of 20 °C, (b) 100 °C, (c) 150 °C, (d) 200 °C, (e) 250 °C and (f) 300 °C. The yield function with the HSGPR-based flow stress model is called at each increment of the FEA to check the yield state of each element integration point and update the flow stresses of the element integration points which have reached plasticity. In order to update the flow stresses, the stiffness of the HSGPR-based flow stress model, that is the gradient of the flow stress model σp with respect to the equivalent plastic strain εp and tem- perature T , respectively, is queried in the FEA32. The corresponding stiffness (i.e. ∂σp/∂εp and ∂σp/∂T ) of the HSGPR-based flow stress model is expressed as: (17) ∂σp ∂εp = ∂φ εp, T ∂εp w + ∂δ εp, T ∂εp · N 0, 12 (17) https://doi.org/10.1038/s41598-022-06870-9 https://doi.org/10.1038/s41598-022-06870-9 Scientific Reports \| (2022) 12:3017 \| www.nature.com/scientificreports/ Figure 8. Flowchart of the stochastic plastic structural analysis steps with the HSGPR-based model. Figure 8. Flowchart of the stochastic plastic structural analysis steps with the HSGPR-based model. (18) ∂σp ∂T = ∂φ εp, T ∂T w + ∂δ εp, T ∂T · N 0, 12 (18) where the detailed expressions and inferences of the gradients ∂φ εp, T /∂εp, ∂φ εp, T /∂T, ∂δ εp, T /∂εp and ∂δ εp, T /∂T are presented in “Appendix B” of the Supplementary Information.hit 33 This procedure can be achieved in the general-purpose finite element software Abaqus33 via its subroutine UHARD. The numerically implemented HSGPR-based stochastic flow stress model can then be used to solve the temperature-dependent plastic problems. Stochastic plastic structural analysis procedure. The Monte Carlo Simulation (MCS) method is applied to achieve the stochastic plastic structural analysis using the HSGPR-based model. The possible flow stress relations estimated by the HSGPR model are generated by following the flow chart in Fig. 1 and samples of other possible basic random variables, such as elastic modulus (E), can also be generated according to their prob- ability distribution. Application in plastic stochastic structural analysis S h SG b d h fl d l d d Boundary condition, loading condition, geometrical size (mm), and mesh distribution of the example model. Figure 10. Four different mesh size strategies for the FEA model of the beam. (a) 10 mm mesh, (b) 5 mm mesh, (c) 1 mm mesh, (d) 0.5 mm mesh. Figure 10 Four different mesh size strategies for the FEA model of the beam (a) 10 mm mesh (b) 5 mm Figure 10. Four different mesh size strategies for the FEA model of the beam. (a) 10 mm mesh, (b) 5 mm mesh, (c) 1 mm mesh, (d) 0.5 mm mesh. (19) ET = 1 −T −Tr 1600 E0 ET = 1 −T −Tr 1600 E0 ET = 1 −T −Tr 1600 E0 (19) where T (K) is the temperature, Tr is the room temperature 293.15 K and the room temperature elastic modulus E0 is a normal random variable with a mean of 69,500 MPa and a standard deviation of 8,548.5 MPa35.tl After the training process, the HSGPR-based flow stress model is implemented into the FEA and the model is validated by the beam problem adopted from36. The geometry, boundary conditions and loading conditions of the beam are depicted in Fig. 9 and the thickness of the beam is 35 mm. The failure strain is set as 0.116. Except for the elastic modulus and flow stress, other uncertainty sources are not taken into account in the current validation process. Abaqus is used to conduct the finite element analysis. The displacement-controlled loading is applied at the middle point of the beam model. The mesh convergence study is conducted with all the parameters being set to their mean values. The linear 4-node doubly curved shell element (S4R) is used in the Abaqus model. As depicted in Fig. 10, four different mesh size strategies are studied in the present work. The load–displacement curve of each case is presented in Fig. 11. The relative errors of the load capacity (i.e. peak load) of the cases with 10 mm, 5 mm and 1 mm mesh relative to the case with 0.5 mm mesh are 1.62%, 0.35% and 0.32% respectively. The case with 5 mm mesh, which could provide sufficient accuracy for estimating the load capacity and acceptable computational efficiency, is used in the current study. The mesh distribution of the beam is presented in Fig. 9. Application in plastic stochastic structural analysis S h SG b d h fl d l d d For each sample, a finite element model is set up and a complete analysis is performed using Abaqus. The procedure of performing the plastic stochastic structural analysis with the HSGPR-based model is presented schematically in Fig. 8. During each iteration, the sampled flow stress and other possible basic random variables are used to update the corresponding value in the Abaqus input files. The number of the MCS, which can result in a converged estimation, is applied in the procedure for the stochastic structural analysis. Numerical examples. Beam problem. As a data-driven material model, the HSGPR-based flow stress model should be compatible with both well-known and unknow (or new) constitutive laws. Here, a well-known flow stress model will be used to validate the developed HSGPR-based flow stress model in the stochastic plastic structural analysis. Instead of using the experimental data, the training data for this example are obtained from the Abaqus built-in Johnson Cook model (Eq. 14), which is the target for the HSGPR-based flow stress model to learn. Tr and Tm of the Johnson Cook model are the deterministic values of 293.15 K and 925 K respectively. The other parameters of the Johnson Cook model are set as the normal random variables to include the effect of the material uncertainty. The means of the parameters c1 , c2 , c3 and c4 of the JC model are 324 MPa, 114 MPa, 0.42 and 1.34, respectively34. The coefficients of variation (COV) of four parameters are set as 0.05, 0.10 and 0.15 to consider three different uncertainty levels. Six temperatures (20, 100, 150, 200, 250, 300 °C) are considered in the training dataset. For each temperature, 20 stress–strain curves (24,000 data points), 100 stress–strain curves (120,000 data points), and 200 stress–strain curves (240,000 data points) are generated for studying the effect of the dataset size. Hence, there are totally nine synthetic flow stress datasets with three uncertainty levels and three dataset sizes generated for training the HSGPR model. The Poisson ratio of the material is set as 0.33 and the elastic modulus is set as a temperature-dependent normal random variable35 and is expressed as https://doi.org/10.1038/s41598-022-06870-9 Scientific Reports \| (2022) 12:3017 \| www.nature.com/scientificreports/ Figure 9. Boundary condition, loading condition, geometrical size (mm), and mesh distribution of the example model. igure 9. Boundary condition, loading condition, geometrical size (mm), and mesh distribution of the example model. Figure 9. Application in plastic stochastic structural analysis S h SG b d h fl d l d d The expectation and standard deviation evaluated by the HSGPR model at the different number of MCS. (Temperature = 175 °C, COV = 0.10, the number of data curves = 100). Figure 12. The expectation and standard deviation evaluated by the HSGPR model at the different number of MCS. (Temperature = 175 °C, COV = 0.10, the number of data curves = 100). than 3%. As presented in Tables 6 and 7, the effect of temperature on the accuracy of the HSGPR model is not obvious at all uncertainty levels, which means that the HSGPR-based flow stress model is suitable for the sto- chastic plastic structural analysis at elevated temperatures with sufficient dataset. In addition, it is worth noting that the HSGPR model can give good estimation on the load capacity at the temperature of 175 °C which is not considered in the synthetic dataset. than 3%. As presented in Tables 6 and 7, the effect of temperature on the accuracy of the HSGPR model is not obvious at all uncertainty levels, which means that the HSGPR-based flow stress model is suitable for the sto- chastic plastic structural analysis at elevated temperatures with sufficient dataset. In addition, it is worth noting that the HSGPR model can give good estimation on the load capacity at the temperature of 175 °C which is not considered in the synthetic dataset. Punch problem. To evaluate the performance of the HSGPR-based model in the loading and unloading pro- cess, the punch problem given by Huang et. al.37 is used as the benchmark test. The exponential flow stress law σp = e1 + e1(e2 + εp)e3 is set as the target model. To consider the material uncertainty, the parameters e1 , e2 and e3 are set as the normal random variables with the mean of 0.05 MPa, 0.00002 and 0.3 respectively and the COV of 0.05. A total of 200 stress–strain curves (40,000 data points) at the room temperature are generated for training the HSGPR model. The Poison ratio is set as 0.33 and the elastic modulus is set as the normal random variable with the mean of 1 MPa and the standard deviation of 0.05 MPa.hlt The HSGPR-based flow stress model obtained after the training process is evaluated by the punch problem37. The geometry of the problem is depicted in Fig. 14. Application in plastic stochastic structural analysis S h SG b d h fl d l d d fih In order to determine the number of MCS, the stochastic structural capacity of the beam is evaluated by the HSGPR model at the different number of iterations. The expectation and standard deviation of the structural capacity with respect to the number of MCS is presented in Fig. 12. It can be noted from the figure that both the expectation and standard deviation given by the MCS converge at the number of 1000. Hence, the number of MCS is set as 1000 for the structure analysis.hl The probability density distributions of the structural load capacities given by the HSGPR-based flow stress model and the targe model at the room temperature and the elevated temperatures are presented in Fig. 13. The corresponding mean and standard deviation of the load capacities are illustrated in Tables 6 and 7, respectively.l It can be observed from Table 6 that the expected capacity given by the HSGPR-based flow stress model have high accuracy with the relative error smaller than 4% even under the high uncertainty level and small dataset size. When the dataset size is large enough, the influence of increasing dataset has little effect on the accuracy of expected capacity. The slight error variation may be due to the model itself. For the variability of the load capacity, the probability density distribution obtained by the HSGPR model coincides with the distribution obtained by the target model at the low uncertainty level (COV = 0.05), as shown in Fig. 13. For the high uncertainty level, the HSGPR model requires larger dataset size to achieve good estimation of the COV of load capacity. With the sufficient material data, the relative error of the standard deviation given by the HSGPR model is smaller Scientific Reports \| (2022) 12:3017 \| https://doi.org/10.1038/s41598-022-06870-9 www.nature.com/scientificreports/ entificreports/ Figure 11. The load–displacement curves of four mesh strategies. Figure 12. The expectation and standard deviation evaluated by the HSGPR model at the different number of MCS. (Temperature = 175 °C, COV = 0.10, the number of data curves = 100). Figure 11. The load–displacement curves of four mesh strategies. Figure 11. The load–displacement curves of four mesh strategies. Figure 11. The load–displacement curves of four mesh strategies. Figure 12. The expectation and standard deviation evaluated by the HSGPR model at the different number of MCS. (Temperature = 175 °C, COV = 0.10, the number of data curves = 100). Figure 12. www.nature.com/scientificreports/ www.nature.com/scientificreports/ h Figure 13. The probability density distribution of the structural capacity given by the HSGPR-based flow stress model and the target model at different dataset size, temperatures and coefficient of variation (COV). (a) COV = 0.05, T = 20 °C, (b) COV = 0.05, T = 175 °C, (c) COV = 0.05, T = 300 °C, (d) COV = 0.10, T = 20 °C, (e) COV = 0.10, T = 175 °C, (f) COV = 0.10, T = 300 °C, (g) COV = 0.15, T = 20 °C, (h) COV = 0.15, T = 175 °C, (i) COV = 0.15, T = 300 °C. Figure 13. The probability density distribution of the structural capacity given by the HSGPR-based flow stress model and the target model at different dataset size, temperatures and coefficient of variation (COV). (a) COV = 0.05, T = 20 °C, (b) COV = 0.05, T = 175 °C, (c) COV = 0.05, T = 300 °C, (d) COV = 0.10, T = 20 °C, (e) COV = 0.10, T = 175 °C, (f) COV = 0.10, T = 300 °C, (g) COV = 0.15, T = 20 °C, (h) COV = 0.15, T = 175 °C, (i) COV = 0.15, T = 300 °C. Figure 13. The probability density distribution of the structural capacity given by the HSGPR-based flow stress model and the target model at different dataset size, temperatures and coefficient of variation (COV). (a) COV = 0.05, T = 20 °C, (b) COV = 0.05, T = 175 °C, (c) COV = 0.05, T = 300 °C, (d) COV = 0.10, T = 20 °C, (e) COV = 0.10, T = 175 °C, (f) COV = 0.10, T = 300 °C, (g) COV = 0.15, T = 20 °C, (h) COV = 0.15, T = 175 °C, (i) COV = 0.15, T = 300 °C. For the deterministic analysis, the obtained load–displacement curve is plotted in Fig. 15. The load displace- ment simulated by using the feed forward neural network (FNN)-based plasticity model by Huang et al.37 is also presented in Fig. 15. It can be observed from Fig. 15 that the HSGPR model and FNN model given by Huang et al.37 have the similar performance with both being close to the target model during the loading and unloading processes. And the average relative error of the HSGPR model is 0.75%. www.nature.com/scientificreports/ However, unlike the HSGPR model, the FNN model cannot capture the material uncertainty and be used in the stochastic plastic structural analysis.fi y , p p gh p ment simulated by using the feed forward neural network (FNN)-based plasticity model by Huang et al.37 is also presented in Fig. 15. It can be observed from Fig. 15 that the HSGPR model and FNN model given by Huang et al.37 have the similar performance with both being close to the target model during the loading and unloading processes. And the average relative error of the HSGPR model is 0.75%. However, unlike the HSGPR model, the FNN model cannot capture the material uncertainty and be used in the stochastic plastic structural analysis. Similar to the beam problem, 1000 Monte Carlo simulations are sufficient to provide the converged estimation for the punch problem as well. By conducting 1000 Monte Carlo simulations using the HSGPR model, the 95% confidence region (i.e. mean ± 2 × standard deviation ) of the load–displacement curve can be obtained and is plotted in Fig. 16. As depicted in Fig. 16, the confidence region given by the HSGPR model is in agreement with the target model and the variation of the load–displacement curve is depicted accurately by the HSGPR model in both the loading and unloading stages.l p y p y Similar to the beam problem, 1000 Monte Carlo simulations are sufficient to provide the converged estimation for the punch problem as well. By conducting 1000 Monte Carlo simulations using the HSGPR model, the 95% confidence region (i.e. mean ± 2 × standard deviation ) of the load–displacement curve can be obtained and is plotted in Fig. 16. As depicted in Fig. 16, the confidence region given by the HSGPR model is in agreement with the target model and the variation of the load–displacement curve is depicted accurately by the HSGPR model in both the loading and unloading stages.l In order to investigate the influence of material uncertainty on the plastic deformation, the equivalent plastic strain of the block after loading is analyzed by using the HSGPR model. Two situations corresponding to the upper bound and the lower bound, as labeled in Fig. 16, are shown in Fig. 17. It can be observed from Fig. Application in plastic stochastic structural analysis S h SG b d h fl d l d d The left side and the top right corner of the block are only fix in the horizontal direction and the bottom of the block is only fix in the vertical direction. During the load- ing stage, the vertical displacement of the top of the block u0 is gradually increased to 0.07 mm, which can be regarded as a quasi-static process. During the unloading stage, the direction of the displacement is reversed until u0 = 0.02 mm. Similar to Huang et. al.37, the block is divided into 100 quadratic quadrilateral elements for the FEA analysis. The load–displacement curve of the node A is recorded. https://doi.org/10.1038/s41598-022-06870-9 Scientific Reports \| (2022) 12:3017 \| www.nature.com/scientificreports/ 17 that the plastic deformation of the upper bound situation in the lower left conner of the block is smaller than Scientific Reports \| (2022) 12:3017 \| https://doi.org/10.1038/s41598-022-06870-9 www.nature.com/scientificreports/ Table 6. The expectation of the structural capacity given by the HSGPR-based flow stress model and the target model at different dataset sizes, temperatures and coefficient of variation (COV). COV Number of data curves T = 20 °C T = 175 °C T = 300 °C Target (kN) HSGPR (kN) Relative error (%) Target (kN) HSGPR (kN) Relative error (%) Target (kN) HSGPR (kN) Relative error (%) 0.05 20 15.80 15.82 0.1 13.38 13.30 0.6 10.47 10.47 0.0 100 15.82 0.1 13.34 0.3 10.49 0.2 200 15.76 0.3 13.37 0.1 10.47 0.0 0.10 20 15.77 15.84 0.4 13.35 13.02 2.5 10.49 10.24 2.4 100 15.84 0.4 13.40 0.4 10.43 0.6 200 15.81 0.3 13.32 0.2 10.40 0.9 0.15 20 15.80 16.36 3.5 13.32 12.91 3.1 10.42 10.11 3.0 100 16.05 1.6 13.12 1.5 10.68 2.6 200 15.81 0.1 13.22 0.8 10.52 1.0 Table 6. The expectation of the structural capacity given by the HSGPR-based flow stress model and the target model at different dataset sizes, temperatures and coefficient of variation (COV). Table 7. The standard deviation of the structural capacity given by the HSGPR-based flow stress model and the target model at different dataset sizes, temperatures and coefficient of variation (COV). COV Number of data curves T = 20 °C T = 175 °C T = 300 °C Target (kN) HSGPR (kN) Relative error (%) Target (kN) HSGPR (kN) Relative error Target (kN) HSGPR (kN) Relative error (%) 0.05 20 0.72 0.74 2.8 0.64 0.61 4.7 0.56 0.53 5.4 100 0.70 2.8 0.66 3.1 0.56 0.0 200 0.71 1.4 0.64 0.0 0.56 0.0 0.10 20 1.41 1.51 7.1 1.30 1.24 4.6 1.08 0.95 12.0 100 1.34 5.0 1.27 2.3 1.16 7.4 200 1.43 1.4 1.27 2.3 1.10 1.9 0.15 20 2.13 1.99 6.6 1.95 1.65 15.4 1.64 2.13 29.9 100 2.21 3.8 2.03 4.1 1.68 2.4 200 2.18 2.3 2.00 2.6 1.63 0.6 Table 7. The standard deviation of the structural capacity given by the HSGPR-based flow stress model and the target model at different dataset sizes, temperatures and coefficient of variation (COV). Figure 14. Punch problem given by Huang et. al.37 (Unit: mm). Figure 14. Punch problem given by Huang et. al.37 (Unit: mm). Figure 14. www.nature.com/scientificreports/ Punch problem given by Huang et. al.37 (Unit: mm). Figure 14. Punch problem given by Huang et. al.37 (Unit: mm). https://doi.org/10.1038/s41598-022-06870-9 Scientific Reports \| (2022) 12:3017 \| www.nature.com/scientificreports/ Figure 15. Deterministic load–displacement curves obtained by the target model, HSGPR model and FNN model given by Huang et. al.37. Figure 15. Deterministic load–displacement curves obtained by the target model, HSGPR model and FNN model given by Huang et. al.37. Figure 15. Deterministic load–displacement curves obtained by the target model, HSGPR model and FNN model given by Huang et. al.37. Figure 16. Stochastic load displacement curves obtained by target model, HSGPR model. Figure 16. Stochastic load displacement curves obtained by target model, HSGPR model. the lower bound situation. However, both the upper bound and the lower bound are the possible situations of the structural responses. The variation of the deformation is caused by the uncertainty inherent in the material. Hence, it can be concluded that the deformation of the structure cannot be described completely without suf- ficiently considering the flow stress uncertainty. the lower bound situation. However, both the upper bound and the lower bound are the possible situations of the structural responses. The variation of the deformation is caused by the uncertainty inherent in the material. Hence, it can be concluded that the deformation of the structure cannot be described completely without suf- ficiently considering the flow stress uncertainty. Data availability Th d d y The raw/processed data required to reproduce these findings cannot be shared at this time due to technica time limitations. However, these data will be shared upon request to the corresponding author. Received: 11 November 2021; Accepted: 3 February 2022 Conclusions Understanding the uncertainty of material flow behavior is important in the stochastic plastic structural analysis. In this paper, a data-driven approach, the heteroscedastic sparse Gaussian process regression (HSGPR)-based flow stress model, is proposed to capture the flow stress behavior and the associated uncertainty directly from the available material data. The established HSGPR-based flow stress model is verified by the experimental data of the Al6061 aluminum alloy and is compared with the conventional GPR model, the ANN model and the Johnson Cook (JC) model. The flow stress of the Al 6061 aluminum alloy at elevated temperatures predicted by the HSGPR model are more accurate than those by the ANN model, the GPR model and the JC model, with the AARE of 6.5% and the MLL of -3.74 on the testing dataset. Besides, unlike the ANN model, which provides only the deterministic flow stress, the flow stress uncertainty is successfully estimated by the HSGPR based model as well.hli ll The HSGPR-based stochastic flow stress model is then implemented into finite element method for stochastic plastic structural analysis. Two numerical examples with synthetic flow stress data, that is the beam problem and the punch problem, are used to verify the accuracy of the HSGPR model in stochastic plastic structural analysis. With the sufficient material data, the expectation and standard deviation of the structural capacity at elevated temperatures have the relative errors less than 1% and 3% respectively in the beam problem. In the punch prob- lem, the variation of the loading and unloading paths given by the HSGPR model is consistent with that given https://doi.org/10.1038/s41598-022-06870-9 Scientific Reports \| (2022) 12:3017 \| www.nature.com/scientificreports/ cientificreports/ Figure 17. The equivalent plastic strains of the block after loading corresponding to (a) the upper bound and (b) the lower bound in Fig. 16. Figure 17. The equivalent plastic strains of the block after loading corresponding to (a) the upper bound an (b) the lower bound in Fig. 16. by the target model. The influence of material uncertainty on the structure plastic deformation is successfully identified by the HSGPR model. by the target model. The influence of material uncertainty on the structure plastic deformation is successfully identified by the HSGPR model. by the target model. The influence of material uncertainty on the structure plastic deformation is successfully identified by the HSGPR model. References References 1. Geng, G., Ding, D., Duan, L. & Jiang, H. A modified Johnson-Cook model of 6061–T6 aluminium profile. Aust. J. Mech. Eng. 1, 1–11. https://doi.org/10.1080/14484846.2020.1721966 (2020).i 1. Geng, G., Ding, D., Duan, L. & Jiang, H. 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https://openalex.org/W3119253981	https://europepmc.org/articles/pmc7827898?pdf=render	English	null	Hypoglycemic Effect of Resveratrol: A Systematic Review and Meta-Analysis	Antioxidants	2,021	cc-by	14,458	Citation: García-Martínez, B.I.; Ruiz-Ramos, M.; Pedraza-Chaverri, J.; Santiago-Osorio, E.; Mendoza-Núñez, V.M. Hypoglycemic Effect of Resveratrol: A Systematic Review and Meta-Analysis. Antioxidants 2021, 10, 69. https://doi.org/10.3390/ antiox10010069 Citation: García-Martínez, B.I.; Ruiz-Ramos, M.; Pedraza-Chaverri, J.; Santiago-Osorio, E.; Mendoza-Núñez, V.M. Hypoglycemic Effect of Resveratrol: A Systematic Review and Meta-Analysis. Antioxidants 2021, 10, 69. https://doi.org/10.3390/ antiox10010069 Keywords: glucose; insulin; glycated hemoglobin; glycemic control; insulin resistance; polypheno- lic compounds Received: 2 December 2020 Accepted: 4 January 2021 Published: 7 January 2021 Review Hypoglycemic Effect of Resveratrol: A Systematic Review and Meta-Analysis ía-Martínez 1, Mirna Ruiz-Ramos 1, José Pedraza-Chaverri 2 , Edelmiro Santiago-Osorio 3 Mendoza-Núñez 1,* Beatriz Isabel García-Martínez 1, Mirna Ruiz-Ramos 1, José Pedraza-Chaverri 2 , Edelmiro Santiago-Osorio 3 and Víctor Manuel Mendoza-Núñez 1,* 1 Research Unit on Gerontology, FES Zaragoza, National Autonomous University of Mexico, gy g y Mexico City 09230, Mexico; isabelgm06@gmail.com (B.I.G.-M.); mirna1411@yahoo.com.mx (M.R.-R.) 2 gy g y Mexico City 09230, Mexico; isabelgm06@gmail.com (B.I.G.-M.); mirna1411@yahoo.com.mx (M.R.-R.) 2 Department of Biology, Faculty of Chemistry, National Autonomous University of Mexico (UNAM), M i Ci 04510 M i d @ Abstract: Resveratrol (RV) is a polyphenolic compound with antioxidant, anti-inﬂammatory, and hypoglycemic properties. Several in vitro and animal model studies have demonstrated the beneﬁcial effects of RV; however, the results in humans are not conclusive. After a search of different databases, 32 studies were selected for this systematic review and 30 were included in the meta-analysis. Studies that evaluated the effect of RV on glucose, insulin, HbA1c, and insulin resistance (HOMA-IR) levels were included. A signiﬁcant decrease of glucose (−5.24 mg/dL, p = 0.002) and insulin levels (−1.23 mIU/L, p = 0.0003) was observed. HbA1c and HOMA-IR did not show signiﬁcant changes. Due to heterogeneity, sub-analyzes were performed. Sub-analysis by dose revealed that glucose levels improve signiﬁcantly after the administration of 500–1000 mg/day of RV (−7.54 mg/dL, p = 0.002), while insulin improves with doses lower than 500 mg/day (−1.43 mIU/L, p = 0.01) and greater than 1000 mg/day (−2.12 mIU/L, p = 0.03). HbA1c and HOMA-IR remained unchanged after sub-analysis by dose. Our ﬁndings suggest that RV improves glucose and insulin levels in subjects with type 2 diabetes mellitus (T2DM) and aged 45–59 years, regardless of the duration of the intervention. HbA1c improves with interventions ≥3 months. HOMA-IR does not exhibit signiﬁcant changes after RV administration. antioxidants antioxidants antioxidants antioxidants 1. Introduction Resveratrol (RV) is a polyphenolic compound that includes two benzene rings con- nected through a methylene, and three hydroxyl groups in its structure. This structure allows the RV molecule to give up electrons to distinct free radicals (FR) and thereby atten- uates the damage to biomolecules. Moreover, RV has anti-inﬂammatory properties due to its ability to block the activation and subsequent translocation of nuclear factor κB (NFκB), which is responsible for the synthesis of pro-inﬂammatory proteins, such as tumor necrosis factor α (TNFα), interleukin 1 (IL1), interleukin 6 (IL6), and prothrombotic molecules [1–3]. RV is found in grapes, peanuts, and blueberries, although the plant Polygonum cuspidatum (Mexican baboo, knotty herb from Japan) is the main natural source of this compound. In the last two decades, multiple investigations have been carried out on the therapeutic properties of RV, which are given by the participation of RV in the signaling pathways that modulate the processes of apoptosis, mitochondrial dysfunction, platelet aggregation, oxidative stress, and inﬂammation [4,5]. In this sense, RV is an attractive compound for the adjunctive treatment of chronic noncommunicable diseases (NCDs), such as diabetes, cardiovascular diseases, arthritis, neurodegenerative disorders, and even cancer [6,7]. Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- ms in published maps and institutio- nal afﬁliations. Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 69. https://doi.org/10.3390/antiox10010069 2 of 21 Antioxidants 2021, 10, 69 Regarding the therapeutic effects of RV, these are strongly related to the activation of sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK). Both proteins act as energy regulators due to their participation in metabolism and mitochondrial function, which makes them a suitable target for the treatment of metabolic diseases, such as type 2 diabetes mellitus (T2DM) [7,8]. Scientiﬁc evidence, obtained from in vitro studies and in animal models, suggests that RV has antioxidant, anti-inﬂammatory, and even anti-cancer properties; however, the results of clinical trials are not conclusive. In this context, some clinical trials suggest that RV exerts beneﬁcial effects on metabolic diseases (obesity, metabolic syndrome, and diabetes), which has been evidenced by its ability to reduce the levels of lipids, glucose, and some adipokines. 2. Materials and Methods The study was carried out according to the guidelines for the presentation of system- atic reviews and meta-analyzes (PRISMA 2009) [14]. 1. Introduction Furthermore, it has been observed that after RV administration, the antioxidant capacity increases and the concentrations of pro-inﬂammatory markers decrease [9–11]. Despite the above, in some investigations carried out in humans, no evidence of the therapeutic effects of RV has been found. Therefore, there is currently no consensus regard- ing the therapeutic beneﬁts of RV and the dose at which they are presented, so research on this compound is still continuing [12,13]. Considering this, the aim of this systematic review and meta-analysis is to show and analyze the ﬁndings on the hypoglycemic effect of different doses of RV from clinical trials and quasi-experimental studies. 2.2. Inclusion Criteria The inclusion criteria were as follows: (a) Blind or double-blind randomized clinical trials (RCTs); (b) the use of RV as a nutritional supplement; (c) placebo controlled; (d) pub- lished in the English language; (e) evaluation of at least one of the following biochemical markers: Serum glucose and insulin levels; HbA1c; and insulin resistance (HOMA-IR); (f) duration of at least 2 weeks; and (g) the participation of adults aged ≥20 years, with- out the distinction of sex, healthy, or with metabolic and/or inﬂammatory conditions, except cancer. 2.1. Search Strategy A literature search was carried out in the following databases: PubMed-Medline; Scopus; Cochrane library; Web of Science; Wiley online library; ScienceDirect; and Lilacs. The search was carried out among all articles published from January 1980 to 31 May 2020. The following search strategy was used: Resveratrol AND (glycemic control OR fasting glucose OR insulin resistance). A National Autonomous University of Mexico (UNAM) thesis search was also carried out to identify unpublished studies that could potentially be included in the review. Titles and abstracts identiﬁed through the search strategy were independently assessed by two reviewers (B.I.G.-M. and M.R.-R.), and discrepancies were resolved by a third reviewer (V.M.M.-N.). Once the titles and abstracts that met the selection criteria had been selected, the full texts of potentially relevant articles for the review were retrieved and an exhaustive review was carried out to select the deﬁnitive studies. 2.5. Data Extraction nd findings of each st (Ⴟ) d ol used, duration of th t t d/ t reviewers (J.P.-C. an ti i i l d d t n di h t ata Extraction Once the studies were chosen, two reviewers (J.P.-C. and E.S.-O.) performed data extraction. Data extracted for the systematic review included the ﬁrst author’s last name, year of publication, study design, dose of resveratrol used, duration of intervention, sam- ple size, characteristics of participants (age, health status, and/or metabolic condition), parameters evaluated, and ﬁndings of each study. For the meta-analysis, the following data were extracted: The means ( e means (Ⴟ) and standard deviations (SD) of the pre- and orresponding to the levels of glucose, insulin, HbA1c, and fference, and their respective SD were calculated in cases port them; for this purpose, appropriate statistical meth- was calculated from the median, the minimum and max- size (n), using the formula Ⴟ = (minimum value + 2n + s calculated with the formula SD = √1/12[Ⴟ2 + (maximum en standard error (SE) was reported, the formula SD = SE D. For calculation of the mean difference, the following in means = meanpost-treatment − meanpre-treatment. n of the corresponding SD, the following formula was − (2 × R × SDpre-treatment × SDpost-treatment)], 8. t report the data in the same units, some conversions were o mg/dL of glucose, the value was multiplied by 18. The ) and standard deviations (SD) of the pre- and post-treatment measurements corresponding to the levels of glucose, insulin, HbA1c, and HOMA-IR. The th status, and/or metabolic condi- dy. For the meta-analysis, the fol- ard deviations (SD) of the pre- and els of glucose, insulin, HbA1c, and ective SD were calculated in cases pose, appropriate statistical meth- e median, the minimum and max- mula Ⴟ = (minimum value + 2n + rmula SD = √1/12[Ⴟ2 + (maximum was reported, the formula SD = SE he mean difference, the following t-treatment − meanpre-treatment. g SD, the following formula was ent × SDpost-treatment)], , SD, mean difference, and their respective SD were calculated in cases in which the studies did not report them; for this purpose, appropriate statistical methods were used [15–17]. The atic review included the first author’s last name, of resveratrol used, duration of intervention, nts (age, health status, and/or metabolic condi- gs of each study. 2.5. Data Extraction nd findings of each st (Ⴟ) d ol used, duration of th t t d/ t reviewers (J.P.-C. an ti i i l d d t n di h t ata Extraction For the meta-analysis, the fol- Ⴟ) and standard deviations (SD) of the pre- and ding to the levels of glucose, insulin, HbA1c, and and their respective SD were calculated in cases m; for this purpose, appropriate statistical meth- lated from the median, the minimum and max- using the formula Ⴟ = (minimum value + 2n + ed with the formula SD = √1/12[Ⴟ2 + (maximum rd error (SE) was reported, the formula SD = SE alculation of the mean difference, the following s = meanpost-treatment − meanpre-treatment. corresponding SD, the following formula was was calculated from the median, the minimum and maximum values, and the sample size (n), using the formula udies were chosen, two reviewers (J.P.-C. and E.S.-O.) performed data xtracted for the systematic review included the first author’s last name, on, study design, dose of resveratrol used, duration of intervention, racteristics of participants (age, health status, and/or metabolic condi- evaluated, and findings of each study. For the meta-analysis, the fol- extracted: The means (Ⴟ) and standard deviations (SD) of the pre- and easurements corresponding to the levels of glucose, insulin, HbA1c, and Ⴟ, SD, mean difference, and their respective SD were calculated in cases dies did not report them; for this purpose, appropriate statistical meth- 5–17]. The Ⴟ was calculated from the median, the minimum and max- d the sample size (n), using the formula Ⴟ = (minimum value + 2n + /4. The SD was calculated with the formula SD = √1/12[Ⴟ2 + (maximum m value)2]. When standard error (SE) was reported, the formula SD = SE to calculate SD. For calculation of the mean difference, the following d: Difference in means = meanpost-treatment − meanpre-treatment. the calculation of the corresponding SD, the following formula was = (minimum value + 2n + maximum value)/4. The SD was calculated with the formula SD = √1/12[ ata Extraction Once the studies were chosen, two reviewers (J.P.-C. and E.S.-O.) performed data ction. Data extracted for the systematic review included the first author’s last name, of publication, study design, dose of resveratrol used, duration of intervention, le size, characteristics of participants (age, health status, and/or metabolic condi- parameters evaluated, and findings of each study. For the meta-analysis, the fol- g data were extracted: The means (Ⴟ) and standard deviations (SD) of the pre- and reatment measurements corresponding to the levels of glucose, insulin, HbA1c, and A-IR. The primary outcomes included the following: outcomes included the following: d i li l l utcomes he primary outcomes included the following: The primary outcomes included the following: outcomes included the following: d i li l l utcomes he primary outcomes included the following: - Serum glucose and insulin levels; osen, two reviewers (J.P.-C. and E.S.-O h i i i l d d h fi ose and insulin levels; entage; The primary outcomes included the follo Serum glucose and insulin levels; - HbA1c percentage; he systematic review includ d E S O f entage; value Serum glucose and insulin HbA1c percentage; - HOMA-IR value. sign, dose of resveratro .P.-C. and E.S.-O.) perfo value. HbA1c percentage; HOMA IR value tool considers seven items for evaluation, includin ence, allocation concealment, blinding of staff and p eria, in addition to assessing their rane collaboration risk of bias as- selected studies, they were reviewed in detail to Risk of Bias and Quality of Studies f Ri k f Bi d Q li f S di 2.6. Assessment of Risk of Bias and Quality of Studies s of results, incomplete results data, selective reports of s. ems for evaluation, including the ment, blinding of staff and partic- e results data, selective reports of nclusion criteria, in addition to assessing their se, the Cochrane collaboration risk of bias as- ders seven items for evaluation, including the cation concealment, blinding of staff and partic- ts, incomplete results data, selective reports of ing the full text of the selected studies, they were reviewed in detail to hat did not meet the inclusion criteria, in addition to assessing their quality. For this purpose, the Cochrane collaboration risk of bias as- as used. This tool considers seven items for evaluation, including the random sequence, allocation concealment, blinding of staff and partic- of the analysis of results, incomplete results data, selective reports of sources of bias. ssessment of Risk of Bias and Quality of Studies After retrieving the full text of the selected studies, they were reviewed in detail to nate those that did not meet the inclusion criteria, in addition to assessing their odological quality. For this purpose, the Cochrane collaboration risk of bias as- ment tool was used. This tool considers seven items for evaluation, including the ation of the random sequence, allocation concealment, blinding of staff and partic- s, blinding of the analysis of results, incomplete results data, selective reports of After retrieving the full text of the selected studies, they were reviewed in detail to eliminate those that did not meet the inclusion criteria, in addition to assessing their methodological quality. For this purpose, the Cochrane collaboration risk of bias assessment tool was used. This tool considers seven items for evaluation, including the generation of the random sequence, allocation concealment, blinding of staff and participants, blinding of the analysis of results, incomplete results data, selective reports of results, and other sources of bias. sion of insulin units from pmol/L to mIU/L was performed by dividing pmol/L ÷ To convert the units of HbA1c, the following formula was used: %HbA1c = /mol/10,929) + 2.15. In some cases, the HOMA-IR index was also calculated, for ot report the data in the same units, some conversions were L to mg/dL of glucose, the value was multiplied by 18. The om pmol/L to mIU/L was performed by dividing pmol/L ÷ st treatment2) (2 R SDpre treatment SDpost treatment)], where R = 0.8. studies did not report the data in the same units, some conversions were used: Dpre-treatment2 + SDpost-treatment2) − (2 × R × SDpre-treatment × SDpost-treatment)], where R = 0.8. u t e o e, o t e ca cu atio o t e co espo di g S , t e o owi g o u a was used: rence = √[(SDpre-treatment2 + SDpost-treatment2) − (2 × R × SDpre-treatment × SDpost-treatment)], SDdifference = √[(SDpre-treatment2 + SDpost-treatment2) −(2 × R × SDpre-treatment × SDpost-treatment)], where R = 0.8. was used: HOMA-IR = [insulin (mIU/L) × glucose d Quality of Studies xt of the selected studies, they were reviewed in detail to meet the inclusion criteria, in addition to assessing their his purpose, the Cochrane collaboration risk of bias as- l d f l l d h g formula was used: %HbA1c = -IR index was also calculated, for R = [insulin (mIU/L) × glucose es, they were reviewed in detail to i i ddi i i h i of glucose, the value was multiplied by 18. The to mIU/L was performed by dividing pmol/L ÷ the following formula was used: %HbA1c = s, the HOMA-IR index was also calculated, for ed: HOMA-IR = [insulin (mIU/L) × glucose of Studies studies did not report the data in the same units, some conversions were nvert mmol/L to mg/dL of glucose, the value was multiplied by 18. The ulin units from pmol/L to mIU/L was performed by dividing pmol/L ÷ t the units of HbA1c, the following formula was used: %HbA1c = 9) + 2.15. In some cases, the HOMA-IR index was also calculated, for wing formula was used: HOMA-IR = [insulin (mIU/L) × glucose where R = 0.8. Because the studies did not report the data in the same units, some conversions were sary. To convert mmol/L to mg/dL of glucose, the value was multiplied by 18. The ersion of insulin units from pmol/L to mIU/L was performed by dividing pmol/L ÷ To convert the units of HbA1c, the following formula was used: %HbA1c = ol/mol/10,929) + 2.15. In some cases, the HOMA-IR index was also calculated, for h the following formula was used: HOMA-IR = [insulin (mIU/L) × glucose dL)]/405 Because the studies did not report the data in the same units, some conversions were necessary. To convert mmol/L to mg/dL of glucose, the value was multiplied by 18. The conversion of insulin units from pmol/L to mIU/L was performed by divid- ing pmol/L ÷ 6945. To convert the units of HbA1c, the following formula was used: %HbA1c = (mmol/mol/10,929) + 2.15. In some cases, the HOMA-IR index was also calcu- lated, for which the following formula was used: HOMA-IR = [insulin (mIU/L) × glucose (mg/dL)]/405. 2.3. Exclusion Criteria The exclusion criteria were as follows: (a) Studies that administered RV in combination with other compounds (e.g., quercetin); (b) studies that administered compounds derived from resveratrol, red wine, or a diet rich in polyphenols; (c) studies without a control group; (d) pilot studies; and (e) research only available in summary, to avoid risk of bias given a lack of information. 3 of 21 Antioxidants 2021, 10, 69 The primary outco Serum glucose and up; (d) pilot studies; a n a lack of informatio derived f control g 2.4. Outcomes following: t i l Outcomes 2.4. Outcomes following: t i l utcomes 2.4. Outcomes The primary outcomes included the following: g y outcomes included the following: o e a d i uli le el Outcomes The primary outcomes included the following: 2.5. Data Extraction nd findings of each st (Ⴟ) d ol used, duration of th t t d/ t reviewers (J.P.-C. an ti i i l d d t n di h t ata Extraction The Ⴟ, SD, mean difference, and their respective SD were calculated in cases ich the studies did not report them; for this purpose, appropriate statistical meth- were used [15–17]. The Ⴟ was calculated from the median, the minimum and max- m values, and the sample size (n), using the formula Ⴟ = (minimum value + 2n + mum value)/4. The SD was calculated with the formula SD = √1/12[Ⴟ2 + (maximum − minimum value)2]. When standard error (SE) was reported, the formula SD = SE was applied to calculate SD. For calculation of the mean difference, the following ula was used: Difference in means = meanpost-treatment − meanpre-treatment. 2 + (maximum value −minimum value)2]. When standard error (SE) was reported, the formula SD = SE × √n was applied to calculate SD. For calculation of the mean difference, the following formula was used: Difference in means = meanpost-treatment −meanpre-treatment. Furthermore, for the calculation of the corresponding SD, the following formula was used: ersion of insulin units from pmol/L to mIU/L was performed by dividing pmol/L ÷ To convert the units of HbA1c, the following formula was used: %HbA1c = ol/mol/10,929) + 2.15. In some cases, the HOMA-IR index was also calculated, for not report the data in the same units, some conversions were /L to mg/dL of glucose, the value was multiplied by 18. The rom pmol/L to mIU/L was performed by dividing pmol/L ÷ post treatment2) (2 R SDpre treatment SDpost treatment)], where R = 0.8. he studies did not report the data in the same units, some conversions were used: SDpre-treatment2 + SDpost-treatment2) − (2 × R × SDpre-treatment × SDpost-treatment)], where R = 0.8. Furthermore, for the calculation of the corresponding SD, the following formula was used: erence = √[(SDpre-treatment2 + SDpost-treatment2) − (2 × R × SDpre-treatment × SDpost-treatment)], SDdifference = √[(SDpre-treatment2 + SDpost-treatment2) −(2 × R × SDpre-treatment × SDpost-treatment)], where R = 0.8. 3.1. Literature Search 3.1. Literature Search 3.1. Literature Search The total number of articles identiﬁed from the database search was 1848, plus 31 the- ses from UNAM. After reviewing the titles and abstracts, duplicate studies were eliminated, as well as those that did not meet the selection criteria, leaving a total of 62 eligible articles, of which the full text was recovered. After reviewing the full text of the 62 preselected studies, 30 of them were eliminated from the qualitative analysis for several reasons (see Appendix A), which are presented in Figure 1, and 32 studies were included in the system- atic review. However, two studies (Bo et al. 2013 and Brasnyó et al. 2011) were discarded from the quantitative analysis because they did not present the necessary information (means before and after treatment or mean difference), so only 30 studies were included in the meta-analysis (Figure 1). Figure 1. Flow diagram of study selection included in the systematic review and meta-analysis. Figure 1. Flow diagram of study selection included in the systematic review and meta-analysis. , 2.7. Statistical Analysis To estimate the overall effect of RV supplementation on glucose, insulin, HbA1c levels, and HOMA-IR values, a random effects model was used. This model considers intra- and inter-study heterogeneity. Heterogeneity was assessed using the I2 test, considering the existence of signiﬁcant heterogeneity if I2 > 50%. Subgroup analyzes were performed, separating by resveratrol dosage (<500 mg/day, 500–1000 mg/day, and >1000 mg/day), health status (with T2DM and without T2DM), duration of the intervention (<3 months and ≥3 months), and age (<45 years, 45–59 years, and >60 years). In addition, sensitivity analyzes were performed to assess the effect of each study on the overall effect. For this, all meta-analyzes were carried out, removing one study at a time. Funnel plots and Egger’s Antioxidants 2021, 10, 69 4 of 21 test were performed to assess publication bias. The value of p < 0.05 was considered as statistically signiﬁcant. The statistical analyzes were performed with Review Manager version 5.3 software from the Cochrane collaboration. 3.2. Study Characteristics Of the included randomized clinical trials, 22 had a parallel double-blind design, 7 a double-blind crossover design, 1 was parallel-blind, and 2 were open-label. The total number of participants included in the meta-analysis was 1651 with different ages and health conditions. The subjects included in each study ranged from 8 to 129, the doses used ranged from 10 to 3000 mg/day, and the duration of the interventions was at least 4 weeks and a maximum of 12 months. The characteristics of the studies included in the present review are shown in Table 1. The results on the assessment of the methodological quality and risk of bias are presented in Figure 2. 5 of 21 Antioxidants 2021, 10, 69 Table 1. Characteristics of clinical trials included in the review. First Author (Year) Study Design RV Dosage Duration Subjects Age Evaluated Parameters Findings Abdollahi et al. (2019) [18] RCT double-blind 1 g/day 8 weeks 71 subjects with T2DM and overweight 50 ± 7 Glucose and insulin levels, HbA1c, HOMA-IR, HOMA-β, QUICKI Signiﬁcant decrease in glucose (p = 0.03) and insulin (p = 0.02), improvement in HOMA-IR (p = 0.01) and QUICKI (p = 0.008). No signiﬁcant changes in HOMA-β and HbA1c after resveratrol administration Asghari et al. (2018) [19] RCT double-blind 600 mg/day 12 weeks 75 subjects with fatty liver 40 ± 7 Glucose and insulin levels HOMA-IR Non-signiﬁcant changes in the glycemic parameters evaluated Banaszewska et al. (2016) [20] RCT double-blind 1.5 g/day 3 months 30 women with POS 27 ± 1 Fasting blood glucose, insulin sensitivity index Signiﬁcant decrease in insulin levels (38%, p = 0.007) and increase in the insulin sensitivity index (66%, p = 0.04) Bashmakov et al. (2014) [21] RCT parallel-blind 100 mg/day 2 months 24 patients with diabetic food 56 ± 9 Glucose and insulin levels, HOMA-IR Tendency of a decrease of glucose levels in both study groups. No changes in insulin levels and HOMA-IR Bhatt et al. (2012) [22] RCT open-label 250 mg/day 3 months 57 subjects with T2DM 57 ± 9 Fasting blood glucose and HbA1c Signiﬁcant decrease in HbA1c levels (p < 0.05) after resveratrol administration Bhatt et al. (2013) [23] RCT open-label 250 mg/day 6 months 57 subjects with T2DM 57 ± 9 HbA1c and glucose levels Non-signiﬁcant decrease in HbA1c and glucose levels after intervention Bo et al. 3.2. Study Characteristics (2016) [24] RCT double-blind 40, 500 mg/day 6 months 179 subjects with T2DM 65 ± 8 Glucose and insulin levels, HOMA-IR, HbA1c, C-peptide Non-signiﬁcant differences between the study groups after intervention Bo et al. (2013) [25] RCT double-blind crossover 500 mg/day 4 weeks 49 healthy smokers 35 ± 9 Glucose and insulin levels HOMA-IR Non-signiﬁcant changes after resveratrol intervention Brasnyó et al. (2011) [26] RCT double-blind 10 mg/day 4 weeks 19 men with T2DM 55 ± 9 Insulin levels, HOMA-IR, HOMA-β No changes in insulin and HOMA-β levels, tendency of a decrease of HOMA-IR in the experimental group Chachay et al. (2014) [27] RCT double-blind 3 g/day 8 weeks 20 men with NAFLD 49 ± 12 HOMA-IR, glucose and insulin levels Resveratrol did not improve glucose, insulin, and HOMA-IR levels Chen (2015) [28] RCT double-blind 600 mg/day 3 months 57 subjects with NAFLD 44 ± 10 Glucose, insulin, C-peptide and HOMA-IR Signiﬁcant decrease in glucose (p = 0.001) and HOMA-IR (p = 0.016). No signiﬁcant changes in insulin and C-peptide levels Dash et al. (2013) [29] RCT double-blind crossover 1–2 g/day 2 weeks 8 overweight and obese subjects 46 ± 3 Glucose and insulin levels, HOMA-IR Non-signiﬁcant changes in evaluated parameters after resveratrol administration De Ligt et al. (2020) [30] RCT double-blind 150 mg/day 6 months 41 overweight individuals 62 ± 1 Glucose and insulin levels, HbA1c Non-signiﬁcant differences in glucose and insulin levels. Signiﬁcant decrease in HbA1c (p = 0.007) after resveratrol administration Table 1. Characteristics of clinical trials included in the review. Antioxidants 2021, 10, 69 6 of 21 Table 1. Cont. First Author (Year) Study Design RV Dosage Duration Subjects Age Evaluated Parameters Findings De Ligt et al. (2018) [31] RCT double-blind crossover 150 mg/day 4 weeks 13 men at high risk of T2DM 66 ± 4 Glucose and insulin levels, HbA1c Non-signiﬁcant differences between the study groups after resveratrol administration Faghihzadeh et al. (2015) [32] RCT double-blind 500 mg/day 12 weeks 50 subjects with NAFLD 45 ± 10 Glucose and insulin levels, HOMA-IR, HOMA-β, QUICKI Non-signiﬁcant changes after the resveratrol intervention Godínez-Salas et al. (2018) [33] RCT double-blind 150 mg/day 3 months 42 subjects with MS 43 ± 1 Glucose and insulin levels Non-signiﬁcant changes in glucose and insulin levels after resveratrol intervention Goh et al. 3.2. Study Characteristics (2014) [34] RCT double-blind 3 g/day 12 weeks 10 subjects with T2DM 56 ± 6 Glucose and insulin levels, HbA1c, HOMA-IR Tendency of a decrease in HbA1c, no signiﬁcant changes in insulin sensitivity Hoseini et al. (2019) [35] RCT double-blind 500 mg/day 4 weeks 56 subjects with T2DM and CD 62 ± 9 Glucose and insulin levels, HOMA-IR, QUICKI Signiﬁcant decrease in glucose, insulin (p = 0.01), and HOMA-IR (p = 0.001); QUICKI increase (p = 0.02) Javid et al. (2016) [36] RCT double-blind 480 mg/day 4 weeks 43 subjects with T2DM and CP 50 ± 8 Glucose and insulin levels, HOMA-IR Signiﬁcant decrease in insulin and HOMA-IR levels (p < 0.05), non-signiﬁcant decrease in glucose levels after resveratrol intervention Kantartzis et al. (2018) [37] RCT double-blind 150 mg/day 12 weeks 105 overweight and obese subjects 48 ± 13 Fasting blood glucose, HbA1c, HOMA-IR No signiﬁcant changes in evaluated parameters Khodabandenlhoo et al. (2018) [38] RCT double-blind 800 mg/day 2 months 45 subjects with T2DM 57 ± 9 Glucose and insulin levels, HbA1c, HOMA-IR, HOMA-β Signiﬁcant decrease in glucose levels (p < 0.05) in experimental group. No changes in HbA1c. No signiﬁcant changes in insulin, HOMA-IR, and HOMA β levels Kjaer et al. (2017) [39] RCT double-blind 1,501,000 mg/day 16 weeks 66 subjects with MS 50 ± 1 Glucose and insulin levels, HOMA-IR No change in glucose and insulin concentrations, no change in HOMA-IR Méndez-del Villar et al.(2014) [40] RCT double-blind 1.5 g/day 3 months 21 subjects with MS 40 ± 5 AUC of glucose and insulin, insulin index Signiﬁcant decrease in insulin AUC and insulin index (p < 0.05) Movahed et al. (2013) [41] RCT double-blind 1 g/day 45 days 64 subjects with T2DM 52 ± 7 Glucose and insulin levels, HOMA-IR, HOMA-β Signiﬁcant decrease (p < 0.05) in glucose, insulin, and HbA1c levels. Improvement of HOMA-IR and HOMA β in experimental group Pollack et al. (2017) [42] RCT double-blindcrossover 2 g/day 6 weeks 30 overweight older adults with impaired glucose tolerance 67 ± 7 Glucose and insulin levels, AUC of glucose and insulin, HbA1c, HOMA-IR No effect of resveratrol on glycemic parameters Poulsen et al. (2013) [43] RCT double-blind 500 mg/day 4 weeks 24 obese subjects 39 ± 3 Glucose and insulin levels, HbA1c, HOMA-IR No changes in evaluated parameters 7 of 21 Antioxidants 2021, 10, 69 Table 1. Cont. 3.2. Study Characteristics First Author (Year) Study Design RV Dosage Duration Subjects Age Evaluated Parameters Findings Sattarinezhad et al. (2019) [44] RCT double-blind 500 mg/day 3 months 60 subjects with T2DM and albuminuria 57 ± 9 Glucose and insulin levels, HbA1c, HOMA-IR Signiﬁcant decrease in evaluated parameters in experimental group (p < 0.05) Seyyedebrahimi et al. (2018) [45] RCT double-blind 800 mg/day 8 weeks 46 subjects with T2DM 58 ± 6 Glucose and insulin levels, HbA1c, HOMA-IR No signiﬁcant changes after resveratrol intervention Thaung et al. (2020) [46] RCT double-blind 150 mg/day 12 months 129 postmenopausal women 64 ± 1 Glucose and insulin levels, HOMA-IR No signiﬁcant changes in evaluated parameters Thazhath et al. (2016) [47] RCT double-blindcrossover 1 g/day 5 weeks 14 subjects with T2DM 68 ± 2 Glucose and HbA1c No signiﬁcant changes in glucose and HbA1c levels Timmers et al. (2016) [48] RCT double-blindcrossover 150 mg/day 4 weeks 16 subjects with T2DM 64 ± 4 Glucose and insulin levels, insulin sensitivity, HbA1c No effect of resveratrol on glycemic parameters Van der Made et al.(2015) [49] RCT double-blindcrossover 150 mg/day 4 weeks 45 overweight and obese subjects 61 ± 7 Glucose and insulin levels, HOMA-IR No changes in HOMA-IR and insulin levels. Glucose levels showed a tendency to decrease Abbreviations: AUC, area under curve; CD, coronary disease; CP, chronic periodontitis; HbA1c, glycated hemoglobin; HOMA-β, insulin resistance of pancreatic cell β; HOMA-IR, insulin resistance; MS, metabolic syndrome; NAFLD, non-alcoholic fatty liver disease; POS, polycystic ovary syndrome; RCT, randomized clinical trials; T2DM, type 2 diabetes mellitus; and QUICKI, Quantitative Insulin Sensitivity Check Index. 8 of 21 Antioxidants 2021, 10, 69 Figure 2. Assessment of the risk of bias and methodological quality of clinical trials included. Most clinical trials display a low risk of bias. Figure 2. Assessment of the risk of bias and methodological quality of clinical trials included. Most clinical trials display a low risk of bias. Figure 2. Assessment of the risk of bias and methodological quality of clinical trials included. Most clinical trials display a low risk of bias. Figure 6. Effect of resveratrol on HOMA-IR. 3.3. Meta-Analysis Thirty articles that reported 32 effect sizes were included for the meta-analysis that evaluated the effect of RV on glucose levels, 24 publications with 26 effect sizes for insulin, 16 articles with 17 effect sizes of RV on HbA1c, and 26 studies with 28 effect sizes on HOMA-IR. There was a statistically significant decrease of glucose (−5.24 mg/dL, p = 0.002; Figure 3) and insulin levels (−1.23 mIU/L, p = 0.0003; Figure 4). HbA1c (Figure 5) and HOMA-IR (Figure 6) did not show signiﬁcant changes. Due to the considerable heterogeneity of the included studies, sub-analyzes were performed (Table 2). Sub-analysis by dose revealed that glucose levels improve signiﬁcantly after the administration of 500–1000 mg/day of RV (−7.54 mg/dL, p = 0.002), while insulin improves with doses lower than 500 mg/day (−1.43 mIU/L, p = 0.01) and with doses greater than 1000 mg/day (−2.12 mIU/L, p = 0.03). HbA1c and HOMA-IR remained unchanged after sub-analysis by dose. 9 of 21 Antioxidants 2021, 10, 69 Figure 3. Effect of resveratrol on glucose levels. Figure 3. Effect of resveratrol on glucose levels. Figure 4. Effect of resveratrol on insulin levels. Figure 4. Effect of resveratrol on insulin levels. 10 of 21 Antioxidants 2021, 10, 69 Figure 5. Effect of resveratrol on HbA1c. Figure 6. Effect of resveratrol on HOMA-IR. 11 of 21 Antioxidants 2021, 10, 69 Table 2. Subgroup analysis conducted to evaluate the hypoglycemic effect of resveratrol. Subgroup No. 3.3. Meta-Analysis of Trials Effect Size 95% CI p Value Heterogeneity (I2) p Value for I2 Glucose Resveratrol dosage (I2 = 76%; p = 0.01) <500 mg/day 13 −5.40 −11.29, 0.49 0.07 97% 0.00001 500–1000 mg/day 13 −7.54 −12.29, −2.79 0.002 89% 0.00001 >1000 mg/day 6 0.82 −2.78, 4.42 0.66 51% 0.00001 Health status (I2 = 91%; p = 0.0008) With T2DM 15 −13.36 −21.09, −5.63 0.0007 96% 0.00001 Without T2DM 17 0.18 −1.52, 1.89 0.83 67% 0.0001 Duration (I2 = 0%; p = 0.89) <3 months 15 −5.29 −9.20, −1.39 0.008 81% 0.00001 ≥3 months 17 −4.83 −9.75, 0.09 0.05 96% 0.00001 Age (I2 = 66%; p = 0.05) <45 years 6 0.08 −4.08, −4.24 0.97 79% 0.0002 45–59 years 16 −11.04 −19.0, −3.07 0.007 96% 0.00001 ≥60 years 10 −2.06 −4.39, 0.27 0.08 73% 0.0001 Insulin Resveratrol dosage (I2 = 0%; p = 0.44) <500 mg/day 9 −1.43 −2.53, −0.32 0.01 90% 0.00001 500–1000 mg/day 12 −0.78 −1.85, 0.30 0.16 83% 0.00001 >1000 mg/day 5 −1.23 −1.90, −0.57 0.03 73% 0.006 Health status (I2 = 0%; p = 0.57) With T2DM 12 −0.94 −1.62, −0.25 0.007 77% 0.00001 Without T2DM 14 −1.39 −2.80, 0.01 0.05 91% 0.00001 Duration (I2 = 0%; p = 0.38) <3 months 13 −0.93 −1.53, −0.33 0.002 69% 0.0001 ≥3 months 13 −1.65 −3.15, −0.16 0.03 93% 0.00001 Age (I2 = 42%; p = 0.18) <45 years 5 −3.60 −7.65, 0.46 0.08 96% 0.00001 45–59 years 13 −0.97 −1.82, −0.12 0.02 82% 0.00001 ≥60 years 8 −0.32 −1.01, 0.36 0.35 57% 0.02 HbA1c Resveratrol dosage (I2 = 0%; p = 0.56) <500 mg/day 7 −0.20 −0.42, 0.02 0.08 99% 0.00001 500–1000 mg/day 8 −0.06 −0.21, 0.10 0.48 69% 0.002 >1000 mg/day 2 −0.25 −1.18, 0.69 0.61 68% 0.08 Health status (I2 = 87%; p = 0.005) With T2DM 12 −0.22 −0.40, −0.04 0.02 97% 0.00001 Without T2DM 5 0.05 −0.01, 0.10 0.11 0% 0.78 Duration (I2 = 0%; p = 0.38) <3 months 9 0.02 −0.08, 0.13 0.66 64% 0.004 ≥3 months 8 −0.29 −0.50, −0.08 0.006 98% 0.00001 Age (I2 = 85%; p = 0.001) <45 years 1 0.05 −0.04, 0.14 0.29 — — 45–59 years 9 −0.34 −0.54, −0.13 0.002 98% 0.00001 ≥60 years 6 0.07 0, 0.15 0.05 0 0.48 HOMA-IR Resveratrol dosage (I2 = 0%; p = 0.62) <500 mg/day 11 −0.22 −0.55, 0.11 0.19 57% 0.01 500–1000 mg/day 12 −0.60 −1.44, 0.24 0.16 97% 0.00001 >1000 mg/day 5 −0.22 −0.74, 0.30 0.42 46% 0.11 Health status (I2 = 64%; p = 0.10) With T2DM 12 −0.83 −1.68, −0.02 0.04 96% 0.00001 Without T2DM 16 −0.08 −0.33, 0.17 0.54 55% 0.004 Duration (I2 = 0%; p = 0.83) <3 months 14 −0.36 −1.11, 0.39 0.35 96% 0.00001 ≥3 months 14 −0.45 −0.91, 0.00 0.05 83% 0.00001 Age (I2 = 0%; p = 0.70) <45 years 5 −0.57 −1.19, 0.04 0.07 68% 0.01 45–59 years 14 −0.39 −1.25, 0.47 0.38 96% 0.00001 ≥60 years 9 −0.28 −0.60, 0.03 0.08 55% 0.02 Abbreviations: CI, conﬁdence interval; HbA1c, glycated hemoglobin; HOMA-IR, insulin resistance (homeostatic model); and T2DM, type 2 diabetes mellitus. 4. Discussion Currently, the incidence of NCDs, such as obesity, diabetes, cardiovascular diseases (CVD), and metabolic syndrome (MS), is increasing and according to the World Health Organization (WHO), is the main cause of death worldwide. The uncontrolled increase in NCDs is related to unhealthy lifestyles, such as diets rich in carbohydrates and fat, sedentary lifestyles, and tobacco and alcohol consumption [50–54]. For this reason, the main strategies applied for the prevention and control of these pathologies focus on achieving a change in lifestyles and improving therapeutic adherence in the population at risk [55–57]. However, it is well-known that the proposed strategies have not been entirely successful and the search for new therapeutic agents has been necessary, among which nutraceuticals stand out. These compounds have aroused great interest among the scientiﬁc community, including phenolic acids, stilbenes, ﬂavonoids, lignans, and curcuminoids, which have been the object of multiple investigations aimed at understand- ing their role in preventing diseases and increasing longevity [58–60]. In this sense, RV has been widely studied. Some research suggests that its use is associated with a lower incidence and better control of a wide variety of NCDs. This occurs due to the antioxidant capacity of RV and its interaction with cell signaling pathways for the modulation of gene expression. However, other investigations show the lack of a therapeutic effect of this nutraceutical [19,22–25,27,29,33–35,61–63]. This means that researchers need to continue conducting clinical trials and analyzing existing ones to identify the efﬁcacy and safety of RV as a complementary treatment for NCDs. This meta-analysis contains 30 articles that study the effects of RV supplementation vs. a placebo on glucose, insulin, HbA1c, and insulin resistance (measured by the HOMA-IR index). These biochemical parameters are important for evaluating the prevention and control of metabolic diseases such as T2DM, obesity, nonalcoholic fatty liver, and MS. For this reason, they are the main biomarkers of outcome in most clinical trials evaluating the effectiveness of RV. Our global results show that RV supplementation vs. a placebo decreases glucose and insulin levels, but has no therapeutic effect on HbA1c and HOMA-IR, which is contrary to what was found in the meta-analysis by Hausenblas et al. [64], who observed a signiﬁcant decrease in HbA1c, without a considerable effect on glucose levels. In addition to this, in the study carried out by Jeyaraman et al. [65], they found that RV did not signiﬁcantly improve HbA1c, glucose, and insulin levels. 3.3. Meta-Analysis In the sub-analysis by health status, we found that the four parameters evaluated im prove signiﬁcantly after the administration of RV in subjects with T2DM (effect size on glu le 2. Subgroup analysis conducted to evaluate the hypoglycemic effect of resveratrol. Abbreviations: CI, conﬁdence interval; HbA1c, glycated hemoglobin; HOMA-IR, insulin resistance (homeostatic model); and T2DM, type 2 diabetes mellitus. Abbreviations: CI, conﬁdence interval; HbA1c, glycated hemoglobin; HOMA-IR, insulin resistance (homeostatic model); and T2DM, type 2 diabetes mellitus In the sub-analysis by health status, we found that the four parameters evaluated im- prove signiﬁcantly after the administration of RV in subjects with T2DM (effect size on glu- cose = −13.36 mg/dL, p = 0.0007; effect size on insulin = −0.94 mIU/L, p = 0.007; effect size Antioxidants 2021, 10, 69 12 of 21 12 of 21 on HbA1c = −0.22%, p = 0.02; effect size of HOMA-IR = −0.83, p = 0.04). The sub-analysis by duration (<3 months or ≥3 months) revealed that glucose levels decrease signiﬁcantly after RV administration for <3 months (−5.29 mg/dL, p = 0.008). Insulin levels improve regardless of the duration of the intervention (effect size at <3 months = −0.93 mIU/L, p = 0.002; effect size at ≥3 months = −1.65 mIU/L, p = 0.03). HbA1c improves signiﬁcantly (effect size = −0.29%, p = 0.006) in interventions lasting ≥3 months. HOMA-IR is not signiﬁcantly modiﬁed, regardless of the duration of the intervention. g y g Finally, the sub-analysis by age revealed that glucose, insulin, and HbA1c levels signiﬁcantly improve in subjects aged 45 to 59 years who use RV (effect size on glu- cose = −11.04 mg/dL, p = 0.007; effect size on insulin = −0.97, p = 0.02; effect size on HbA1c = −0.34%, p = 0.002). However, HOMA-IR exhibited no signiﬁcant changes. 4.1. Sub-Analysis by RV Dosage After performing the stratiﬁed analysis by dose, a positive and statistically signiﬁcant effect of RV on glucose levels was found at doses of 500–1000 mg/day, while the effect of RV on insulin was signiﬁcant after consuming doses of less than 500 mg/day and greater than 1000 mg/day. In the systematic review and meta-analysis carried out by Zhu et al. [68], they found that, at doses of less than 100 mg/day, there are no changes in glucose levels, but higher doses (even 1 g) are capable of decreasing glucose levels, which partially coincides with our results. This is due to the fact that Zhu et al. only included subjects with T2DM, while in our study, subjects with and without T2DM were included. It has been shown that the efﬁcacy of RV may differ according to the administered dose, because the molecular target changes. In addition, it has been proposed that RV could have a dose–response effect (hormesis), so, at low doses, it triggers a stimulating response of some metabolic pathways, and at high doses, it causes the inhibition of the same pathways [69]. SIRT1 is known to play an important role in AMPK activation to improve mitochon- drial function and stimulate glucose utilization, as well as protect cells against metabolic decline. In this regard, both in vitro and in vivo studies have shown that moderate doses of RV activate SIRT1 and this, in turn, activates AMPK. In contrast, high doses activate AMPK independently of SIRT1, but do not improve mitochondrial function or protect against metabolic deterioration [70]. It has also been observed that in murine models, low doses of RV improve the insulin sensitivity and decrease its secretion by parts of the pancreatic β cells in the long term, while high doses have the same effect in the short term; however, high doses of RV cause nephrotoxicity [71]. In our meta-analysis, we found that high and low doses of RV exert similar effects on insulin levels. However, the variability in the duration of the interventions and in the health conditions of the participants does not allow us to establish if this result is due to the biological effects of RV occurring in a dose-dependent manner or a consequence of the metabolic conditions of cells, since, depending on the cellular needs, RV activates different molecules and signaling pathways, which translates into different biological effects [7,8]. 4.1. Sub-Analysis by RV Dosage In addition, it should be emphasized that changes in insulin levels after RV administration, although statistically signiﬁcant, do not necessarily represent a clinically important change. Due to this, it is necessary to carry out more research on the biological effects of RV to determine if these are presented in a dose-dependent manner in humans, since, so far, many of the results in animal models have not been reproduced in humans. For this reason, it is very difﬁcult to propose a therapeutic dose of RV. Regarding the insulin resistance markers (HOMA-IR) and HbA1c, in this review, no signiﬁcant changes were observed in these parameters, which, in addition to being consistent among most of the publications included, coincides with that reported by Zhu et al. [68]. 4. Discussion Among the biochemical parameters most used in research, due to their reliability in evaluating the therapeutic efﬁcacy of different nutraceuticals in the control of metabolic diseases, are HbA1c, insulin resistance (calculated by the HOMA-IR index), fasting glucose, and insulin. On the one hand, HbA1c is formed when glucose binds to an amino group of the β chain of hemoglobin through a non-enzymatic reaction that is inﬂuenced by the concentration of glucose in the blood, so that a state of hyperglycemia is manifested as a high percentage of HbA1c [66]. On the other hand, it is known that insulin is the most important regulator in glucose and lipid metabolism, so insulin resistance is a distinctive feature of obesity, T2DM, and cardiovascular diseases [67]. 13 of 21 13 of 21 Antioxidants 2021, 10, 69 The evidence from our meta-analysis shows that RV consumption does not improve HbA1c and insulin resistance, since, in most of the included studies, there were no signiﬁ- cant changes in these parameters. Given the above, our results suggest that RV administra- tion is not effective for prolonged glycemic control (around 90–120 days). However, there is considerable heterogeneity between the studies, which is attributed to the wide variation of RV dosage, duration of administration, and number of participants. Furthermore, some studies were at risk of bias in selection and blinding, due to the open and single-blind design [23,26,29,30,39,43,46,48]. Considering the general results and the inﬂuence of heterogeneity, a subgroup analysis was performed, stratifying the publications included by dose, health status, duration of intervention, and age of the participants. 4.2. Sub-Analysis by Health Condition According to the analysis by the presence or absence of T2DM, we observed that RV consumption had a positive effect on the four measured parameters (glucose, insulin, 14 of 21 14 of 21 Antioxidants 2021, 10, 69 HOMA-IR, and HbA1c), in favor of the subjects with T2DM, which was consistent with the majority of the results from clinical trials conducted in diabetic subjects that were included in the meta-analysis (Abdollahi et al.; Bhatt et al.; Hoseini et al.; Javid et al.; Khodabandenlhoo et al.; Movahed et al.; and Sattarinezhad et al.) [18,22,35,36,38,41,44]. They observed a signiﬁcant decrease in glycemic control markers after RV consumption in diabetic subjects. These results are consistent with the meta-analysis by Liu et al. [72], where they found that RV consumption signiﬁcantly reduced glucose, insulin, insulin resistance, and HbA1c levels in participants with T2DM. HOMA-IR, and HbA1c), in favor of the subjects with T2DM, which was consistent with the majority of the results from clinical trials conducted in diabetic subjects that were included in the meta-analysis (Abdollahi et al.; Bhatt et al.; Hoseini et al.; Javid et al.; Khodabandenlhoo et al.; Movahed et al.; and Sattarinezhad et al.) [18,22,35,36,38,41,44]. They observed a signiﬁcant decrease in glycemic control markers after RV consumption in diabetic subjects. These results are consistent with the meta-analysis by Liu et al. [72], where they found that RV consumption signiﬁcantly reduced glucose, insulin, insulin resistance, and HbA1c levels in participants with T2DM. p p The hypoglycemic effect of RV has been attributed to its antioxidant and anti- inﬂammatory properties. It is known that molecular targets include SIRT1, AMPK, nuclear factor kappa β, and transcription factor Nrf2, among others [73]. It has been demonstrated in several in vitro experiments and in vivo in diabetic animal models that RV increases glucose uptake, utilization, and storage, at the same time that it restores insulin signaling pathways and increases its sensitivity [74–76]. 4.2. Sub-Analysis by Health Condition The proposed mechanisms are the following: • Increases the expression of GLUT4 (an insulin-dependent glucose transporter) and improves glucose uptake; p g p • Activation of SIRT1, which modulates different metabolic pathways, as follows: (i) It deacetylates the FOXO 1 protein, inhibiting its activity and suppressing the apoptosis of pancreatic β cells; (ii) it reduces the expression of the nuclear factor kappa β, which translates into a decrease in the activity of inﬂammation markers and oxidative stress, responsible for the production of advanced glycation end products (AGE); (iii) it activates AMPK, which regulates various intracellular processes, such as energy metabolism, mitochondrial functions, and cellular homeostasis. AMPK inactivity is correlated with insulin resistance and tissue damage caused by hyperglycemia; and (iv) it activates FOXO 3 expression, thereby suppressing the production of reactive oxygen species and improving regulation in manganese superoxide dismutase (MnSOD) expression; • Decreases the expression of the AGE receptor (RAGE) that contributes to insulin resistance by modifying its receptor proteins, by phosphorylating the serine/threonine segment, causing insulin resistance. Therefore, the decrease in the production and activity of AGE improves insulin signaling; • Activation of factor Nrf2, which is a transcription factor that coordinates the activation of a wide range of genes of antioxidant systems, thereby increasing the activity of the antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), and catalase. In the meta-analysis carried out by Liu et al. [72], non-diabetic subjects who consumed RV did not show a signiﬁcant decrease in the glycemic control parameters. In this meta- analysis, we found similar results. The same has been reported in other investigations for healthy animal models [76]. In these studies, it was has been observed that RV admin- istration does not have a signiﬁcant effect on glucose, the lipid proﬁle, and the insulin sensitivity, although the cellular mechanisms are not entirely clear [9,11]. These results can be explained considering that, in normal physiological conditions, glucose and insulin concentrations are in an acceptable range. Therefore, there are no metabolic alterations and RV consumption does not activate the molecular targets or metabolic pathways that are affected due to the presence of T2DM. In this sense, the results suggest that RV does not cause hypoglycemia in healthy people, although more quality clinical trials are required to evaluate the effects of RV consumption in healthy people. 4.4. Sub-Analysis by Age Three groups were formed according to the age of participants: Those (i) under 45 years old; (ii) from 45 to 59 years old; and (iii) over 60 years old. Signiﬁcant changes in favor of RV were only presented for glucose, insulin, and HbA1c levels in the studies that included subjects aged 45 to 59 years, while the HOMA-IR index did not have signiﬁcant changes in any group. These results are in contrast to the ﬁndings of Crandall et al. [79] and Witte et al. [80], who found that RV administration in older adults improves the insulin sensitivity, plasma glucose, and glucose metabolism. However, in the clinical trials included in our meta- analysis, which were conducted in subjects under 45 years of age (Asghari et al.; Bo et al.; Godínez-Salas et al.; Poulsen et al. [19,25,33,43]), it was observed that glycemic control markers did not change. Moreover, among clinical trials with people older than 60 years, only Hoseini et al. [35] reported a signiﬁcant change in glucose levels after an intervention with 500 mg/day of RV for 4 weeks. Most of the studies where the age of the participants ranged between 45 and 59 years found signiﬁcant changes in the biomarkers of glycemic control, except those with low doses of RV (Kantartzis et al. [37]) or short intervention periods (Dash et al. [29]). RV is a nutraceutical widely studied for the control of metabolic diseases due to its antioxidant and anti-inﬂammatory properties. Its role has been demonstrated in preclinical studies, but its effects in humans are controversial. This is probably due to its unfavorable pharmacokinetics and its low bioavailability, which could be inﬂuenced by the intestinal microbiota [81,82]. In addition, the genetic inﬂuence is an important factor for the individ- ual response to RV [83]. RV has been shown to activate the expression of SIRT1, which is a histone deacetylase that plays a crucial role in glucose metabolism, lipids, the inﬂammatory process, and antioxidant defenses [74,75,84]. In vivo investigations have indicated that, in aging, the activity of SIRT1 is decreased [85], which could cause a poor response of the body to the administration of RV in older adults. 4.3. Sub-Analysis by Duration of Intervention Analysis by duration of the intervention (studies with an intervention <3 months and studies with an intervention ≥3 months) revealed a positive effect on glucose when the intervention was less than three months. HbA1c showed a signiﬁcant decrease when the intervention had a duration of more than three months, while the effect on insulin was Antioxidants 2021, 10, 69 15 of 21 15 of 21 positive in both interventions (<3 months and ≥3 months). However, the HOMA-IR index had no signiﬁcant effect regarding the duration of the intervention. positive in both interventions (<3 months and ≥3 months). However, the HOMA-IR index had no signiﬁcant effect regarding the duration of the intervention. g g g The discrepancy in glucose and HbA1c results is due to the serum glucose levels reﬂecting a very short period of glucose metabolism and being inﬂuenced by diet in the short term. In contrast, HbA1c reﬂects glucose metabolism for a period ranging from 90 to 120 days, which is why it is considered a highly reliable marker of long-term glycemic control. In this regard, the results of different clinical trials included in this meta-analysis show that the intervention time plays an important role in glycemic control. Abdollahi et al. [18] observed that the administration of 1 g/day of RV for 8 weeks is not enough to have a positive effect on HbA1C, despite lowering glucose levels, as did Thazhath et al. [47], who reported that 5 weeks of treatment with 1 g/day of RV has no effect on HbA1c levels in diabetic patients. On the other hand, Bhatt et al. [22] reported that 3 months of supplementation with 250 mg/day of RV signiﬁcantly reduces HbA1c, while Sattarinezhad et al. [44] found that 500 mg/day of RV for 3 months triggers a signiﬁcant decrease in HbA1C, insulin, and the HOMA-IR index. Our results are consistent with the study by Timmers et al. [77] carried out in obese subjects. This study reported that RV consumption for a period of 30 days improves glucose homeostasis and insulin resistance because it mimics the effects of caloric restriction. Meanwhile, the meta-analysis carried out by Guo et al. [78], who evaluated the effects of VR intervention on risk factors for NCDs, showed that a 3-month intervention signiﬁcantly reduces low-density lipoproteins (LDL-cholesterol) and HbA1c levels. 4.4. Sub-Analysis by Age The controversy about the biological effects of RV in humans justiﬁes the continuity of research, and it is necessary to know the efﬁcacy and safety of RV in the prevention and treatment of high prevalence metabolic diseases, most of which are related to oxidative stress and inﬂammatory process. Another important factor to elucidate is the metabolic Antioxidants 2021, 10, 69 16 of 21 16 of 21 pathways that it activates and how age, health status, dose, and time of treatment inﬂuence these. 4.5. Limitations This meta-analysis was not registered in PROSPERO; however, the guidelines estab- lished in PRISMA were followed. Conﬂicts of Interest: The authors declare no conﬂict of interest. doi:10.1111/dom.13409 Theodotou, et al. Exp Ther Med. 2018; 18: 559–56 p doi:10.3892/etm.2019.7607 Vatavuk-Serrati, et al. Rev Soc Cardiol Estado de São Voduc, et al. Appl. Physiol. Nutr. Metab. 2014. 39:1183–1 doi:10.3945/ajcn.115.122937 doi:10.3945/ajcn.115.122937 Ornstrup, et al. J Clin Endocrinol Metab. 2014;99:4720–4729, Ornstrup, et al. J Clin Endocrinol Metab. 2014;99:4720–4729, Ornstrup, et al. J Clin Endocrinol Metab. 20 doi:10.3390/ijms18030554 doi:10.3390/ijms18030554 j Maginley, et al. J Investig Med. 2019;67:793, doi:10.1136/jim-2019-001036.20 j Mahmood, et al. J. Pharm Sci Res. 2018;10(5):999–1005. Mahmood, et al. J. Pharm Sci Res. 2018;10(5):999–1005. Milton-Laskibar, et al. IUBMB. 2016, doi:10.1002/biof.1347 Most et al. Am J Clin Nutr 2016;104:215–27, 5. Conclusions 17 of 21 17 of 21 Antioxidants 2021, 10, 69 Appendix A. Studies Excluded from the Systematic Review and Meta-Analysis Study Reason for Exclusion Abdollahi, et al. BMJ. 2019;9:e026337, doi:10.1136/ bmjopen-2018-026337 It is a protocol Asghari, et al. Adv Phar Bull. 2018, 8(2), 307–317, doi:10.15171/apb.2018.036 They do not evaluate glycemic parameters Bo, et al. Acta Diabetol. 2018;55:331–3402018, doi:10.1007/s00592-017-1097-4 They do not report pre- and post-treatment means of glycemic parameters Brenjian, et al. Am J Reprod Immunol. 2020;83:e13186, doi:10.1111/aji.13186 They do not evaluate glycemic parameters Cao, et al. Exp Ther Med. 2018; 15: 576–584, doi:10.3892/etm.2017.5400 They do not evaluate glycemic parameters Crandall, et al. J Gerontol A Biol Sci Med Sci. 2012;67:1307–1312, doi:10.1093/gerona/glr235 It is a pilot study Foroghi, et al. IJEM. 2018;20:169-176. Language other than English Gospin, et al. J Investig Med 2016;64:800–825, doi:1 0.1136/jim-2016-000080.35 Only abstract available Huhn, et al. 2018. NeuroImage Doi:10.1016/j.neuroimage.2018.03.023 They use a combination of resveratrol with quercetin Kjaer, et al. The Prostate. 2015;75:1255–1263, doi:10.1002/pros.23006 They do not evaluate glycemic parameters Knop, et al. Diabet Med. 2013;61:1886 Doi:10.1111/dme.12231 They do not evaluate glycemic parameters Köbe, et al. Front. Neurosci. 2017. 11:105. Doi:10.3389/fnins.2017.00105 They use a combination of resveratrol with quercetin Konings, et al. Int J Obes. 2014;38:470–473. Doi:10.1038/ijo.2013.155 They do not evaluate glycemic parameters Korsholm, et al. Int. J. Mol. Sci. 2017, 18, 554; doi:10.3390/ijms18030554 They do not evaluate glycemic parameters Maginley, et al. J Investig Med. 2019;67:793, doi:10.1136/jim-2019-001036.20 Only abstract available Mahmood, et al. J. Pharm Sci Res. 2018;10(5):999–1005. They do not evaluate glycemic parameters Milton-Laskibar, et al. IUBMB. 2016, doi:10.1002/biof.1347 Study carried out on animals Most et al. Am J Clin Nutr 2016;104:215–27, doi:10.3945/ajcn.115.122937 They use a combination of resveratrol with epigallocatechin Ornstrup, et al. J Clin Endocrinol Metab. 2014;99:4720–4729, doi:10.1210/jc.2014-2799 They do not evaluate glycemic parameters Pankaj, et al. Biochem Bioph Res Co. 2015. 10.1016/j.bbrc.2015.10.126 Study carried out on animals Poulsen, et al. Diabetes Obes Metab. 2018;20:2504–2509, doi:10.1111/dom.13409 They do not report pre- and post-treatment means of glycemic parameters Theodotou, et al. Exp Ther Med. 2018; 18: 559–565, doi:10.3892/etm.2019.7607 The comparison group is not a placebo Van der Made, et al. Nutrients. 2017;9,596, doi:10.3390/nu9060596 They do not evaluate glycemic parameters Vatavuk-Serrati, et al. Rev Soc Cardiol Estado de São Paulo-Supl-2019;29(1):88–93 Language other than English Voduc, et al. Appl. Physiol. Nutr. Metab. 2014. 39:1183–1188. Doi.org/10.1139/apnm-2013-0547 It is a pilot study Walker, et al. J Clin Transl Res. 2019;4:122–135. doi:10.1210/jc.2014-2799 Pankaj, et al. Biochem Bioph Res Co. 2015. Doi:10.1038/ijo.2013.155 Doi:10.1038/ijo.2013.155 j Korsholm, et al. Int. J. Mol. Sci. 2017, 18, 554; 5. Conclusions The ﬁndings of this meta-analysis suggest that RV signiﬁcantly improves glucose and insulin levels in subjects with T2DM and aged 45–59 years, regardless of the duration of the intervention. Meanwhile, HbA1c improves signiﬁcantly with interventions whose duration is greater than 3 months. Nevertheless, the insulin resistance measured by HOMA-IR does not display signiﬁcant changes after RV administration. Regarding the dose used, the results do not allow a therapeutic dose to be suggested. Therefore, more clinical trials are required to identify how the RV dosage, duration of the interventions, health status, and age of the subjects inﬂuence the biological effects of RV, since, due to the heterogeneity presented by the available publications, the results are not conclusive. In this sense, it would be convenient to carry out studies that compare the effects of RV in young vs. old adults, in addition to studies comparing small vs. large doses. Long-term follow-up studies (>12 months) with intermediate measurements in the short and medium term (1, 3, 6, and 12 months, for example) could also be carried out, in order to observe and compare the effects of RV at different doses and durations. Finally, it is recommended that future clinical trials analyze and compare the results of subjects with different health conditions, such as diabetes, hypertension, dyslipidemias, and metabolic syndrome, since this will allow more clarity about the hypoglycemic efﬁcacy of RV, as well as the proposal of a therapeutic dose, depending on the patient’s conditions (age and health status). In addition to this, long-term research will be useful for obtaining data about the safety of this compound over long periods of time. Author Contributions: Conceptualization, V.M.M.-N. and J.P.-C.; methodology, B.I.G.-M. and M.R.- R.; software, B.I.G.-M.; validation, E.S.-O. and V.M.M.-N.; formal analysis, B.I.G.-M. and M.R.-R.; investigation, E.S.-O. and J.P.-C.; resources, E.S.-O.; data curation, J.P.-C. and V.M.M.-N.; writing— original draft preparation, B.I.G.-M. and M.R.-R.; writing—review and editing, V.M.M.-N.; visualiza- tion, E.S.-O.; supervision, J.P.-C.; project administration, V.M.M.-N.; funding acquisition, M.R.-R. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (DGAPA-UNAM) (PAPIIT IN308120); Posgrado en Ciencias Biológicas, UNAM; and Consejo Nacional de Ciencia y Tecnología (Beca de Posgrado, CVU 706595), Programa para la Investigación Bibliográﬁca Cientíﬁca sobre Salud (PIBCIS) de la FES Zaragoza, UNAM. 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https://openalex.org/W2979025845	https://europepmc.org/articles/pmc6779791?pdf=render	English	null	Dynamic Alterations of Spontaneous Neural Activity in Parkinson's Disease: A Resting-State fMRI Study	Frontiers in neurology	2,019	cc-by	6,695	Keywords: Parkinson’s disease, resting-state fMRI, dynamic brain activity, amplitude of low-frequency ﬂuctuations, support vector machine Dynamic Alterations of Spontaneous Neural Activity in Parkinson’s Disease: A Resting-State fMRI Study Chao Zhang 1†, Binru Dou 1†, Jiali Wang 1, Kai Xu 1, Haiyan Zhang 2, Muhammad Umair Sami 1, Chunfeng Hu 1, Yutao Rong 1, Qihua Xiao 3, Nan Chen 4 and Kuncheng Li 4 Chao Zhang 1†, Binru Dou 1†, Jiali Wang 1, Kai Xu 1, Haiyan Zhang 2, Muhammad Umair Sami 1, Chunfeng Hu 1, Yutao Rong 1, Qihua Xiao 3, Nan Chen 4 and Kuncheng Li 4 Chao Zhang 1†, Binru Dou 1†, Jiali Wang 1, Kai Xu 1, Haiyan Zhang 2, Muhammad Umair Sami 1, Chunfeng Hu 1, Yutao Rong 1, Qihua Xiao 3, Nan Chen 4 and Kuncheng Li 4 1 Department of Radiology, Afﬁliated Hospital of Xuzhou Medical University, Xuzhou, China, 2 Department of Radiology, Afﬁliated 2 Hospital of Xuzhou Medical University, Xuzhou, China, 3 Department of Neurology, Afﬁliated Hospital of Xuzhou Medical University, Xuzhou, China, 4 Department of Radiology, Xuanwu Hospital, Capital Medical University, Beijing, China Objective: To investigate the dynamic amplitude of low-frequency ﬂuctuations (dALFFs) in patients with Parkinson’s disease (PD) and healthy controls (HCs) and further explore whether dALFF can be used to test the feasibility of differentiating PD from HCs. Citation: Zhang C, Dou B, Wang J, Xu K, Zhang H, Sami MU, Hu C, Rong Y, Xiao Q, Chen N and Li K (2019) Dynamic Alterations of Spontaneous Neural Activity in Parkinson’s Disease: A Resting-State fMRI Study. Front. Neurol. 10:1052. doi: 10.3389/fneur.2019.01052 ORIGINAL RESEARCH published: 01 October 2019 doi: 10.3389/fneur.2019.01052 Methods: Twenty-eight patients with PD and 28 demographically matched HCs underwent resting-state functional magnetic resonance imaging (rs-fMRI) scans and neuropsychological tests. A dynamic method was used to calculate the dALFFs of rs-fMRI data obtained from all subjects. The dALFF alterations were compared between the PD and HC groups, and the correlations between dALFF variability and disease duration/neuropsychological tests were further calculated. Then, the statistical differences in dALFF between both groups were selected as classiﬁcation features to help distinguish patients with PD from HCs through a linear support vector machine (SVM) classiﬁer. The classiﬁer performance was assessed using a permutation test (repeated 5,000 times). Edited by: Wenbin Guo, Central South University, China Reviewed by: Xiuqin Jia, Capital Medical University, China Tianmei Si, Peking University Sixth Hospital, China Li Wang, Capital Medical University, China Correspondence: Kai Xu xukaixz@163.com Edited by: Wenbin Guo, Central South University, China Reviewed by: Xiuqin Jia, Capital Medical University, China Tianmei Si, Peking University Sixth Hospital, China Li Wang, Capital Medical University China Correspondence: Kai Xu xukaixz@163.com Results: Signiﬁcantly increased dALFF was detected in the left precuneus in patients with PD compared to HCs, and dALFF variability in this region was positively correlated with disease duration. Our results show that 80.36% (p < 0.001) subjects were correctly classiﬁed based on the SVM classiﬁer by using the leave-one-out cross-validation method. †These authors have contributed equally to this work Specialty section: This article was submitted to Applied Neuroimaging, a section of the journal Frontiers in Neurology Conclusion: Patients with PD exhibited abnormal dynamic brain activity in the left precuneus, and the dALFF variability could distinguish PD from HCs with high accuracy. Our results showed novel insights into the pathophysiological mechanisms of PD. Received: 07 June 2019 Accepted: 17 September 2019 Published: 01 October 2019 Keywords: Parkinson’s disease, resting-state fMRI, dynamic brain activity, amplitude of low-frequency ﬂuctuations, support vector machine INTRODUCTION Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by progressive impairment of motor function and widespread non-motor symptoms, which aﬀects patients’ quality of life and is, hence, a signiﬁcant social burden (1–3). At present, the pathophysiological mechanism of PD is not fully understood, and it is still a neuroimaging challenge to form a October 2019 \| Volume 10 \| Article 1052 Frontiers in Neurology \| www.frontiersin.org Resting-State fMRI Study of Parkinson’s Disease Zhang et al. deﬁnitive diagnosis at the early stage of the disease (1). Magnetic resonance imaging (MRI) has made great contributions in the clinical evaluation of PD (4, 5). Conventional MRI has been used to exclude secondary parkinsonism caused by neoplasms, vascular parkinsonism, and multiple sclerosis among others. The common imaging features of primary PD include iron deposition and substantia nigra atrophy (6, 7). However, several new reports have revealed that the cerebral region is widely involved in patients with PD. Therefore, it is important to explore novel imaging features that could help eﬀectively identify PD. exists with substantial ﬂuctuations either (24, 25). Furthermore, support vector machine (SVM) is one of the most widely used supervised machine-learning approaches that can enable individual-level classiﬁcation and prediction with high accuracy (26, 27). Uddin et al. (28) applied SVM to distinguish patients with autism from normal individuals with a classiﬁer accuracy of over 80%. Accordingly, SVM has been proposed as an eﬀective tool for diagnostic application in the clinic. To date, no studies have evaluated the combined eﬀects of dynamic ALFF and supervised machine-learning approaches on PD. Based on previous ﬁndings that a static ALFF has greater heterogeneity in PD and the proven property of dynamic methods, we hypothesized that dynamic intrinsic local spontaneous neuronal activity will show greater variability in subjects with PD than HCs, and that it may be another powerful index of rs-fMRI in exploring the underlying mechanisms of PD. We aimed to identify the cerebral regions that displayed abnormal dynamic local neuronal activity based on the voxel level of the whole brain. Furthermore, we applied SVM to observe whether the aberrant dALFF could be used as a feature to distinguish PD from HCs. This study may improve our understanding of the potential pathophysiological mechanism of PD. Moreover, we hope that this research can contribute to the clinical diagnosis of PD at an individual level. INTRODUCTION g g p y y In recent years, advanced neuroimaging techniques have allowed us to noninvasively explore the nature of the human brain in an eﬃcient manner (8–10). Resting-state functional MRI (rs-fMRI) is an established tool to investigate the intrinsic neuronal activity of the human brain by measuring the amplitude of spontaneous low-frequency ﬂuctuations (ALFFs) of blood- oxygen-level dependent (BOLD) signals (11–13). ALFF has been widely used to investigate regional brain activity in neurological diseases. Abnormal ALFFs in PD have been detected in extensive brain regions and act as an important characteristic related to subtypes of motor symptom or comorbidities (14). When compared with healthy controls (HCs), patients with tremor- dominant PD exhibited increased ALFF in the right cerebellar posterior lobe, while those with PD with postural instability/gait diﬃculty exhibited decreased ALFF in the bilateral putamen and cerebellar posterior lobe (15). PD patients with depression had signiﬁcantly lower ALFF in the prefrontal cortex and anterior cingulated cortex than PD patients without depression (16). PD patients with apathy showed lower ALFF in the left orbital middle frontal gyrus and bilateral superior frontal gyrus (17) than PD patients without apathy. Further, PD patients with visual hallucinations showed lower ALFF in both lingual gyrii/cunei and greater ALFF in the temporo-parietal regions, medial temporal gyrus, and cerebellum than those with non-visual hallucinations and HCs (18). By measuring the local spontaneous brain activities, these studies provided satisfactory evidence that widespread cerebral regions were involved in PD, which greatly contributed to the understanding the neurobiological foundations of such disorders. However, the results of these studies were limited in that their focus on abnormal brain activities in PD with diﬀerent motor/non-motor symptoms was based solely on group-level analysis; thus far, to our best knowledge, no study has used these abnormal features to distinguish PD with speciﬁc symptoms at an individual level. Frontiers in Neurology \| www.frontiersin.org Subjects This study was carried out in accordance with the tenets of the Helsinki Declaration and approved by the local ethics committee of Xuzhou Aﬃliated Hospital, Xuzhou Medical University. Written informed consent was obtained from all subjects before participation in the study. p p y The project used a convenience sample of 31 hospitalized patients with PD who met the UK Bank diagnostic criteria for PD (29). All patients underwent neuropsychological tests such as the Mini Mental Status Examination (MMSE) and the Montreal Cognitive Assessment (MoCA) and motor impairment evaluation including the Uniﬁed Parkinson’s Disease Rating Scale (UPDRS) and the Hoehn and Yahr (H-Y) stage when patients were oﬀmedication. The included patients had no history of mental illness or other neurological diseases. The exclusion criteria of the participants were MRI-conﬁrmed brain abnormalities such as trauma, stroke, tumor, and infection and contraindications to MRI such as claustrophobia and implanted metal devices. In addition, the subjects with a history of drug and/or alcohol abuse and syncope were also excluded. All patients underwent routine treatment, and none of them received any other relevant interventions. Thirty-two age- and sex-matched healthy volunteers were included as HCs. Three patients and four HCs were excluded because of head motion artifacts. Finally, 28 patients with PD (15 male and 13 female, 59.17 ± 9.72 years old) and 28 HCs (14 male and 14 female, 58.18 ± 6.46 years old) were included for analysis. There were no signiﬁcant intergroup diﬀerences with respect to age and sex (Table 1). All patients underwent functional MRI scanning when they were oﬀmedication; all the HCs also underwent the same protocol Previous studies on aberrant static ALFF in PD are inconsistent as they report both lower and higher local brain activity in various cerebral areas. However, a static state analysis of ALFF ignores the dynamic characteristics of brain activity during the whole scanning period. Evidences from rs-fMRI that employ a sliding window approach have eﬀectively detected dynamic functional connectivity features with higher sensitivity than the static state method (12, 19, 20). Recent studies reported that dynamic rs-fMRI analysis strategy not only made good contributions to human-brain exploration but also played an important role in studying the pathogenesis of schizophrenia (21–23). Nevertheless, few reports have focused on time-varying local spontaneous neuronal activity in PD, as ALFF itself October 2019 \| Volume 10 \| Article 1052 2 Resting-State fMRI Study of Parkinson’s Disease Zhang et al. Dynamic ALFF Analysis y y The analysis of dynamic amplitude of low-frequency ﬂuctuations (dALFF) was carried out using Temporal Dynamic Analysis (TDA) toolkits based on DPABI (34). Before dALFF calculation, functional volumes were bandpass ﬁltered (0.01–0.08 Hz) to minimize the inﬂuences of low-frequency drifts and ﬂuctuations of the signal. The sliding window is an important parameter to capture dynamic spontaneous neural activities, and the proper window length is critical for dynamic analysis. Too small a window length may not allow robust estimation of dynamic changes, and too long a window length may not be able to detect dynamic activity. Previous studies provided the range of the appropriate window length as 10–75 TR, step = 1 TR) (12, 35). To maximize the statistical power, a moderate sliding window length of 50 TR (step = 1 TR) was selected. The post-processed 175 volumes of each subject were segmented into 126 windows in all. The ALFF was calculated in each sliding window. The standard deviation (SD) of ALFF values of each voxel across 126 windows was further calculated to assess the variability of ALFF. We also calculated the static ALFF containing the whole sliding window. for MRI scanning and neuropsychological tests. All subjects were right-handed. for MRI scanning and neuropsychological tests. All subjects were right-handed. Statistical Analysis Two-sample t-test was used to observe intergroup diﬀerences in age and MoCA/MMSE scores. Sex-based group diﬀerence was evaluated using the chi-square test. A general linear mode (GLM) with age, sex, and mean FD as covariates was used to compare the diﬀerence of dALFF/ALFF between the PD and HC groups. Multiple comparisons were corrected using Gaussian Random-Field (GRF) method (voxel level, p < 0.001; cluster level, p < 0.05). Partial correlation analysis was calculated between dALFF variability and disease duration/MoCA/MMSE/UPDRS/H-Y with age and sex as covariates (p < 0.05). All statistical analyses were performed using SPSS version 16 (SPSS Inc., Chicago, IL, USA). MRI Data Acquisition All participants were scanned in a 3.0 Tesla MRI scanner (GE Medical Systems, Signa HD, Waukesha, WI) with an eight- channel head coil. During the scan, comfortable foam pads were used to stabilize the head of each subject to minimize head motion, and all subjects wore earplugs to reduce the noise from the MRI machine. Then, an echo-planar imaging sequence was employed to acquire resting BOLD images. The parameters of the protocol are as follows: time of repetition, 2,000 ms; time of echo, 30 ms; ﬁeld of view, 220 mm × 220 mm; slice thickness, 3 mm; slice gap, 1 mm; voxel size, 3.4 mm × 3.4 mm × 4.0 mm; number of slices, 36; ﬂip angle, 90◦; and total volume of each subject, 185. Subjects TABLE 1 \| Demographics and clinical data. Variable PD (N = 28) HCs (N = 28) P Sex (M/F) 15/13 14/14 0.789# Age (years) 59.17 ± 9.72 58.18 ± 6.46 0.794 Duration of PD (years) 8.46 ± 2.92 N/A N/A UPDRS-III 29.1 ± 8.70 N/A N/A H-Y 2.02 ± 0.71 N/A N/A MoCA 24.39 ± 2.52 25.86 ± 1.73 0.015* MMSE 27.64 ± 1.25 27.71 ± 1.24 0.831* CV values 0.18 ± 0.04 0.13 ± 0.12 <0.001* PD, Parkinson’s disease; HCs, healthy controls; M, male; F, female; UPDRS-III, uniﬁed Parkinson’s disease rating scale; H-Y, Hoehn and Yahr disability scale; MoCA, Montreal Cognitive Assessment; MMSE, Mini-Mental Status Examination. Data are presented as the range and mean ± SD. #The p-value was obtained using a chi-square test. *The p-value was obtained by a general linear mode analysis. TABLE 1 \| Demographics and clinical data. there is still some controversy regarding removal of the global signal (31–33). Preprocessing of rs-fMRI Data p g The rs-fMRI data preprocessing were carried out using data Processing and Analysis for (Resting-State) Brain Imaging (DPABI) (http://www.rfmri.org/dpabi) (30). The ﬁrst 10 time points of each subject were excluded to stabilize the status and allow participants to adapt to the scanning condition. Slice timing was carried out on the remaining 175 volumes to correct time diﬀerences. Realignment was performed to correct head motion by using a Friston-24 model for individual-level correction, and any subject with a head maximum displacement >2 mm, maximum rotation >2.0◦, or mean framewise displacement (FD) >0.3 was excluded. In our study, mean FD was set as a covariate for further group-level statistics to minimize the potential inﬂuences of head motion. Several covariates such as the Friston 24 head-motion parameters, cerebrospinal ﬂuid signal, and white matter signal were regressed. Then, the processed volumes were normalized to the standard Montreal Neurological Institute (MNI) echo planar imaging (EPI) template with a voxel size of 3 mm × 3 mm × 3 mm. Finally, functional volumes were smoothed with 6-mm full width at half maximum. We did not carry out global signal regression of our data given that Frontiers in Neurology \| www.frontiersin.org Differences in ALFF/Dynamic ALFF and Correlational Analysis Region Cluster size (voxel) MNI (x,y,z) t-value Left Precuneus 94 (−12, −42, 60) 4.34 Support Vector Machine Analysis Support Vector Machine Analysis The intergroup dALFF diﬀerence was used as the classiﬁcation feature in this study. We then trained the SVMs by providing labeled observations, for which the classiﬁcation results were known. To overcome the limitations of our samples, the leave- one-out cross-validation (LOOCV) method was applied to estimate the generalization ability of our classiﬁer. To verify the ability of the validation strategy, we also made a classiﬁcation comparison by introducing 10-fold cross-validations. Then, the total accuracy, sensitivity, and speciﬁcity were obtained to assess classiﬁer performance. A permutation test was used to evaluate the statistical signiﬁcance of this classiﬁcation accuracy (36). The permutation test was repeated 5,000 times, and during each time, the classiﬁer randomly reallocated labels of PD and HC to the training subjects and repeated the entire classiﬁcation process. The p-value was obtained after the total permutation was accomplished. October 2019 \| Volume 10 \| Article 1052 Frontiers in Neurology \| www.frontiersin.org 3 Resting-State fMRI Study of Parkinson’s Disease Zhang et al. DISCUSSION Upon literature review, we observed that only few studies employed a TDA method to explore the neural-activity characteristics of PD. The present study showed the following ﬁndings: (1) the dALFF of patients with PD compared to HCs was notably diﬀerent in the left precuneus; (2) a signiﬁcant correlation between CV of dALFF in the left precuneus and the course of the disease was found in PD; (3) dALFF in the left precuneus showed high accuracy in distinguishing between patients with PD and HCs. Differences in ALFF/Dynamic ALFF and Correlational Analysis The intergroup diﬀerences in dALFF are shown in Figure 1 and Tables 1, 2. Compared with HCs, signiﬁcantly increased coeﬃcient of variation (CV) of dALFF was noted in the left precuneus of PD patients (p < 0.001). In addition, we found that the CV of dALFF was positively correlated with disease duration (p < 0.001, r = 0.800) (Figure 1), and no signiﬁcant correlation was found between dALFF variability and MoCA/MMSE/UPDRS/H-Y scores (Supplementary Material). There were no signiﬁcant intergroup diﬀerences in ALFF. Figure 1 and Tables 1, 2. Compared with HCs, signiﬁcantly increased coeﬃcient of variation (CV) of dALFF was noted in the left precuneus of PD patients (p < 0.001). In addition, we found that the CV of dALFF was positively correlated with disease duration (p < 0.001, r = 0.800) (Figure 1), and no signiﬁcant correlation was found between dALFF variability and MoCA/MMSE/UPDRS/H-Y scores (Supplementary Material). There were no signiﬁcant intergroup diﬀerences in ALFF. Classiﬁcation Results Classiﬁcation results are shown in Figure 2. The accuracy of linear SVM classiﬁer using LOOCV achieved an accuracy of 80.36%, sensitivity of 85.71%, and speciﬁcity of 75% (p < 0.001, was notably diﬀerent in the left precuneus; (2) a signiﬁcant correlation between CV of dALFF in the left precuneus and the course of the disease was found in PD; (3) dALFF in the left precuneus showed high accuracy in distinguishing between patients with PD and HCs. Prior studies have noted the importance of cerebral static local neural activities in PD (4, 14, 15). To our best knowledge, dynamic changes in spontaneous neural activity has been very TABLE 2 \| dALFF alterations between PD groups and HCs. Region Cluster size (voxel) MNI (x,y,z) t-value Left Precuneus 94 (−12, −42, 60) 4.34 FIGURE 1 \| Increased CV of dALFF in the left precuneus displayed in coronal (A), sagittal (B), transverse (C), and three-dimensional view (D). Box plots with Whiskers (min–max) show the CV values in the left precuneus of the two groups (E), and scatterplots show the relationship between the CV in the precuneus of the PD group and the disease duration (F). CV, coefﬁcient of variation; dALFF, dynamic spontaneous low-frequency ﬂuctuation. Prior studies have noted the importance of cerebral static local neural activities in PD (4, 14, 15). To our best knowledge, dynamic changes in spontaneous neural activity has been very TABLE 2 \| dALFF alterations between PD groups and HCs. RESULTS non-parametric permutation approach). The receiver operating characteristic (ROC) curve of the classiﬁer was 0.82. A 10-fold validation was also employed in our study to verify the reliability of the classiﬁcation method, which generated a classiﬁer accuracy of 71.43%. Demographics and Clinical Data The details of age, sex, and MoCA/MMSE scores are listed in Table 1. The results showed no signiﬁcant diﬀerence in age (p = 0.652), sex (p = 0.789), and MMSE (p = 0.831) between the PD and HC groups. However, the MoCA score of the PD group was signiﬁcantly lower than that of the HCs (p < 0.05). Classiﬁcation Results Classiﬁcation results are shown in Figure 2. The accuracy of linear SVM classiﬁer using LOOCV achieved an accuracy of 80.36%, sensitivity of 85.71%, and speciﬁcity of 75% (p < 0.001, FIGURE 1 \| Increased CV of dALFF in the left precuneus displayed in coronal (A), sagittal (B), transverse (C), and three-dimensional view (D). Box plots with Whiskers (min–max) show the CV values in the left precuneus of the two groups (E), and scatterplots show the relationship between the CV in the precuneus of the PD group and the disease duration (F). CV, coefﬁcient of variation; dALFF, dynamic spontaneous low-frequency ﬂuctuation. FIGURE 1 \| Increased CV of dALFF in the left precuneus displayed in coronal (A), sagittal (B), transverse (C), and three-dimensional view (D). Box plots with Whiskers (min–max) show the CV values in the left precuneus of the two groups (E), and scatterplots show the relationship between the CV in the precuneus of the PD group and the disease duration (F). CV, coefﬁcient of variation; dALFF, dynamic spontaneous low-frequency ﬂuctuation. October 2019 \| Volume 10 \| Article 1052 4 Frontiers in Neurology \| www.frontiersin.org Resting-State fMRI Study of Parkinson’s Disease Zhang et al. FIGURE 2 \| Classiﬁcation accuracy of altered dynamic ALFF in the left precuneus obtained by the leave-one-out (red line) and nested 10-fold (blue line) cross validation methods in PD groups, respectively. FIGURE 2 \| Classiﬁcation accuracy of altered dynamic ALFF in the left precuneus obtained by the leave-one-out (red line) and nested 10-fold (blue line) cross validation methods in PD groups, respectively. poorly researched. The rs-fMRI analysis was based on the hypothesis that brain activity was in a stationary state during the entire scanning period; thus, the dynamic characteristics could not be identiﬁed. Dynamic algorithm was proven to represent the time-dependent characteristics of brain activity under the given scanning period. A recent study captured abnormal dALFF/ Regional Homogeneity (ReHo) in stroke patients by using TDA and compared it with HCs; they further found that variability in brain activity could be used to evaluate patients’ motor function (24). Liu et al. (12) found abnormal functional network connection (FNC) through dynamic instead of static state. Dynamic FNC was signiﬁcantly correlated with the frequency of epileptic seizures and the course of the disease. Frontiers in Neurology \| www.frontiersin.org Classiﬁcation Results In addition, morphological changes of the left precuneus were also found in PD with cognitive impairment and isolated apathy through voxel- based morphometry (44, 45). Therefore, the left precuneus is undoubtedly an important and vulnerable structure in patients with PD. Our study has some limitations. First, all patients were on medication. Although patients underwent fMRI scanning while they were oﬀmedication, the eﬀects of the long-term treatment could not be completely ruled out. Second, the classiﬁcation power based on the 28 PD patients was still not strong enough, and we just used SVM in the same sample to testify the classiﬁcation accuracy. Third, the patients did not undergo comprehensive cognitive scales testing, which could have prevented a more accurate detection of cognitive performance. Future research should include a larger sample size and another independent test sample should be recruited for testifying classiﬁcation accuracy. In our study, the higher variability of dynamic local brain activity level in the left precuneus was positively correlated with the course of PD. This meant that the degree of variation was signiﬁcantly increased with an extended disease course, which reﬂected the increased or decreased brain activity at diﬀerent sliding windows during the whole scanning period. These ﬁndings revealed a localized brain function impairment over time in PD. However, the abnormal dALFF did not correlate with UPDRS/H-Y scores in the present ﬁndings, likely because the heterogeneous motor symptoms in PD were associated with integration of multiple cerebral region function, rather than being determined by a single brain region impairment (46, 47). Unlike previous reports, the present study did not ﬁnd signiﬁcant intergroup diﬀerences with respect to ALFF, either because the sample size in our study was relatively small or because the result was not powerful enough to pass the multiple comparison correction of the present statistical methods. In fact, the dynamic features were concealed under the static analysis that represented a measure of the average amplitude of local activity across diﬀerent scanning time points within the whole scan (12). Thus, static rs-fMRI may not be as sensitive as dynamic analysis to detect neural-activity changes. Our study indicated that dynamic analysis could completely unearth information of brain activity. In addition, the present result suggested that the left precuneus was an important structure involved in PD, and higher dALFF in this region was a promising imaging marker reﬂecting the disease duration. Classiﬁcation Results Furthermore, dynamic FNC could distinguish patients with idiopathic generalized epilepsy with generalized tonic–clonic seizures from controls with an accuracy of 77.91% through linear SVM classiﬁer (p < 0.001). Dynamic algorithm showed the capability to characterize neural activity of the human brain by identifying speciﬁc function signatures. result was partially consistent with previous reports that the left precuneus was a key cerebral region in patients with PD. Precuneus, which mainly constitutes the medial and posterior part of the parietal lobe, contributes to motor and cognitive tasks, and has been reported as displaying the highest resting metabolic rate among all cerebral regions (37). Interestingly, the precuneus consumed 35% more glucose than other brain regions in the resting brain (38). Perfusion single photon-emission computed tomography (SPECT) and [18F]ﬂuorodeoxyglucose positron emission tomography ([18F]FDG-PET) proved that the precuneus was the most remarkable area of hypometabolism in the posterior cortical region (39). Another [18F]FDG-PET- based study on PD found that the metabolic capability of the left precuneus decreased with disease progression (40). Similarly, a more recent research using the arterial spin labeling (ASL) technique showed that cerebral blood ﬂow (CBF) in the left precuneus signiﬁcantly reduced in the PD group when compared with HCs (41). These functional imaging studies supported the view that the left precuneus might be more prone to attack in neuropsychiatric disorders. Additional rs-fMRI studies showed that the left precuneus was closely Our dynamic algorithm showed that the dALFF in the left precuneus in PD was notably diﬀerent than HCs. The October 2019 \| Volume 10 \| Article 1052 5 Resting-State fMRI Study of Parkinson’s Disease Zhang et al. nested 10-fold cross-validation method was used to assess the classiﬁer’s performance; the accuracy was 71.43%. These ﬁndings showed that SVM could achieve better classiﬁcation capability with LOOCV, and the results also provided evidence that patients with PD could be distinguished from HCs at the individual level when using dALFF variation in the left precuneus. These results support the hypothesis that the dALFF could identify individual PD patients. associated with motor and non-motor symptoms in PD. Hu et al. (42) found that increased local brain activity in the left precuneus was related to the Hamilton Depression Rating Scale score, by using static brain activity analysis. Thibes et al. (43) used a brain connection algorithm and showed that the left precuneus was a critical node connecting with speciﬁc cerebral regions in PD. CONCLUSION To our best knowledge, this is the ﬁrst study to attempt to investigate the dynamic spontaneous neural activities in patients with PD. Our results provided evidence that dynamic analysis was more sensitive to detect alteration of brain activity than a static method. In addition, the CV of dALFF was found to be correlated with the course of the disease, which may ultimately contribute to identifying PD at the individual level. Thus, our results provide novel insights on the pathophysiological mechanisms of PD. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. Classiﬁcation Results Besides, our ﬁndings did not show a correlation between dALFF and MoCA/MMSE tests. This may be because the MoCA and MMSE scales were mainly appropriate for cognitive screening, and our study lacked detailed assessment of cognitive performance compared to previous studies (48). ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethical committee of Xuzhou Aﬃliated Hospital. The patients/participants provided their written informed consent to participate in this study. The imaging diagnosis of PD remains a challenge even now, as a conﬁrmed diagnosis in most patients is still made depending on the clinical symptoms (49). An assessment of the iron content and volume of substantia nigra may be useful indicators to identify PD and evaluate the disease progression (6, 7). However, this approach has not been widely applied in the clinical management of patients with PD. Previous reports demonstrated that SVM was a powerful tool utilizing imaging features to distinguish PD patients from HCs. In our study, we tested the inter-group diﬀerence of dALFF in the left precuneus as a classiﬁcation feature to discriminate PD from HCs through a linear SVM classiﬁer. 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https://openalex.org/W2118045122	http://umu.diva-portal.org/smash/get/diva2:390899/FULLTEXT01	English	null	Effectiveness of maternal referral system in a rural setting: a case study from Rufiji district, Tanzania	BMC health services research	2,010	cc-by	8,261	© 2010 Pembe et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The functional referral system is important in backing-up antenatal, labour and delivery, and postnatal services in the primary level of care facilities. The aim of this study was to evaluate the effectiveness of the maternal referral system through determining proportion of women reaching the hospitals after referral advice, appropriateness of the referral indications, reasons for non-compliance and to find out if compliance to referrals makes a difference in the perinatal outcome. Methods: A follow-up study was conducted in Rufiji rural district in Tanzania. A total of 1538 women referred from 18 primary level of care facilities during a 13 months period were registered and then identified at hospitals. Those not reaching the hospitals were traced and interviewed. Results: Out of 1538 women referred 70% were referred for demographic risks, 12% for obstetric historical risks, 12% for prenatal complications and 5.5% for natal and immediate postnatal complications. Five or more pregnancies as well as age <20 years were the most common referral indications. The compliance rate was 37% for women referred due to demographic risks and more than 50% among women referred in the other groups. Among women who did not comply with referral advice, almost half of them mentioned financial constraints as the major factor. Lack of compliance with the referral did not significantly increase the risk for a perinatal death. Conclusion: Majority of the maternal referrals were due to demographic risks, where few women complied. To improve compliance to maternal referrals there is need to review the referral indications and strengthen counseling on birth preparedness and complication readiness. Effectiveness of maternal referral system in a rural setting: a case study from Rufiji district, Tanzania ndrea B Pembe1,2, Anders Carlstedt2,3†, David P Urassa4†, Gunilla Lindmark2†, Lennarth Nyström5 http://www.diva-portal.org http://www.diva-portal.org This is the published version of a paper published in BMC Health Services Research. Citation for the original published paper (version of record): Pembe, A., Carlstedt, A., Urassa, D., Lindmark, G., Nyström, L. et al. (2010) Effectiveness of maternal referral system in a rural setting: a case study from Rufiji district, Tanzania. Access to the published version may require subscription. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-39339 Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Open Access Correspondence: andreapembe@yahoo.co.uk † Contributed equally 1Department of Obstetrics and Gynaecology, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania Full list of author information is available at the end of the article Background There are 56 primary level of care facilities of which four are rural health centres (RHCs) owned by the government, 47 government dispensaries and five private dispensaries. pregnant women attending at least once but on the other hand only 47% of deliveries take place in health facilities [6]. Based on the demographic and health sur- vey in 2005 the maternal mortality ratio (MMR) was estimated at 578 per 100,000 live births [6], however, the United Nations arrived at a much higher estimate (950) based on adjustments made on the data collection methods [1]. The Reproductive and Child Health Card 4 (RCHC-4) of the Ministry of Health and Social Welfare (MoHSW) of Tanzania has guidelines for referrals of pregnant women to the hospital. These guidelines do not make a difference between historical risk factors and actual complications in the present pregnancy. Studies in Tan- zania and elsewhere have shown that compliancy to referral advice is low [7-10]. For women to comply with referral they have to understand that something is wrong with the pregnancy. Other factors which may hinder acceptance of referral advice include lack of reli- able transport, costs involved and perceived quality of care at the hospitals [7,11,12]. The primary levels of care facilities are the main access point for maternal and child health services in rural areas. They are staffed primarily by clinical officers who are prescribers and most common the in-charge of the facilities, nurses and/or maternal and child health (MCH) aides as trained maternal service providers. In some dispensaries nurse auxiliaries with one year train- ing or on job training on provision of maternal services are providing maternal care. The rural health centers are larger than the dispensaries and have in addition beds for admitting patients. There is no user fee for antenatal and delivery care services except at Mchukwi mission hospital where investigations and operative pro- cedures are charged. Women book for antenatal care at the health facility nearest to them. All the primary level of care facilities provide antenatal and delivery care for low risk women and are supposed to refer women according to referral indications stipulated in the Minis- try of Health and Social Welfare RCHC-4. The aim of the study was to evaluate the effectiveness of the maternal referral system in a rural district. Background complication occurs. The shift has been due to the fact that most maternal deaths occur during labour, delivery and the first day postpartum. The complications leading to these deaths are unpredictable but can be treated if diagnosed early [4,5]. The extent to which this approach will reduce maternal and neonatal mortality is not known as the capacity at the primary level of care to deal with complications is limited by the availability of skilled human resources and facilities. Moreover, acces- sibility to the hospitals during obstetric emergencies remains difficult in most low-resource countries. The WHO estimates that in 2005, 536 000 maternal deaths occurred due to complication of pregnancy and childbirth and more than half of these occurred in sub- Saharan Africa [1]. Furthermore 3.7 million deaths of newborns occur in the first 28 days of life, 50% of these deaths occur in the first 24 hours of life and 75% in the first week of life. There are 32 stillbirths per 1000 deliv- eries of which 24-37% are intrapartum deaths [2,3]. In the last decade the approach to reduce maternal and perinatal mortality has shifted from the risk approach involving identification of high risk pregnancies which can develop complications to provision of skilled care during delivery and emergency obstetric care when a Tanzania is a low-resource country which has pyrami- dal shaped health care infrastructure with dispensaries and health centres as primary level of care facilities at the base and referral hospitals at the apex. The district hospitals are the first referral hospitals for maternal care where most obstetric intervention including surgery and blood transfusion are available. There is high utilization of antenatal care services with more than 94% of * Correspondence: andreapembe@yahoo.co.uk † Contributed equally 1Department of Obstetrics and Gynaecology, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania Full list of author information is available at the end of the article Page 2 of 9 Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 close by and first referral for all primary level of care facilities. Caesarean section and blood transfusion can only be provided at the hospitals, while vacuum extrac- tion rarely can be provided at primary level of care or hospitals. Background The evaluation was performed through determining the pro- portion of women reaching the hospitals after being referred, appropriateness of the referral indications, rea- sons for non-compliance to the referral and to find out if compliance to referrals makes a difference in the peri- natal outcome. Only the district hospital has an ambulance which serves all the primary level of care facilities with emer- gency referrals to the district hospital. Another vehicle owned by the Mchukwi hospital shuttle three times per day between the hospital and the nearby towns. In emergency referrals health workers assist the woman and family to arrange for transport by calling for the ambulance from the district hospital, but they have to pay for fuel. Sample size and data collection According to Kielmann et al 1995 and UNICEF 1997, a random sample of 25% to 30% of the health facilities in a district of an average size is usually adequate and fea- sible to represent a district health service situation [14,15]. All four RHCs and 14 randomly selected dispen- saries in the flood plains and plateau zones among those with five or more deliveries per month were included in the study. The delta zone was not included due to diffi- culties in accessing the area. The primary levels of care facilities included covered 54% of the population in the district. The sample size of referred women was calculated using the soft ware Epi Info 6. Based on a study in Gutu, Zimbabwe [16] with an antenatal and delivery referral rate of 36%, a desired precision of 5%, 95% con- fidence interval, and a power of 90% a sample size of 364 referred women could have been sufficient. How- ever to be able to compare with other studies on the use of obstetric care and captured variation of maternal referrals during rain and dry seasons, all maternal refer- rals to the hospitals from 1 June 2007 to 30 June 2008 were recorded. A parallel data collection system was established since the routine data collection indicated the risks but no information whether the women were referred to hospi- tal or not. Health workers at the primary level of care facilities received refresh training on the RCHC-4 with an emphasis on the referral indications. Accurate recording of all women referred to hospital during preg- nancy, delivery and after delivery was emphasized. Dur- ing the training, it was emphasized that all health workers should stick to the national guidelines on refer- ral indications and the health workers should repeat advising the women on referral in the subsequent visits. Information on women’s socio-demographic characteris- tics and indications for referral were collected. If a woman had more than one indication for referral, the one associated with worse outcome or needing urgent attention based on obstetrician assessment was taken. A research identification number tag was stapled on the woman’s antenatal card. Women that were referred were identified by the trained health workers at the hos- pitals, who recorded the treatments and outcome of deliveries if delivery takes place at the hospital. Methods Study area d According to the projection from the 2002 population and housing census the population in Rufiji district in 2007 was estimated at 240,000 with a yearly growth rate of 2.5% [13]. Geographically the Rufiji river intersects the district from West to East dividing it into flood plain, coastal-delta, and plateau zones. The district experiences a heavy rainy season from February to May and a less intense one from October to December. The majority of the population are peasants growing cassava, rice, sorghum and maize as food crops. Cashew nuts and coconuts are the main cash crops available. Com- monly their farms are located some distance from the family home and residents shift to temporary dwellings at the farms during the heavy rain season. Transport in the district includes canoes, boats, motor vehicles and bicycles. Most of the roads in the district are unpaved and difficult to pass especially during the rainy season. The RCHC-4 is divided into three sections; the preg- nancy care, delivery and immediately after delivery care, and follow up after delivery care. In the first part, the prenatal care, there are three categories of referral indi- cations named A, B and C. A woman with one of the indications in category A should be referred for further investigations while one with indication in category B, should be referred for delivery. A woman requiring referral in category C is supposed to be referred imme- diately to hospital. The referral indications in category A, B and C are presented in the table below (Table 1). In the natal and immediate postnatal period women with referral indication should as well be referred imme- diately to the hospital. Indications during this period include spontaneous rupture of membranes without labour, labour before 34 weeks, labour for >12 hours/ obstructed, abnormal lie or presentation of the baby, vaginal bleeding, variability of foetal heart beats ( < 120 The district has two hospitals; the government owned district hospital located in the district town Utete south of the river and the non profit mission hospital Mchukwi located in the northern part of the river. The hospitals serve as a first line centre for women living Page 3 of 9 Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Pembe et al. Methods Study area d BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Table 1 Referral indications during pregnancy care according to RCHC-4, Tanzania Category A Age below 20 years Ten or more years since last pregnancy Previous caesarean sectiona Previous stillbirth/perinatal death (within one week) Three or more consecutive abortions Intercurrent illnesses (heart disease, diabetes mellitus, tuberculosis) Category B ≥5 pregnancies Height <150 cm Pelvic deformity First pregnancy at 35 or more years Previous caesarean sectiona or vacuum delivery Postpartum haemorrhage in previous delivery Retained placenta in the previous delivery Category C Blood pressure ≥140/90 mmHg Haemoglobin less than 60% (8.5 gm/dl) Albumin in urine Sugar in urine Gestational age more than 40 weeks Intrauterine foetal death Abnormal lie after 36 weeks Oedema of the legs, face and hands Suspected twin pregnancy Fundal height too big or too small for gestation age Danger signsb aCaesarean section is in category A and B. bDanger signs are not stated Table 1 Referral indications during pregnancy care according to RCHC-4, Tanzania Table 1 Referral indications during pregnancy care according to RCHC-4, Tanzania delivery actually come back to the primary level of care in labour for delivery. It is up to the discretion of the health worker to decide to re-refer the women or con- duct the delivery. Table 1 Referral indications during pregnancy care according to RCHC-4, Tanzania Category A Age below 20 years Ten or more years since last pregnancy Previous caesarean sectiona Previous stillbirth/perinatal death (within one week) Three or more consecutive abortions Intercurrent illnesses (heart disease, diabetes mellitus, tuberculosis) Category B ≥5 pregnancies Height <150 cm Pelvic deformity First pregnancy at 35 or more years Previous caesarean sectiona or vacuum delivery Postpartum haemorrhage in previous delivery Retained placenta in the previous delivery Category C Blood pressure ≥140/90 mmHg Haemoglobin less than 60% (8.5 gm/dl) Albumin in urine Sugar in urine Gestational age more than 40 weeks Intrauterine foetal death Abnormal lie after 36 weeks Oedema of the legs, face and hands Suspected twin pregnancy Fundal height too big or too small for gestation age Danger signsb aCaesarean section is in category A and B. bDanger signs are not stated Category A Results A total of 5596 women booked for antenatal care in the selected primary levels of care. There were 1538 (28%) women referred to hospitals and out of these 1079 (70%) were referred due to demographic risks, 186 (12%) due to obstetric historical risks, 189 (12%) with prenatal complication and 84 (5.5%) with natal and immediate postnatal complications. The median age of referred women was 24 years (Range: 14-48) and the median gravidity was 3 (Range: 1-15). A majority of women were married/cohabiting (71%), peasants (90%) and had primary education (54%). The structural quality of the hospitals was assessed. Information on qualified staff for provision of emer- gency obstetric care, functioning operative theatre, blood transfusion facilities, evacuation facilities, func- tioning vacuum extractor and availability of antibiotics, oxytocics and anticonvulsants. Majority (70%) of the women were referred due to demographic risks. Five or more pregnancies (34%) and age <20 years (30%) were the most common indications contributing to 65% of all referrals. In the group of obstetric historical risks, previous caesarean section made up 65% of all referrals in the group. Low haemo- globin (2.7%), elevated blood pressure (2.3%) and abnor- mal lie/presentation (2.1%) were the most common indications contributing to 58% of all referrals in the prenatal complications group. Labour for >12 hours/ obstructed contributed to 4.2% of all women referred from the primary level of care facilities and 78% of women referred in the group with natal complications. Sample size and data collection The data collection forms were reviewed by the first author in each primary level of care and the health workers com- pleted missing information. aCaesarean section is in category A and B. bDanger signs are not stated or >160 beats per minute), elevated body temperature of >38 Centigrade, eclampsia or blood pressure ≥140/90 mmHg, haemoglobin <60% (8.5 gm/dl), small pelvis or big baby, meconeum, retained placenta, severe perineal tear and blood loss ≥500 mls. During the first visit women are screened for referral indications in category A and B. For those identified as in need of referral a tick is made on a specific box on the antenatal card and the woman is informed of the need to go to hospital for further assessment or for delivery. During the subsequent antenatal care visits women given referral advice should be emphasized on the referral advice given. Those referred for delivery are advised to stay near the hospitals at the late months of pregnancy despite there are no maternity waiting homes near the hospitals. Women and their families have to arrange for a place to stay either in guesthouses or by their relatives. If a woman develops any of the indica- tions for referral in category C during her prenatal care, she is referred immediately to hospital. Some women given a referral to hospital for further assessment or Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Page 4 of 9 The woman was regarded complied with referral advice when she reached the hospital after being referred from the primary level of care facility due to any of the indications. Women who did not reach the hospitals were identified by comparing the register books in the primary level of care and the hospitals. These women were traced by the primary level of care providers in their respective catchment’s areas. Those women contacted were asked about reasons for not going to the hospital and their pregnancy outcome. If the mother had deceased, a relative or anybody who was with the mother during the incident was interviewed. All participants agreed voluntarily to participate in the study after being informed of the aim and the consent sought from them. Statistical analysis The referral indications in category A and B in the RCHC-4 card is a mixture of demographic and obstetri- cal risks, we re-grouped the referral indications in these two categories to make the group containing only the demographic risks and a second group with risks from the obstetrical history and risks related to delivery. The final groups were the demographic risk factors, obstetric historical risks, prenatal complications, natal complica- tions and immediately postnatal complications. Factors which may have higher risks of perinatal mortality were calculated. All factors in the demographic risk and obstetric historical risks groups were used in the calcu- lation of risk of perinatal death. The prenatal and natal complications groups were calculated together and the risk factors included were haemoglobin <60%, blood pressure ≥140/90 mmHg, abnormal lie/presentation, vaginal bleeding, large fundal height for gestation age, labour for >12 hours/obstructed and eclampsia. The risk of perinatal death was not calculated in the postnatal complications group as complications in this group do not affect perinatal outcome. The software SPSS was used for statistical analysis. The risk for perinatal death for women not complying with the referral in relation to those complying was calculated using odds ratio (OR) and 95% confidence intervals (CI). Reaching the hospital after being given referral advice from the primary level of care facility was regarded as compliance. Out of 1538 women referred 45% complied. The compliance rate for women referred due to demo- graphic risks, obstetric historical risks, prenatal compli- cations and natal complication was 37%, 65%, 56% and 78% respectively. In the obstetric historical risks group women with previous caesarean section (69%), height <150 cm (65%) and history of stillbirth/perinatal death (58%) had the highest compliance rate. Other indication with high compliance rate were blood pressure ≥140/90 mmHg, abnormal lie/presentation and uterus too big for gestational age in the prenatal complication group and labour for >12 hours/obstructed in the natal complica- tion group. The lowest compliance rate was found for ≥5 pregnancies, history of severe bleeding after delivery, ≥10 years since last delivery, intercurrent illnesses and age <20 years (Table 2). Ethical consideration Financial constraint was the major reason for not complying in 53% in demographic risks, 54% in obstetric historical risks, 46% in prenatal complications, and 70% in the natal complications groups. Other reasons were difficulty acquiring transport, a labour that started sud- denly, delivering on the way to hospital and lack of accompaniment to the hospital. Out of 431 women The protocol was reviewed and approved by the Muhimbili University of Health and Allied Sciences, the Senate research and Publication committee as part of the on going studies on quality assessment and monitor- ing of maternal referrals in the district. Permission to conduct the study was obtained from the Rufiji District Medical Officer and District Executive Director’s office. Page 5 of 9 Pembe et al. cSelf request, wanted bilateral tubal ligation, severe abdominal pain, pendulous abdomen, abdominal mass, paraumbilical hernia Indications not in the RCHC 4 bHeart disease, diabetes mellitus, tuberculosis, HIV, mental illness, epilepsy bHeart disease, diabetes mellitus, tuberculosis, HIV, mental illness, epilepsy cSelf request, wanted bilateral tubal ligation, severe abdominal pain, pendulous abdomen, abdominal mass, paraumbilical hernia Ethical consideration BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Page 6 of 9 Table 3 Reasons for not attending referral hospital according to referral indication (n = 522) Reason Group of referral indication Total Demographic risks Obstetric historical risks Prenatal complications Natal complications n % n % n % n % Financial difficulties 229 53 19 54 21 46 7 70 276 Difficult to get transport 17 3.9 2 5.7 2 4.3 1 10 22 Thought it was not necessary 15 3.5 2 5.7 0 0 0 0 17 Labour started suddenly 14 3.2 2 5.7 1 2.2 0 0 17 Delivered on the way to hospital 10 2.3 1 2.9 4 8.7 0 0 15 No one to accompany her to hospital 12 2.8 0 0 1 2.2 0 0 13 Delayed to go to hospital 12 2.8 0 0 0 0 0 0 12 The spouse was away on safari 5 1.2 0 0 4 8.7 1 10 10 Aborted 4 0.9 0 0 2 4.3 0 0 6 Labour started at night 4 0.9 0 0 0 0 0 0 4 Othera 6 1.4 1 2.9 1 2.2 1 10 9 No reason given 103 24 8 23 10 22 0 0 121 Total 431 99.9 35 99.9 46 100.3 10 100 522 aOthers includes feared to deliver in hospital, spouse died, refused or sick, no one to care her children at home, disabled, or away on travel Table 3 Reasons for not attending referral hospital according to referral indication (n = 522) Total aOthers includes feared to deliver in hospital, spouse died, refused or sick, no one to care her children at home, disabled, or away on travel died of severe postpartum haemorrhage, she was referred on the basis of ≥5 pregnancies. Two women did not comply to the referral advice, one was referred due to threaten abortion and she died two weeks later in the village, and another woman was referred due to ≥5 pregnancies but it was not known when and why she died. traced in the demographic risks group 15 (3.5%) thought it was not necessary to comply with referral advice given (Table 3). Delivery outcome was available for 653 women who complied with the referral advice and 516 who did not comply. Ethical consideration BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Table 2 Number of women referred, proportional complied, and proportional traced after not complying with referr advice according to indications (n = 1538) Indication for referral Referred Complied Did not comply Traced Lost to follow-up Number n (%) n (%) n (%) Demographic risk factors ≥5 pregnancies 529 146 (28) 257 (49) 126 (24) Age <20 years 465 222 (48) 141 (30) 102 (22) First pregnancya 37 12 (32) 15 (41) 10 (27) Intercurrent illnessesb 15 7 (47) 5 (33) 3 (20) ≥10 years since last delivery 13 5 (38) 5 (38) 3 (23) History of severe bleeding after delivery 6 2 (33) 3 (50) 1 (17) ≥3 consecutive abortions 5 0 3 (60) 2 (40) Pregnancy at ≥35 yearsa 2 1 (50) 1 (50) 0 (0) First pregnancy at ≥35 years 1 1 (100) Other indicationsc 6 4 (67) 1 (17) 1 (17) Total 1079 400 (37) 431 (40) 248 (23) Obstetric historical risks Previous caesarean section 115 79 (69) 18 (16) 18 (16) Height <150 cm 48 31 (65) 12 (25) 5 (10) Stillbirth/Perinatal death 19 11 (58) 1 (5.3) 7 (37) Pelvic deformity 4 0 4 (100) 0 (0) Total 186 121 (65) 35 (19) 30 (16) Prenatal complications Haemoglobin < 60% (8.5 gm/dl) 42 18 (43) 13 (31) 11 (26) Blood pressure ≥140/90 mmHg 35 23 (66) 6 (17) 6 (17) Abnormal lie/presentation at >36 weeks 33 18 (55) 10 (30) 5 (15) Vaginal bleeding 27 12 (44) 8 (30) 7 (26) Uterus too big for date 26 20 (77) 4 (15) 2 (7.7) Oedema of hands and face 16 9 (56) 3 (19) 4 (25) Intrauterine foetal death 4 3 (75) 1 (25) 0 (0) Albumin in urine 2 1 (50) 0 (0) 1 (50) Pregnancy >40 weeks 2 1 (50) 0 (0) 1 (50) Threaten abortion 2 1 (50) 1 (50) 0 (0) Total 189 106 (56) 46 (24) 37 (20) Natal complication Labours for >12 hours/obstructed 64 51 (80) 8 (13) 5 (7.8) Eclampsia 10 7 (70) 1 (10) 2 (20) Spontaneous rupture of membrane without labour 7 5 (71) 1 (14) 1 (14) Cord presentation 1 1 (100) Total 82 64 (78) 10 (12) 8 (9.8) Immediate postnatal complication Blood loss ≥500 ml 1 1 (100) Retained placenta 1 1 (100) Total 2 2 (110) Total 1538 693 (45) 522 (34) 323 (21) aIndications not in the RCHC-4 bHeart disease, diabetes mellitus, tuberculosis, HIV, mental illness, epilepsy Table 2 Number of women referred, proportional complied, and proportional traced after not complying with referral advice according to indications (n = 1538) omen referred, proportional complied, and proportional traced after not complying with referral ndications (n = 1538) Pembe et al. Ethical consideration BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Page 7 of 9 Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Table 5 Risks of perinatal death among women who did not comply with the referral in comparison with women who complied by referral indications Table 5 Risks of perinatal death among women who did not comply with the referral in comparison with women who complied by referral indications Compliance to referral Perinatal death OR 95% CI Yes No Demographic risks Complied 14 386 1 Did not comply 15 416 1 0.48-2.1 Obstetric historical risks Complied 3 118 Did not comply 0 35 Prenatal and natal complicationsa Complied 9 137 1 Did not comply 2 48 1.6 0.34-7.8 a3 women were referred of intrauterine foetal deaths. These are excluded in the prenatal and natal complication group. Odds ratio (OR) with 95% confidence intervals (CI). become pregnant early in their life and many have sev- eral pregnancies. Most of the demographic and histori- cal referral indications have been shown to poorly predict occurrence of complications and bad delivery outcome [17,19-21]. Moreover, if all complied with the referral advice, they could overburden the hospitals with otherwise normal deliveries that may lead to a misuse of resources and hampering quality of the maternal care provided. become pregnant early in their life and many have sev- eral pregnancies. Most of the demographic and histori- cal referral indications have been shown to poorly predict occurrence of complications and bad delivery outcome [17,19-21]. Moreover, if all complied with the referral advice, they could overburden the hospitals with otherwise normal deliveries that may lead to a misuse of resources and hampering quality of the maternal care provided. Compliance to referral advice was lower in the demo- graphic risks group (37%) compared to other risk groups which had more than half of women complying. Low level of referral compliance has been reported from Tanzania and other countries [7,16,22]. A previous qua- litative study in the same district showed that the com- munity does not often agree with referral advice based on age <20 years and ≥5 pregnancies without previous complication [12]. In our study the low compliance to these referral indications supports the findings in the qualitative study. Despite the fact that many women did not comply only few (3.3%) thought the referral was not necessary. Ethical consideration the workers to be adequate at Mchukwi hospital, while at Utete hospital it was perceived that more nursing staff and doctors were needed. In both hospitals there was sufficient equipment and facilities, including operat- ing theatres, operative instruments, blood transfusion facilities, antibiotics and oxytocics. Mchukwi hospital had magnesium sulphate solution and diazepam, while Utete had diazepam only for management of eclamptic fits. None of the hospitals had a functioning vacuum extractor. Another reason for low level of compliance in the demographic risks group might be due to the criteria for identifying women with risks on the first visit: tick the boxes for the risk and advise the women to go for further assessment or a hospital delivery. This informa- tion is given at the first visit. It can be questioned if this is the right time but then the woman and her family can start to prepare for potential referral. The impor- tance of referral should however be emphasized in every subsequent visit by the health worker otherwise the potential risk factor can be forgotten or not considered seriously by the mother and her family. The same reminder should be given for the obstetric historical risks group although in this group women’s previous bad experience prompt them to take the referral advice seriously and act upon it. Ethical consideration Among women who did not comply, there was no difference in sociodemographic and obstetric charac- teristics between those found on follow-up and those lost to follow-up. A higher proportion of women referred in the demographic risks group and who did not comply with referral advice delivered at home com- pared to other referral indication groups in which they delivered more in the primary level of care facilities (Table 4). Overall there was no elevated risk for perinatal death among women not complying with the referral advice. Though not significant, perinatal death was elevated in the prenatal and natal complications group in women not complying to referral advice (Table 5). During the study period there were 1608 hospital deliveries, 840 in Utete hospital and 768 in Mchukwi hospital. In each hospital there were four doctors (one medical officer and three assistant medical officers) who provided care to women with obstetric complications. There were three nurse midwives at Mchukwi hospital working in the labour ward and two at Utete hospital. There were a number of MCH Aides and nurse assis- tants working in the labour ward with the nurse mid- wives. The number of qualified staff was perceived by There were six maternal deaths. Four complied with the referral and they died at the hospital. Among these, three women died of severe pre-eclampsia/eclampsia. The first woman was referred due to previous caesarean section, the second woman had elevated blood pressure and generalized oedema, and the third one had elevated blood pressure, generalized oedema, twin pregnancy and antepartum haemorrhage. The fourth of these women among women complied and did not comply with to referral advice (N = 1169) Table 4 Place of delivery among women complied and did not comply with to referral advice (N = 1169) Referral indication Total Complied Did not comply Home/en route PLCF Hospital Home/en route PLCF Demographic risks 804 5 (0.62) 23 (2.9) 349 (43) 266 (33) 161 (20) Historical obstetric risks 152 0 (0) 2 (1.3) 115 (76) 11 (7.2) 24 (16) Prenatal complications 140 1 (0.71) 4 (2.9) 91 (65) 20 (14) 24 (17) Natal complications 73 0 (0) 0 (0) 63 (86) 4 (5.5) 6 (8.2) Total 1169 6 (0.51) 29 (2.5) 618 (53) 301 (26) 215 (18) Percentages in brackets PLCF - Primary level of care facility Page 7 of 9 Pembe et al. Author details 1 1Department of Obstetrics and Gynaecology, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania. 2Department of Women’s and Children’s Health, International Maternal and Child Health (IMCH), Uppsala University, Sweden. 3Department of Surgery, Central Hospital, Karlstad, Sweden. 4Department of Community Health, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania. 5Department of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden. Discussion Community-based loans and insurance schemes can be used to ease the cost the family incur during referral [25-27]. Hoffman et al suggest the use of motorcycle ambulances at health centres in resource- poor countries as a cheap and time-reducing option to increase timely access for emergency obstetric care [28]. Another way of increasing referral acceptance for women with high risk pregnancies is the use of mater- nity waiting homes close to the hospitals to reduce access barriers [29]. However, a recent Cochrane review has concluded that there is limited evidence of the use of maternity waiting homes in improving maternal and newborn outcomes [30]. make decisions as well as a person to accompany the woman to the hospital, and allocate funds to be used during referral. Community-based loans and insurance schemes can be used to ease the cost the family incur during referral [25-27]. Hoffman et al suggest the use of motorcycle ambulances at health centres in resource- poor countries as a cheap and time-reducing option to increase timely access for emergency obstetric care [28]. Another way of increasing referral acceptance for women with high risk pregnancies is the use of mater- nity waiting homes close to the hospitals to reduce access barriers [29]. However, a recent Cochrane review has concluded that there is limited evidence of the use of maternity waiting homes in improving maternal and newborn outcomes [30]. Authors’ contributions ABP, AC, DPU, GL, LN and ED participated in the design of the study. ABP, AC, DPU and ED involved in data collection. ABP performed statistical analysis and drafted the first manuscript. All authors read, commented on and approved the final manuscript. ABP, AC, DPU, GL, LN and ED participated in the design of the study. ABP, AC, DPU and ED involved in data collection. ABP performed statistical analysis and drafted the first manuscript. All authors read, commented on and approved the final manuscript. analysis and drafted the first manuscript. All authors read, commented on and approved the final manuscript. Acknowledgements S i l th k t t g Special thanks go to the District Medical Officer, Dr. Said Mkikima for his assistance in supervision of data collection. This work was supported by the Swedish International Development Cooperation Agency (Sida/SAREC) through Muhimbili University of Health and Allied Sciences (MUHAS), reproductive health research programme. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Few women were referred because of anaemia (2.7%) and elevated blood pressure (2.3%) in spite of the high prevalence of anaemia and severe anaemia in pregnant women (60% and 27% respectively) [31]. This can be due to poor ability of health workers to screen for these con- ditions leading to underestimation of the problems [31,32]. More than failure to detect the complications, there may be failure of health workers to refer women with identified risks or complications. Majoko et al [16] reported that nurse midwives in Zimbabwe did not refer 59% of women with previous complications recom- mended for hospital assessment. It was further noted that 52% of women with elevated blood pressure (≥140/90 mmHg) were not referred. Studies in Zaire have shown that health workers are more willing to refer women with prenatal complications but are reluctant to refer women with risk factors as they are not perceived to predict adverse pregnancy outcome [19,22]. Thus there may be many women not referred to hospitals due to failure of health workers to identify the risks or due to altered per- ceptions visavis how identified risks affect the outcomes. Received: 18 April 2010 Accepted: 3 December 2010 Published: 3 December 2010 Received: 18 April 2010 Accepted: 3 December 2010 Published: 3 December 2010 Received: 18 April 2010 Accepted: 3 December 2010 Published: 3 December 2010 Conclusions The existing maternal referral system in Rufiji rural dis- trict is less effective as majority of the women referred from the primary level of care facilities to the hospitals did not comply. Most of the women were referred due to demographic risk factors and hence a call for review of the national maternal referral indications. Moreover, there is need of strengthening birth preparedness and complication readiness to improve compliance. Further qualitative studies employing in-depth interviews con- cerning non-compliance to maternal referrals could reveal more information on experiences and perceptions of the women and their families on the referrals. We calculated the risk of perinatal death among women not complying with the referral advice despite knowing that the results might be inconclusive based on small sample and perinatal death being a rare event. The results of a non-elevated risk of perinatal death among non-compliant women to referral advice in the prenatal and natal complications group could be explained by the delays in reaching hospitals. Another reason in the demographic risks group may be that women started labour at home and went to hospital bypassing the primary level of care when there were more serious complications. Studies in Africa have shown that bypassing of the primary level of care facil- ities and going directly to hospitals is common [7,16]. The absence of vacuum extractors in the hospitals is another bottleneck which may hamper reducing perina- tal deaths as cases of foetal distress in the second stage of labour can not be delivered promptly. References 1. WHO: Maternal mortality in 2005: Estimates developed by WHO, UNICEF, UNFPA and the World Bank. Geneva: World Health Organization; 2007, Report No.: 978 92 4 159621 3. 1. WHO: Maternal mortality in 2005: Estimates developed by WHO, UNICEF, UNFPA and the World Bank. Geneva: World Health Organization; 2007, Report No.: 978 92 4 159621 3. 2. Lawn JE, Cousens S, Zupan J: 4 million neonatal deaths: When? Where? Why? Lancet 2005, 365:891-900. y , 3. Stanton C, Lawn JE, Rahman H, Wilczynska-Ketende K, Hill K: Stillbirth rates: delivering estimates in 190 countries. Lancet 2006, 367:1487-94. 4. Ronsmans C, Graham WJ: Maternal mortality: who, when, where, and why. Lancet 2006, 368:1189-200. 3. Stanton C, Lawn JE, Rahman H, Wilczynska-Ketende K, Hill K: Stillbirth rates: delivering estimates in 190 countries. Lancet 2006, 367:1487-94. 4. Ronsmans C, Graham WJ: Maternal mortality: who, when, where, and why. Lancet 2006, 368:1189-200. Campbell OM, Graham WJ: Strategies for reducing maternal mortality: getting on with what works. Lancet 2006, 368:1284-99. NBS: Tanzania Demographic and Health Survey 2004/5. Dar es Salaam, Tanzania: National Bureau of Statistics (NBS) and ORC Macro 2005. Jahn A, Kowalewski M, Kimatta SS: Obstetric care in southern Tanzania: does it reach those in need? Trop Med Int Health 1998, 3:926-32. Urassa DP, Carlstedt A, Nystrom L, Massawe SN, Lindmark G: Are process indicators adequate to assess essential obstetric care at district level?–a case study from Rufiji district, Tanzania. Afr J Reprod Health 2005, 9:100-11. Belghiti A, De Brouwere V, Kegels G, Van Lerberghe W: Monitoring unmet obstetric need at district level in Morocco. Trop Med Int Health 1998, 3:584-91. 0. Gupta J, Gupta H: Perceptions of and constraints upon pregnancy-related referrals in rural Rajasthan, India. Health Serv Manage Res 2000, 13:6-15. Campbell OM, Graham WJ: Strategies for reducing maternal mortality: getting on with what works. Lancet 2006, 368:1284-99. NBS: Tanzania Demographic and Health Survey 2004/5. Dar es Salaam, Tanzania: National Bureau of Statistics (NBS) and ORC Macro 2005. Jahn A, Kowalewski M, Kimatta SS: Obstetric care in southern Tanzania: does it reach those in need? Trop Med Int Health 1998, 3:926-32. Urassa DP, Carlstedt A, Nystrom L, Massawe SN, Lindmark G: Are process indicators adequate to assess essential obstetric care at district level?–a case study from Rufiji district, Tanzania. Afr J Reprod Health 2005, 9:100-11. Discussion In this study we found that 28% of women who were booked for antenatal care in the primary levels of care facilities were referred to hospitals. More than two thirds of women referred to the hospitals are in the group of demographic risk factors. Compliance to the referral advice in this group of demographic risks was poor compared to the obstetric historical risks, prenatal, natal and postnatal complications groups. Common rea- sons mentioned for not complying with the referral advice included financial constraints and difficulty acquiring transport. The two hospitals were considerably well equipped to provide comprehensive emergency obstetric care except for the lack of vacuum extractors. Financial difficulty and transport problems were the main reason for not complying with the the referral advice. This is in accordance with other studies in devel- oping countries where geographical and financial diffi- culties deter women from going for the referral [8,20,23,24]. We found that many women referred due to prenatal and natal complications were able to mobi- lize resources and went to hospital. To improve the referral compliance it is necessary to focus on the refer- ral indications which are more predictive of occurrence of the complications and are as well acceptable to women and the community. To increase women’s access to hospital, birth preparedness and emergency readiness should be emphasized. This entails that the woman and her family to devise a plan for delivery at the hospital by identifying a mode of transport, choosing a person to About one quarter of women booked for antenatal care were referred to hospital during pregnancy, delivery or after delivery. This proportion is lower compared to other studies in developing countries which have shown higher proportions of women needing referral [16-19]. The most common referral indications were ≥5 preg- nancies and age <20 years and these accounted for two thirds of all the referrals. Similar findings were found from studies in other parts of sub-Saharan Africa [16,19]. The reason for a high prevalence of these risk factors is that most women in sub-Saharan Africa Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Page 8 of 9 Page 8 of 9 make decisions as well as a person to accompany the woman to the hospital, and allocate funds to be used during referral. Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Page 9 of 9 11. Kowalewski M, Jahn A, Kimatta SS: Why do at-risk mothers fail to reach referral level? Barriers beyond distance and cost. Afr J Reprod Health 2000, 4:100-9. 11. Kowalewski M, Jahn A, Kimatta SS: Why do at-risk mothers fail to reach referral level? Barriers beyond distance and cost. Afr J Reprod Health 2000, 4:100-9. doi:10.1186/1472-6963-10-326 Cite this article as: Pembe et al.: Effectiveness of maternal referral system in a rural setting: a case study from Rufiji district, Tanzania. BMC Health Services Research 2010 10:326. doi:10.1186/1472-6963-10-326 Cite this article as: Pembe et al.: Effectiveness of maternal referral system in a rural setting: a case study from Rufiji district, Tanzania. BMC Health Services Research 2010 10:326. 12. Pembe AB, Urassa DP, Darj E, Carlstedt A, Olsson P: Qualitative study of maternal referrals in rural Tanzania: Decision making and acceptance of referral advice. Afr J Reprod Health 2008, 12:120-31. 13. NBS: 2002 Population and Housing Census general report. Dar es Salaam, Tanzania: Government Printers; 2003. 13. NBS: 2002 Population and Housing Census general report. Dar es Salaam, Tanzania: Government Printers; 2003. 14. Kielmann A, Janovsky K, Annett K: Protocol for rapid data collection and analysis. Assessing District Health Needs, Services and Systems. London: Macmillan Education LTD; 1995. 14. Kielmann A, Janovsky K, Annett K: Protocol for rapid data collection and analysis. Assessing District Health Needs, Services and Systems. London: Macmillan Education LTD; 1995. 15. UNICEF, UNFPA, WHO: Guidelines for Monitoring the Availability and Use of Obstetric Services. New York: UNICEF; 1997. 15. UNICEF, UNFPA, WHO: Guidelines for Monitoring the Availability and Use of Obstetric Services. New York: UNICEF; 1997. 16. Majoko F, Nystrom L, Munjanja SP, Lindmark G: Effectiveness of referral system for antenatal and intra-partum problems in Gutu district, Zimbabwe. J Obstet Gynaecol 2005, 25:656-61. 17. Kulmala T, Vaahtera M, Rannikko J, Ndekha M, Cullinan T, Salin ML, Ashorn P: The relationship between antenatal risk characteristics, place of delivery and adverse delivery outcome in rural Malawi. Acta Obstet Gynecol Scand 2000, 79:984-90. 18. Majoko F, Nystrom L, Munjanja S, Lindmark G: Usefulness of risk scoring at booking for antenatal care in predicting adverse pregnancy outcome in a rural African setting. J Obstet Gynaecol 2002, 22:604-9. 19. Mugisho E, Dramaix M, Porignon D, Mahangaiko E, Hennart P, Buekens P: [Referral and delivery outcomes in Kivu, Democratic Republic of Congo]. Rev Epidemiol Sante Publique 2003, 51:237-44. 20. References Belghiti A, De Brouwere V, Kegels G, Van Lerberghe W: Monitoring unmet obstetric need at district level in Morocco. Trop Med Int Health 1998, 3:584-91. . Gupta J, Gupta H: Perceptions of and constraints upon pregnancy-related referrals in rural Rajasthan, India. Health Serv Manage Res 2000, 13:6-15. Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 31. Urassa DP, Carlstedt A, Nystrom L, Massawe SN, Lindmark G: Quality assessment of the antenatal program for anaemia in rural Tanzania. Int J Qual Health Care 2002, 14:441-8. 31. Urassa DP, Carlstedt A, Nystrom L, Massawe SN, Lindmark G: Quality assessment of the antenatal program for anaemia in rural Tanzania. Int J Qual Health Care 2002, 14:441-8. 32. Urassa DP, Nystrom L, Carlstedt A, Msamanga GI, Lindmark G: Management of hypertension in pregnancy as a quality indicator of antenatal care in rural Tanzania. Afr J Reprod Health 2003, 7:69-76. 32. Urassa DP, Nystrom L, Carlstedt A, Msamanga GI, Lindmark G: Management of hypertension in pregnancy as a quality indicator of antenatal care in rural Tanzania. Afr J Reprod Health 2003, 7:69-76. Pembe et al. BMC Health Services Research 2010, 10:326 http://www.biomedcentral.com/1472-6963/10/326 McDonagh M: Is antenatal care effective in reducing maternal morbidity and mortality? Health Policy Plan 1996, 11:1-15. 21. 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https://openalex.org/W4379056266	http://www.journalofdairyscience.org/article/S0022030223002916/pdf	English	null	Application of single-step single nucleotide polymorphism best linear unbiased predictor model with unknown-parent groups for type traits in Japanese Holsteins	Journal of dairy science	2,023	cc-by	11,682	J. Dairy Sci. 106:4847–4859 https://doi.org/10.3168/jds.2022-22541 © 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). J. Dairy Sci. 106:4847–4859 https://doi.org/10.3168/jds.2022-22541 © 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). © 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Associat This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Received July 17, 2022. Accepted January 30, 2023. Corresponding author: t0ohsawa@nlbc.go.jp ABSTRACT The reliability of the predictions for the validation cows was Key words: single-step genomic evaluation, single nucleotide polymorphism best linear unbiased predictor, unknown-parent groups, type trait, Holstein Application of single-step single nucleotide polymorphism best linear unbiased predictor model with unknown-parent groups for type traits in Japanese Holsteins Takefumi Osawa,1 Yutaka Masuda,2 Junichi Saburi,1 and Keita Hirumachi1 1National Livestock Breeding Center, Nishigo-mura, Fukushima, 961-8511, Japan 2Rakuno Gakuen University, Ebetsu, Hokkaido, 069-8501, Japan ABSTRACT calculated as the square of the correlation between Yadj and GEBV divided by heritability. The predictive abil- ity was highest in the SCY group and lowest in the CY group. However, minimal difference was found in predictive abilities with or without UPG models using different parameters of residual polygenic variance. The regression coefficients approached 1.0 as the parameter of residual polygenic variance increased, but regression coefficients were mostly similar regardless of the use of UPG across the groups of genotyped animals. The ssSNPBLUP model, including UPG, was demonstrated as feasible for implementation in the national evalua- tion of type traits in Japanese Holsteins. The objectives of this study were to investigate the computational performance and the predictive ability and bias of a single-step SNP BLUP model (ssSN- PBLUP) in genotyped young animals with unknown- parent groups (UPG) for type traits, using national genetic evaluation data from the Japanese Holstein population. The phenotype, genotype, and pedigree data were the same as those used in a national genetic evaluation of linear type traits classified between April 1984 and December 2020. In the current study, 2 data sets were prepared: the full data set containing all en- tries up to December 2020 and a truncated data set ending with December 2016. Genotyped animals were classified into 3 types: sires with classified daughters (S), cows with records (C), and young animals (Y). The computing performance and prediction accuracy of ssSNPBLUP were compared for the following 3 groups of genotyped animals: sires with classified daughters and young animals (SY); cows with records and young animals (CY); and sires with classified daughters, cows with records, and young animals (SCY). In addition, we tested 3 parameters of residual polygenic variance in ssSNPBLUP (0.1, 0.2, or 0.3). Daughter yield devia- tions (DYD) for the validation bulls and phenotypes adjusted for all fixed effects and random effects other than animal and residual (Yadj) for the validation cows were obtained using the full data set from the pedigree- based BLUP model. The regression coefficients of DYD for bulls (or Yadj for cows) on the genomic estimated breeding value (GEBV) using the truncated data set were used to measure the inflation of the predictions of young animals. The coefficient of determination of DYD on GEBV was used to measure the predictive ability of the predictions for the validation bulls. J. Dairy Sci. 106:4847–4859 https://doi.org/10.3168/jds.2022-22541 © 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). J. Dairy Sci. 106:4847–4859 https://doi.org/10.3168/jds.2022-22541 © 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). INTRODUCTION Currently, genomic evaluation systems for dairy cattle have been implemented in many countries us- ing large-scale SNP genotypes. For this purpose, most countries have applied a multistep method (VanRaden, 2008) that typically uses deregressed EBV as a pseudo- phenotype obtained via pedigree-based genetic evalua- tions. This method requires a set of genotyped animals, referred to as a reference population, that have reliable pseudo-phenotypes to estimate SNP effects or genomic EBV (GEBV). In this approach, genomic predic- tion is an extra step added to a conventional genetic evaluation. One possible disadvantage of the multistep method is a preselection bias in conventional EBV (Patry and Ducrocq, 2011). When young animals are selected for many generations based on GEBV from a multistep model, the conventional EBV will be biased because genomic preselection is not accounted for in the conventional evaluation. The single-step genomic BLUP model (ssGBLUP) provides GEBV for genotyped and nongenotyped animals (Aguilar et al., 2010), and it can account for genomic preselection in young animals; therefore, the 4847 4848 4848 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP GEBV is expected to be unbiased (Masuda et al., 2018). This model integrates the genomic relationship matrix G and pedigree relationship matrix A into the hybrid matrix H and replaces the matrix A in the BLUP equations. The matrix H may not be easily determined in an actual population. Because the scales of G and A may not be identical, G should be aligned to the relationship matrix for genotyped animals, A22, or vice versa, using scaling factors. In addition, when including a substantial number of genotyped animals, computing costs will be very high for calculating the inverse of G, which is required in the mixed model equations in ssGBLUP. Therefore, efficient techniques such as the algorithm for proven and young animals (Misztal et al., 2014; Fragomeni et al., 2015) should be used to avoid the high computing costs required for large-scale genotype data, such as a national-level database. GEBV is expected to be unbiased (Masuda et al., 2018). This model integrates the genomic relationship matrix G and pedigree relationship matrix A into the hybrid matrix H and replaces the matrix A in the BLUP equations. The matrix H may not be easily determined in an actual population. MATERIALS AND METHODS Liu et al. (2014, 2016) developed a single-step SNP BLUP model (ssSNPBLUP) that is theoretically equivalent to ssGBLUP. It does not require G and its inverse, and it can be relatively easily introduced into computer programs to solve traditional BLUP equa- tions. Initially, most studies focused on ssGBLUP, but recently, studies have increasingly focused on ssSNPB- LUP. Osawa and Saburi (2018) applied this method to a repeatability model for 305-d milk yields in Japanese Holstein cows with approximately 5,500 genotyped animals. Konstantinov and Goddard (2020) applied the method to multivariate models, that is, a random regression model and a multiple trait model in Aus- tralian dairy cattle. Alkhoder et al. (2022) compared ssSNPBLUP and a routine multistep model for confor- mation traits in German Holsteins and demonstrated the superiority of ssSNPBLUP. INTRODUCTION Because the scales of G and A may not be identical, G should be aligned to the relationship matrix for genotyped animals, A22, or vice versa, using scaling factors. In addition, when including a substantial number of genotyped animals, computing costs will be very high for calculating the inverse of G, which is required in the mixed model equations in ssGBLUP. Therefore, efficient techniques such as the algorithm for proven and young animals (Misztal et al., 2014; Fragomeni et al., 2015) should be used to avoid the high computing costs required for large-scale genotype data, such as a national-level database. for past selections that cannot be accounted for by in- complete pedigree relationships with missing parents. The UPG model for pedigree-based BLUP (Quaas, 1988) was extended by Misztal et al. (2013), who ap- plied the Quaas and Pollak (1981) (QP) transforma- tion to ssGBLUP. Vandenplas et al. (2021) proposed the application of QP transformation to ssSNPBLUP, but this method has not been tested thoroughly in the Japanese dairy populations. Therefore, we investigated the computational perfor- mance and the predictive ability and bias of the ssSN- PBLUP model with UPG for type traits of genotyped young animals, using national genetic evaluation data from the Japanese Holstein population. MATERIALS AND METHODS Journal of Dairy Science Vol. 106 No. 7, 2023 Data The phenotype and pedigree data used in this study were the same as those used in the national genetic evaluation of linear type traits for Japanese Holsteins, as carried out by the National Livestock Breeding Cen- ter (NLBC, Nishigo-mura, Fukushima, Japan). The phenotypes of the first 3 lactations were provided by the Holstein Cattle Association of Japan (HCAJ, Nakano- ku, Tokyo). Cows were classified between April 1984 and December 2020. In this study, 2 data sets were pre- pared to validate the predictive ability of ssSNPBLUP. One data set contained records for type traits of the full data set up to December 2020, and the other was the truncated data set with the type traits ending with December 2016. The number of records and animals in the full and truncated data sets are shown in Table 1. Thirteen linear type traits (stature, chest width, body depth, angularity, rump angle, rear leg side view, foot angle, fore udder attachment, rear udder height, rear udder width, udder support, udder depth, and front teat placement) were analyzed in this study. All traits were scored on a 9-point scale by HCAJ classifiers and adjusted for heterogeneous variance in a contemporary group (Strabel et al., 2006). The heritability and re- peatability of these 13 traits are presented in Table 2. Generally, ssSNPBLUP is reported to have poorer convergence than ssGBLUP (Taskinen et al., 2017) when the preconditioned conjugate gradient (PCG) method is used. Vandenplas et al. (2018, 2019) developed special preconditioners for SNP effects in the PCG method to be applied for large-scale genomic evaluation. The national genetic evaluation of dairy cattle usu- ally considers unknown-parent groups (UPG), known as phantom parents or genetic groups, to compensate Table 1. Number of records and animals in the full and truncated data sets Item Full data set Truncated data set Number of records 1,880,607 1,687,715 First parity 1,248,182 1,106,504 Second parity 412,428 379,847 Third parity 219,997 201,364 Pedigree 2,810,212 2,440,784 Number of cows with records 1,476,987 1,325,097 Number of sires with classified daughters 10,706 9,464 Table 1. Number of records and animals in the full and truncated data sets Journal of Dairy Science Vol. 106 No. 7, 2023 4849 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP Table 2. Single-Step SNP BLUP Model The genomic model for type traits was similar to the single-trait repeatability model used in routine national genetic evaluation by NLBC in Japan (http://www .nlbc.go.jp/en/sec03.html). y = Xb + Zpp + Wu + e, [1] [1] where y is the vector of observations (pre-adjusted for heterogeneous variances), b is the vector of fixed effects, p is the vector of permanent environmental effects, u is the vector of additive genetic effects, and e is the vector of random residuals. The matrices X, Zp, and W are the incidence matrices for b, p, and u, respectively. An where y is the vector of observations (pre-adjusted for heterogeneous variances), b is the vector of fixed effects, p is the vector of permanent environmental effects, u is the vector of additive genetic effects, and e is the vector of random residuals. The matrices X, Zp, and W are the incidence matrices for b, p, and u, respectively. An Data The numbers of genotyped animals of SY, CY, and SCY were 68,209, 104,086, and 109,766 in the full data set, and 47,367, 49,613, and 54,264 in the truncated data set, respectively (Table 3). The trend in the number of genotyped animals is shown in Figure 1. Most cows had been genotyped in the preceding 5 years, resulting in a significant reduction in the number of genotyped cows with records in the truncated data set up to 2016. The study included a total of 109,766 genotyped ani- mals and the details are shown in Table 3. Genotyped animals were divided into 2 groups based on the date of birth. One group included the full data set of ani- mals born in November 2020 or earlier, and the other comprised the truncated data set with animals born in November 2016 or earlier. Most sires with classi- fied daughters were genotyped with a medium-density SNP chip equivalent to the Illumina BovineSNP 50K array, whereas most of the cows with records and the young animals were genotyped with a low-density SNP chip equivalent to the Illumina BovineSNP 7K array. Animals with lower density genotypes were imputed to 50K. Genotype imputing was performed with a program developed in house based on the method of VanRaden et al. (2011). Animals with call rates of <0.90 were excluded, and the number of SNP markers used in this study was 42,275, which was the same used in routine national genomic evaluation by NLBC in Japan. Data Heritabilities and repeatabilities of linear type traits Trait Heritability Repeatability Stature   0.51 0.66 Chest width   0.26 0.39 Body depth   0.35 0.51 Angularity   0.20 0.30 Rump angle   0.42 0.64 Rear leg side view   0.21 0.36 Foot angle   0.06 0.13 Fore udder attachment   0.22 0.40 Rear udder height   0.27 0.45 Rear udder width   0.21 0.35 Udder support   0.20 0.36 Udder depth   0.46 0.62 Front teat placement   0.39 0.52 young animals in this analysis included males without own daughter 7 yr of age or less and females without own record less than 4 yr of age or less. The numbers of genotyped animals are shown in Table 3. We created 3 data sets, each with identical phenotypes and pedigrees but with a different group of genotyped animals. Then, ssSNPBLUP was applied to each data set, and we compared computing performance and prediction ac- curacy among the 3 groups (scenarios). The first group consisted of sires with classified daughters and young animals (SY), the second group consisted of cows with records and young animals (CY), and the third group consisted of sires with classified daughters, cows with records, and young animals (SCY; i.e., all genotyped animals). The numbers of genotyped animals of SY, CY, and SCY were 68,209, 104,086, and 109,766 in the full data set, and 47,367, 49,613, and 54,264 in the truncated data set, respectively (Table 3). The trend in the number of genotyped animals is shown in Figure 1. Most cows had been genotyped in the preceding 5 years, resulting in a significant reduction in the number of genotyped cows with records in the truncated data set up to 2016. young animals in this analysis included males without own daughter 7 yr of age or less and females without own record less than 4 yr of age or less. The numbers of genotyped animals are shown in Table 3. We created 3 data sets, each with identical phenotypes and pedigrees but with a different group of genotyped animals. Then, ssSNPBLUP was applied to each data set, and we compared computing performance and prediction ac- curacy among the 3 groups (scenarios). The first group consisted of sires with classified daughters and young animals (SY), the second group consisted of cows with records and young animals (CY), and the third group consisted of sires with classified daughters, cows with records, and young animals (SCY; i.e., all genotyped animals). Journal of Dairy Science Vol. 106 No. 7, 2023 Genotyping Scenario Three groups were defined for genotyped animals: sires with classified daughters (S), cows with records (C), and young animals (Y). Note that the definition of Table 3. Number of genotyped animals with different groups of genotyped animals (SY, CY, and SCY) in the full and truncated data sets Item Full data set Truncated data set Number of genotyped animals     Sire with classified daughters (S) 5,680 4,651 Cow with records (C) 41,557 6,897 Young animal (Y) 62,529 42,716 Male 5,930 4,032 Female 56,599 38,684 Genotyping scenario     Sire with classified daughters and young animal (SY) 68,209 47,367 Cow with records and young animal (CY) 104,086 49,613 Sire with classified daughters, cow with records, and young animal (SCY) 109,766 54,264 e 3. Number of genotyped animals with different groups of genotyped animals (SY, CY, and SCY) in the nd truncated data sets 4850 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP Figure 1. The number of genotyped animals (sires with classified daughters, cows with records, and young animals) by birth year. The dotted line indicates the cutting date, the last day of truncated data. 4850 Figure 1. The number of genotyped animals (sires with classified daughters, cows with records, and young animals) by birth year. The dotted line indicates the cutting date, the last day of truncated data. Figure 1. The number of genotyped animals (sires with classified daughters, cows with records, and young animals) by birth year. The dotted line indicates the cutting date, the last day of truncated data. mals when genotypes were centered using the observed allele frequencies instead of the base population allele frequencies. The system of equations for ssSNPBLUP can be written as follows: additional fixed covariate, the so-called J factor (Hsu et al., 2017), was included in ssSNPBLUP. Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP and A A A A A −=       1 11 12 21 22 , where superscripts 1, 2, and g refer to groups of non- genotyped animals, genotyped animals, and SNP ef- fects, respectively, ˆu1 is the vector of additive genetic effects of nongenotyped animals, ˆu2 is a vector of the additive genetic effects of genotyped animals, ˆg is a vector of SNP effects, W1 and W2 are incidence matri- ces for the additive genetic effects of nongenotyped and genotyped animals, respectively, R I = σe 2 , and A11, A12, A21, and A22 are submatrices of the inverse of the pedi- where superscripts 1, 2, and g refer to groups of non- genotyped animals, genotyped animals, and SNP ef- fects, respectively, ˆu1 is the vector of additive genetic effects of nongenotyped animals, ˆu2 is a vector of the additive genetic effects of genotyped animals, ˆg is a vector of SNP effects, W1 and W2 are incidence matri- ces for the additive genetic effects of nongenotyped and genotyped animals, respectively, R I = σe 2 , and A11, A12, A21, and A22 are submatrices of the inverse of the pedi- Implementation of Genetic Groups in the ssSNPBLUP Model Vandenplas et al. (2021) applied a QP transformation (Quaas and Pollak, 1981) to ssSNPBLUP to incorpo- rate UPG into Equation [2]. This method is equivalent to the system of equations derived by Misztal et al. (2013) for ssGBLUP. Journal of Dairy Science Vol. 106 No. 7, 2023 Genotyping Scenario The purpose of including it was to correct for the difference of genetic levels between the genotyped and nongenotyped ani- X R X X R Z X R W X R W 0 Z R X Z R Z I Z R W Z ′ ′ ′ ′ ′ ′ ′ − − − − − − − − + 1 1 1 1 1 2 1 1 2 1 1 p p p p p p σ p p u u u g ′ ′ ′ ′ ′ R W 0 W R X W R Z W R W W R − − − − − − − + 1 2 1 1 1 1 1 1 1 2 11 2 12 2 1 2 σ σ σ Σ Σ Σ − − − − − − − − + 1 2 1 2 21 2 1 2 2 22 2 2 2 1 2 2 X W R Z W R W 0 0 ′ ′ p u u u g u g u g σ σ σ σ σ Σ Σ Σ Σ Σ σu gg −           2 1 2 Σ ˆ ˆ ˆ ˆ ˆ b p u u g   =       − − − − X R y Z R y W R y W R y 0 ′ ′ ′ ′ 1 1 1 1 2 1 p  [2] [2] Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP 4851 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP with Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ −=       = 1 11 12 1 21 22 2 1 2 11 12 21 0 g g g g gg A A A A A A Z Z A B Z A 22 22 1 22 1 22 1 1 1 1 1 0 1 1 1 1 + −       − − − + ′ ′ − − − − k k k k k 22 1 −      Z and A A A A A −=       1 11 12 21 22 , with gree relationship matrix A−1 for all animals, A22 1 − is the inverse of the pedigree relationship matrix for geno- typed animals (A22). Z is a design matrix of regression coefficients for genotyped animals at all SNP markers [2 − 2pj, 1 − 2pj, or −2pj, for genotypes AA, AB, or BB of the jth SNP marker, respectively, as in VanRaden (2008), where pj represents the observed allele frequency of the jth SNP marker]; B is 2 1 1 1 p p j j m j = − ∑ − ( )     I; and σp 2, σu 2, and σe 2 are permanent environmental, additive genetic, and residual variances, respectively. The pa- rameter of residual polygenic variance (k) ranges from 0 to 1, and 3 values (0.1, 0.2, and 0.3) were tested in this study. Liu et al. (2014) presented a calculation procedure that separated the estimation process of SNP effects from Equation [2]; this approach was used by Osawa and Saburi (2018). However, we solved Equa- tion [2] without separating it, as in Konstantinov and Goddard (2020). Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP with = − − − − −′ − ′ −′ A A A Q A Q A A A Q A Q Q A Q A Q A 11 12 11 1 12 1 21 22 21 1 22 2 1 11 2 21 1 0 0 12 2 22 1 0 0 0 0 0 − ′       + ′ − Q A Q A Q 0 0 0 0 0 1 1 1 1 1 0 22 1 22 1 2 22 1 k k k −       − −       − − − − A A Q A Z − −       ′ −       ′ ′ − − 1 1 1 1 1 2 22 1 2 22 1 2 2 22 k k k Q A Q A Q Q A− − − − − − − +    ′ ′ ′ 1 22 1 22 1 2 1 22 1 0 1 1 1 1 1 Z Z A Z A Q B Z A Z k k k k    , The system of Equations [2] or [3], which is written as Cx = b, can be written as M−1Cx = M−1b with the preconditioner matrix M in the PCG. Specifically, M causes the condition number of the left-hand side matrix to be small and improves the convergence. Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP Before running PCG itera- tions, we first calculated A Z 22 1 − , 1 1 1 1 22 1 − + ′ − − k k B Z A Z, A Q 22 1 2 − , ′ − Q A Q 2 22 1 2, and ′ − Q A Z 2 22 1 , using indirect multiplica- tion algorithms of A Q 22 1 − that do not require calculations of the inverse A22 1 − (Masuda et al., 2017; Strandén et al., 2017). The matrix products were stored as double pre- cision real numbers in random access memory (RAM). This program was written in Fortran 90/95 and com- piled using the Intel Fortran Compiler 2019 (Intel Cor- poration) with the Basic Linear Algebra Subprograms (BLAS), Linear Algebra Package (LAPACK), and di- rect sparse solver PARDISO provided by the Intel Math Kernel Library 2019. Parallel computing using OpenMP was implemented in this program. Eight OpenMP threads were used for all the computations. All analyses were performed on a Linux server with 768 GB of RAM and 2 Intel Xeon Gold 6252 (2.10 GHz) processors with 48 physical cores in total. Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP As a result of the preanalysis, the preconditioner matrix M was chosen, as suggested by Konstantinov and God- dard (2020): C =       C C C C oo og go gg , M = ( )       diag C C oo gg 0 0 , C B Z A Z gg a k k = − +       ′ − − − σ 2 1 22 1 1 1 1 , C =       C C C C oo og go gg , M = ( )       diag C C oo gg 0 0 , where superscript t refers to genetic groups, ˆt is a vec- tor of genetic group effects, and Q is a matrix that as- signs animals to genetic groups. In this study, 81 ge- netic groups were defined based on the birth year, sex, and selection path used in the national routine genetic evaluation in Japan. The orders of ′ − Q A Q 2 22 1 2 and ′ − Q A 2 22 1Z in all genotyping scenarios were 81 by 81 and 81 by 42,275, respectively, and the order of A Q 22 1 2 − was 68,209 by 81, 104,086 by 81, and 109,766 by 81 in SY, CY, and SCY, respectively. and C B Z A Z gg a k k = − +       ′ − − − σ 2 1 22 1 1 1 1 , where C is the left-hand side matrix in Equations [2] or [3], and subscripts g and o refer to parts of the SNP effects and other effects, respectively. In this study, the convergence criterion was defined as \|\| \|\| \|\| \|\| / b Cx b − < − 2 2 14 10 , where \|\| \|\| ⋅ is the 2-norm. Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP 4852 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP with Q Q Q =       1 2 , ˆ ˆ ˆ , a Q t u 1 1 1 = + ˆ ˆ ˆ , a Q t u 2 2 2 = + and Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ QP QP QP QP t QP g QP QP QP t QP g QP t QP t Q − = 1 11 12 1 1 21 22 2 2 1 2 P tt QP tg QP g QP g QP gt QP gg Σ Σ Σ Σ Σ 1 2       = − − − − −′ − ′ −′ A A A Q A Q A A A Q A Q Q A Q A Q A 11 12 11 1 12 1 21 22 21 1 22 2 1 11 2 21 1 0 0 12 2 22 1 0 0 0 0 0 − ′       + ′ − Q A Q A Q 0 0 0 0 0 1 1 1 1 1 0 22 1 22 1 2 22 1 k k k −       − −       − − − − A A Q A Z − −       ′ −       ′ ′ − − 1 1 1 1 1 2 22 1 2 22 1 2 2 22 k k k Q A Q A Q Q A− − − − − − − +    ′ ′ ′ 1 22 1 22 1 2 1 22 1 0 1 1 1 1 1 Z Z A Z A Q B Z A Z k k k k    , 1999; Tsuruta et al., 2001). Implementation of Genetic Groups in the ssSNPBLUP Model The QP-transformed system of Equation [2] is as follows: X R W 0 0 ′ −1 2 Z R W 0 0 p u QP u Q ′ − − 1 2 2 12 2 σ σ Σ Σ P t u QP g u QP u 1 2 1 2 1 2 2 22 2 σ σ σ − − − + Σ Σ W R W ′ Σ Σ Σ Σ Σ QP t u QP g u QP t u QP tt u QP tg 2 2 2 2 2 2 2 σ σ σ σ − − − Σ Σ Σ u QP g u QP gt u QP gg 2 2 2 2 σ σ σ − −    X R W 0 0 ′ −1 2 Z R W 0 0 p u QP u Q ′ − − 1 2 2 12 2 σ σ Σ Σ P t u QP g u QP u 1 2 1 2 1 2 2 22 2 σ σ σ − − − + Σ Σ W R W ′ Σ Σ Σ Σ Σ QP t u QP g u QP t u QP tt u QP tg 2 2 2 2 2 2 2 σ σ σ σ − − − Σ Σ Σ u QP g u QP gt u QP gg 2 2 2 2 σ σ σ − −    X R X X R Z X R W X R W 0 0 Z R X Z R Z I Z R W ′ ′ ′ ′ ′ ′ ′ − − − − − − − − + 1 1 1 1 1 2 1 1 2 1 1 p p p p p p σ Z R W 0 0 W R X W R Z W R W p p u QP u QP u Q ′ ′ ′ ′ − − − − − − + 1 2 1 1 1 1 1 1 1 2 11 2 12 2 σ σ σ Σ Σ Σ P t u QP g p u QP u QP u 1 2 1 2 1 2 1 2 21 2 1 2 2 22 2 σ σ σ σ − − − − − − + Σ Σ Σ W R X W R Z W R W ′ ′ ′ Σ Σ Σ Σ Σ Σ QP t u QP g u QP t u QP t u QP tt u QP tg u 2 2 2 2 1 2 2 2 2 2 σ σ σ σ σ σ − − − − − 0 0 0 0 Σ Σ Σ Σ QP g u QP g u QP gt u QP gg 1 2 2 2 2 σ σ σ − −       ′       = ′ − ˆ ˆ ˆ ˆ ˆ ˆ b p a a t g X R Z R 1 2 1y p − − − ′ ′       1 1 1 2 1 0 0 y W R y W R y [3] [3] Journal of Dairy Science Vol. 106 No. 7, 2023 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP Daughter yield deviations (DYD) for the validation bulls as well as phenotypes adjusted for all fixed and random effects other than animal and residual (Yadj) for the validation cows were obtained using the full data set from the pedigree-based BLUP model with UPG. Note than Yadj for cows with multiple records used the average value for each animal. In addition, DYD were estimated us- ing the method described by VanRaden and Wiggans (1991). LUP, and the memory requirement in the iteration phase was also increased from 0.8 GB in pedigree-based BLUP to 51 to 65 GB in ssSNPBLUP due to the need to create and store matrices related to SNP markers of genotyped animals. The ssSNPBLUP model also re- quired more iterations and time per iterations owing to more complex equations than pedigree-based BLUP. Nevertheless, the computing time was still acceptable for routine national genetic evaluation. Similar to the results of Vandenplas et al. (2021), implementing UPG via QP transformation slightly increased the calcula- tion time. The SY group (sires with classified daugh- ters and young animals) with the lowest number of genotyped animals (n = 68,209) had the shortest cal- culation time (approximately 4 s per iteration), where- as the SCY group (sires with classified daughters, cows with records, and young animals) with the highest number of genotyped animals (n = 109,766) had a slightly longer calculation time (approximately 5 s per iteration round). In contrast, for the CY group (cows with records and young animals) with a smaller num- ber of genotyped animals (n = 104,086) than SCY, the calculation time (approximately 13 s per iteration round) increased significantly. This was due to the number of nonzero elements of A00 (superscript 0 re- fers to nongenotyped ancestors of genotyped animals) in indirect multiplication of A22 1 −Q (Masuda et al., 2017) for CY was much larger than that in the other groups. The numbers of nonzero elements of A00 in SY, CY, and SCY were 645,866, 1,443,820, and 907,914, respec- tively. In CY, genotyped sires were treated as nongeno- typed animals, and their ancestors added more nonzero elements to A00 than SY and SCY. Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP LUP, and the memory requirement in the iteration phase was also increased from 0.8 GB in pedigree-based BLUP to 51 to 65 GB in ssSNPBLUP due to the need to create and store matrices related to SNP markers of genotyped animals. The ssSNPBLUP model also re- quired more iterations and time per iterations owing to more complex equations than pedigree-based BLUP. Nevertheless, the computing time was still acceptable for routine national genetic evaluation. Similar to the results of Vandenplas et al. (2021), implementing UPG via QP transformation slightly increased the calcula- tion time. The SY group (sires with classified daugh- ters and young animals) with the lowest number of genotyped animals (n = 68,209) had the shortest cal- culation time (approximately 4 s per iteration), where- as the SCY group (sires with classified daughters, cows with records, and young animals) with the highest number of genotyped animals (n = 109,766) had a slightly longer calculation time (approximately 5 s per iteration round). In contrast, for the CY group (cows with records and young animals) with a smaller num- ber of genotyped animals (n = 104,086) than SCY, the calculation time (approximately 13 s per iteration round) increased significantly. This was due to the number of nonzero elements of A00 (superscript 0 re- fers to nongenotyped ancestors of genotyped animals) in indirect multiplication of A22 1 −Q (Masuda et al., 2017) for CY was much larger than that in the other groups. The numbers of nonzero elements of A00 in SY, CY, and SCY were 645,866, 1,443,820, and 907,914, respec- tively. In CY, genotyped sires were treated as nongeno- typed animals, and their ancestors added more nonzero elements to A00 than SY and SCY. As a result, the computational cost of A x y 00 1 ( ) = − with PARDISO was estimated to have increased more in CY than To validate GEBV, we defined the validation bulls as genotyped young bulls that had no daughters in the truncated data set but had at least 15 daughters in 10 herds in the full data set. The validation cows were genotyped heifers in the truncated data set, but had their own records in the full data set. Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP As a result, the computational cost of A x y 00 1 ( ) = − with PARDISO was estimated to have increased more in CY than The regression coefficients of DYD for bulls (or Yadj for cows) based on EBV or GEBV were used to mea- sure the inflation of the predictions of young animals. The coefficient of determination of DYD for EBV or GEBV was used to measure the reliability of the pre- dictions for the validation bulls. The reliability of the predictions for the validation cows was calculated as the square of the correlation between Yadj and GEBV divided by heritability. Journal of Dairy Science Vol. 106 No. 7, 2023 Validation We also adopted the PCG to solve the above equa- tions. A solver supporting the ssSNPBLUP model was developed to process large amounts of data in routine national genetic evaluations. The computer program used the iteration on data algorithm (Lidauer et al., Three types of GEBV (SY, CY, and SCY) were cal- culated for each trait. In addition, the EBV obtained using pedigree-based BLUP with the truncated data set was calculated and used as a control. Journal of Dairy Science Vol. 106 No. 7, 2023 4853 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP of 13 traits. as peak resident set size (“high water mark,” VmHWM) obtained from the Linux/proc virtual file system. Computational Performance and Convergence Table 4 shows an overview of the convergence in the pedigree-based BLUP and ssSNPBLUP models for the 13 type traits. This shows a result with the parameter of residual polygenic variance = 0.1. As this parameter increased, the number of iterations decreased. The peak memory requirement was increased from 0.8 GB in pedigree-based BLUP to 62 to 98 GB in ssSNPB- Table 4. Computational performance in pedigree-based BLUP and single-step (ss)SNPBLUP models (residual polygenic variance = 0.10) Group1 UPG2 Number of iterations3 Time per iteration4 (s) Calculation time3 (s) Peak memory5 (GB) Memory in iterative phase6 (GB) PED   No 148–377 0.3 67–134 0.8 0.8 PED   Yes 188–321 0.4 84–123 0.8 0.8 SY   No 545–806 3.8 3,967–5,398 62 51 SY   Yes 342–1,007 3.9 3,569–5,491 64 52 CY   No 470–807 12.9 10,299–14,471 93 63 CY   Yes 446–980 13.1 10,537–16,471 95 63 SCY   No 492–839 5.4 5,802–7,305 96 65 SCY   Yes 459–1,027 5.6 5,673–10,018 98 65 1PED = pedigree-based BLUP; SY = ssSNPBLUP consisting of sires with classified daughters and young animals; CY = ssSNPBLUP consisting of cows with records and young animals; SCY = ssSNPBLUP consisting of sires with classified daughters, cows with records, and young animals. 2UPG = unknown-parent group. 3R f 13 t it e in pedigree-based BLUP and single-step (ss)SNPBLUP models (residual polygenic variance = 0.10) Table 4. Computational performance in pedigree-based BLUP and single-step (ss)SNPBLU Wall clock time. Average of 13 traits. 5Peak memory is defined as peak resident set size (“high water mark,” VmHWM) obtained from the Linux/proc virtual file system. Wall clock time. Average of 13 traits. 5Peak memory is defined as peak resident set size (“high water mark,” VmHWM) obtained from the Linux/proc virtual file system. Journal of Dairy Science Vol. 106 No. 7, 2023 4854 SCY. This outcome is not an issue in the real-world genomic evaluation in Japan because CY was consid- ered only as a scenario of comparison in this study; this group will not be used in practice. In our program, memory requirements were higher than in the study by 1 1 1 1 22 1 − + ′ − − k k B Z A Z, A Q 22 1 2 − , ′ − Q A Q 2 22 1 2, and ′ − Q A Z 2 22 1 equations were dense and stored as double precision real numbers in the memory. Computational Performance and Convergence PED_NOUPG and PED_UPG = pedigree-based BLUP without th unknown-parent group (UGP) and with UPG; SY_NOUPG and SY_UPG = ssSNPBLUP consisting of sires with classified daughters and youn animals without UGP and with UPG; CY_NOUPG and CY_UPG = ssSNPBLUP consisting of cows with records and young animals withou UGP and with UPG; SCY_NOUPG and SCY_UPG = ssSNPBLUP consisting of sires with classified daughters, cows with records, and youn animals without UGP and with UPG; sta = stature; cwi = chest width; bde = body depth; ang = angularity; ran = rump angle; rls = rear le side view; fan = foot angle; fua = fore udder attachment; ruh = rear udder height; ruw = rear udder width; usu = udder support; ude = udde depth; ftp = front teat placement. Figure 2. Coefficients of determination (R2) for the validation bulls (n = 764) between daughter yield deviations (DYD) from pedigree- based BLUP in the full data set and EBV from pedigree-based BLUP (PED) as well as genomic estimated breeding value from single-step (ss) SNPBLUP by groups of genotyped animals in the truncated data set. PED_NOUPG and PED_UPG = pedigree-based BLUP without the unknown-parent group (UGP) and with UPG; SY_NOUPG and SY_UPG = ssSNPBLUP consisting of sires with classified daughters and young animals without UGP and with UPG; CY_NOUPG and CY_UPG = ssSNPBLUP consisting of cows with records and young animals without UGP and with UPG; SCY_NOUPG and SCY_UPG = ssSNPBLUP consisting of sires with classified daughters, cows with records, and young animals without UGP and with UPG; sta = stature; cwi = chest width; bde = body depth; ang = angularity; ran = rump angle; rls = rear leg side view; fan = foot angle; fua = fore udder attachment; ruh = rear udder height; ruw = rear udder width; usu = udder support; ude = udder depth; ftp = front teat placement. SCY. This outcome is not an issue in the real-world genomic evaluation in Japan because CY was consid- ered only as a scenario of comparison in this study; this group will not be used in practice. In our program, memory requirements were higher than in the study by Vandenplas et al. (2020) because the A Z 22 1 − , SCY. Computational Performance and Convergence A22 1 −Z required the most memory with 21 5 GB (size of 68 209 by 42 275) 32 8 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP Figure 2. Coefficients of determination (R2) for the validation bulls (n = 764) between daughter yield deviations (DYD) from pedigree- based BLUP in the full data set and EBV from pedigree-based BLUP (PED) as well as genomic estimated breeding value from single-step (ss) SNPBLUP by groups of genotyped animals in the truncated data set. PED_NOUPG and PED_UPG = pedigree-based BLUP without the unknown-parent group (UGP) and with UPG; SY_NOUPG and SY_UPG = ssSNPBLUP consisting of sires with classified daughters and young animals without UGP and with UPG; CY_NOUPG and CY_UPG = ssSNPBLUP consisting of cows with records and young animals without UGP and with UPG; SCY_NOUPG and SCY_UPG = ssSNPBLUP consisting of sires with classified daughters, cows with records, and young animals without UGP and with UPG; sta = stature; cwi = chest width; bde = body depth; ang = angularity; ran = rump angle; rls = rear leg side view; fan = foot angle; fua = fore udder attachment; ruh = rear udder height; ruw = rear udder width; usu = udder support; ude = udder depth; ftp = front teat placement. 4854 SCY. This outcome is not an issue in the real-world genomic evaluation in Japan because CY was consid- d l f h d 1 1 1 1 22 1 − + ′ − − k k B Z A Z, A Q 22 1 2 − , ′ − Q A Q 2 22 1 2, and ′ − Q A Z 2 22 1 Figure 2. Coefficients of determination (R2) for the validation bulls (n = 764) between daughter yield deviations (DYD) from pedigree based BLUP in the full data set and EBV from pedigree-based BLUP (PED) as well as genomic estimated breeding value from single-step (ss SNPBLUP by groups of genotyped animals in the truncated data set. Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP Implementing a compressed form (e.g., the Plink 1 bi- nary form) for storing the genotyped matrix in RAM and computational strategies for the multiplication of matrix by a vector such as A22 1 −Z x y = on the fly devel- oped by Vandenplas et al. (2020) is expected to reduce the required RAM and improve computational effi- ciency. accurate and the CY group was the least accurate. This result likely occurred because the number of genotyped cows with records in the truncated data set was similar to the number of genotyped sires with classified daugh- ters, whereas the reliability of cows with records was lower than that of proven sires. Moreover, not much difference was found in the predictive abilities between the models with and without UPG using different parameters of residual polygenic variance. In Japan, Holsteins must have pedigree registration records be- fore the animals are genotyped. Thus, most genotyped animals have known parents. In addition, only pedigree registered cows have type traits. These factors may account for little differences between evaluations with and without UPG. Correlations and reliabilities of predictions for cows from PED and ssSNPBLUP based on the 3 groups of genotyped animals are shown in Table 5. Similar to the predictive abilities for the validation bulls, no difference was found in the predictive abilities of the validation cows owing to differences in UPG and residual poly- genic variance; only the results with UPG are shown. The average (ranges) of correlations across traits were 0.31 (0.10–0.50) for PED, 0.39 (0.13–0.60) for SY, 0.38 (0.13–0.59) for CY, and 0.40 (0.13–0.61) for SCY. The correlations of GEBV from ssSNPBLUP increased by an average of 0.08 compared with PED. The average (ranges) of reliabilities were 0.34 (0.15–0.50) for PED, 0.53 (0.28–0.72) for SY, 0.51 (0.27–0.69) for CY, and Predictive Abilities of Validation Bulls and Cows The coefficients of determination (R2) of DYD for EBV [pedigree-based BLUP (PED)] or GEBV (SY, CY, and SCY) are shown in Figure 2. The R2 of EBV from the PED was low (0.06–0.31), whereas the R2 of GEBV from ssSNPBLUP was higher than that of PED by an average of 0.20. The SCY group was the most l f D i S i V l 106 N 7 2023 Table 5. Correlations and validation reliabilities1 (in parentheses) of EBV from pedigree-based BLUP (PED) and genomic EBV from single-step (ss)SNPBLUP [residual polygenic variance (k) = 0.10] by groups of genotyped animals for the validation cows (n = 22,594)2,3 Trait4 PED SY CY SCY sta 0.50 (0.50) 0.60 (0.70) 0.59 (0.69) 0.61 (0.72) cwi 0.27 (0.29) 0.34 (0.43) 0.33 (0.42) 0.34 (0.45) bde 0.36 (0.36) 0.45 (0.58) 0.44 (0.56) 0.46 (0.60) ang 0.25 (0.30) 0.30 (0.45) 0.30 (0.44) 0.31 (0.47) ran 0.38 (0.34) 0.49 (0.58) 0.49 (0.56) 0.51 (0.61) rls 0.21 (0.21) 0.30 (0.44) 0.29 (0.40) 0.31 (0.46) fan 0.10 (0.15) 0.13 (0.28) 0.13 (0.27) 0.13 (0.29) fua 0.32 (0.46) 0.40 (0.72) 0.38 (0.66) 0.41 (0.75) ruh 0.34 (0.43) 0.41 (0.62) 0.40 (0.59) 0.42 (0.64) ruw 0.24 (0.26) 0.30 (0.43) 0.30 (0.42) 0.31 (0.47) usu 0.23 (0.28) 0.31 (0.49) 0.31 (0.48) 0.32 (0.52) ude 0.48 (0.49) 0.57 (0.71) 0.56 (0.69) 0.58 (0.73) ftp 0.36 (0.33) 0.45 (0.51) 0.44 (0.49) 0.46 (0.53) Average 0.31 (0.34) 0.39 (0.53) 0.38 (0.51) 0.40 (0.56) 1Validation reliability = the square of correlation divided by the heritability. 2All results were calculated by the model that included unknown-parent groups. 3PED = pedigree-based BLUP; SY = ssSNPBLUP consisting of sires with classified daughters and young ani- mals; CY = ssSNPBLUP consisting of cows with records and young animals; SCY = ssSNPBLUP consisting of sires with classified daughters, cows with records, and young animals. 4sta = stature; cwi = chest width; bde = body depth; ang = angularity; ran = rump angle; rls = rear leg side view; fan = foot angle; fua = fore udder attachment; ruh = rear udder height; ruw = rear udder width; usu = udder support; ude = udder depth; ftp = front teat placement. Table 5. Computational Performance and Convergence This outcome is not an issue in the real-world genomic evaluation in Japan because CY was consid- ered only as a scenario of comparison in this study; this group will not be used in practice. In our program, memory requirements were higher than in the study by Vandenplas et al. (2020) because the A Z 22 1 − , Journal of Dairy Science Vol. 106 No. 7, 2023 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP 4855 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP 109,766 by 42,275) in SY, CY, and SCY, respectively. The next highest was 1 1 1 1 22 1 − + ′ − − k k B Z A Z, with 13.3 GB (size of 42,275 by 42,275) for all analyses. Matrices related to genetic groups, A Q 22 1 2 − , ′ − Q A Q 2 22 1 2, and ′ − Q A Z 2 22 1 , were less than 1 GB. Peak memory was reached during the construction of A Z 22 1 − , but memory decreased dur- ing the iterative phase. For example, in SCY, the peak memory was approximately 100 GB, and the memory in the iterative phase was reduced to 68 GB. These memory requirements were acceptable for routine na- tional evaluations in Japan. Our program has not been fully optimized, and room remains for improvement. Implementing a compressed form (e.g., the Plink 1 bi- nary form) for storing the genotyped matrix in RAM and computational strategies for the multiplication of matrix by a vector such as A22 1 −Z x y = on the fly devel- oped by Vandenplas et al. (2020) is expected to reduce the required RAM and improve computational effi- ciency. 109,766 by 42,275) in SY, CY, and SCY, respectively. The next highest was 1 1 1 1 22 1 − + ′ − − k k B Z A Z, with 13.3 GB (size of 42,275 by 42,275) for all analyses. Matrices related to genetic groups, A Q 22 1 2 − , ′ − Q A Q 2 22 1 2, and ′ − Q A Z 2 22 1 , were less than 1 GB. Peak memory was reached during the construction of A Z 22 1 − , but memory decreased dur- ing the iterative phase. For example, in SCY, the peak memory was approximately 100 GB, and the memory in the iterative phase was reduced to 68 GB. These memory requirements were acceptable for routine na- tional evaluations in Japan. Our program has not been fully optimized, and room remains for improvement. Journal of Dairy Science Vol. 106 No. 7, 2023 1Validation reliability = the square of correlation divided by the heritability. Predictive Abilities of Validation Bulls and Cows Correlations and validation reliabilities1 (in parentheses) of EBV from pedigree-based BLUP (PED) and genomic EBV from single-step (ss)SNPBLUP [residual polygenic variance (k) = 0.10] by groups of genotyped animals for the validation cows (n = 22,594)2,3 Trait4 PED SY CY SCY sta 0.50 (0.50) 0.60 (0.70) 0.59 (0.69) 0.61 (0.72) cwi 0.27 (0.29) 0.34 (0.43) 0.33 (0.42) 0.34 (0.45) bde 0.36 (0.36) 0.45 (0.58) 0.44 (0.56) 0.46 (0.60) ang 0.25 (0.30) 0.30 (0.45) 0.30 (0.44) 0.31 (0.47) ran 0.38 (0.34) 0.49 (0.58) 0.49 (0.56) 0.51 (0.61) rls 0.21 (0.21) 0.30 (0.44) 0.29 (0.40) 0.31 (0.46) fan 0.10 (0.15) 0.13 (0.28) 0.13 (0.27) 0.13 (0.29) fua 0.32 (0.46) 0.40 (0.72) 0.38 (0.66) 0.41 (0.75) ruh 0.34 (0.43) 0.41 (0.62) 0.40 (0.59) 0.42 (0.64) ruw 0.24 (0.26) 0.30 (0.43) 0.30 (0.42) 0.31 (0.47) usu 0.23 (0.28) 0.31 (0.49) 0.31 (0.48) 0.32 (0.52) ude 0.48 (0.49) 0.57 (0.71) 0.56 (0.69) 0.58 (0.73) ftp 0.36 (0.33) 0.45 (0.51) 0.44 (0.49) 0.46 (0.53) Average 0.31 (0.34) 0.39 (0.53) 0.38 (0.51) 0.40 (0.56) 4sta = stature; cwi = chest width; bde = body depth; ang = angularity; ran = rump angle; rls = rear leg side view; fan = foot angle; fua = fore udder attachment; ruh = rear udder height; ruw = rear udder width; usu = udder support; ude = udder depth; ftp = front teat placement. 4856 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP Figure 3. Regression coefficients of daughter yield deviations (DYD) from pedigree BLUP in the full data set on EBV from pedigree-based BLUP (PED), and genomic EBV from single-step (ss)SNPBLUP based on groups of genotyped animals in the truncated data set for the valida- tion bulls (n = 762). Predictive Abilities of Validation Bulls and Cows PED = pedigree-based BLUP; SY = ssSNPBLUP consisting of sires with classified daughters and young animals; CY = ssSNPBLUP consisting of cows with records and young animals; SCY = ssSNPBLUP consisting of sires with classified daughters, cows with records, and young animals; NOUPG and UPG = without unknown-parent group (UGP) and with UPG; 10, 20, and 30 = assumed proportion of polygenic residual variance = 0.10, 0.20, and 0.30; sta = stature; cwi = chest width; bde = body depth; ang = angularity; ran = rump angle; rls = rear leg side view; fan = foot angle; fua = fore udder attachment; ruh = rear udder height; ruw = rear udder width; usu = udder support; ude = udder depth; ftp = front teat placement. 4856 0 56 (0 29 0 75) for SCY Correlations and reliabilities across country evaluation (MACE) In the multistep Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP Figure 3. Regression coefficients of daughter yield deviations (DYD) from pedigree BLUP in the full data set on EBV from pedigree-based BLUP (PED), and genomic EBV from single-step (ss)SNPBLUP based on groups of genotyped animals in the truncated data set for the valida- tion bulls (n = 762). PED = pedigree-based BLUP; SY = ssSNPBLUP consisting of sires with classified daughters and young animals; CY = ssSNPBLUP consisting of cows with records and young animals; SCY = ssSNPBLUP consisting of sires with classified daughters, cows with records, and young animals; NOUPG and UPG = without unknown-parent group (UGP) and with UPG; 10, 20, and 30 = assumed proportion of polygenic residual variance = 0.10, 0.20, and 0.30; sta = stature; cwi = chest width; bde = body depth; ang = angularity; ran = rump angle; rls = rear leg side view; fan = foot angle; fua = fore udder attachment; ruh = rear udder height; ruw = rear udder width; usu = udder support; ude = udder depth; ftp = front teat placement. Figure 3. Regression coefficients of daughter yield deviations (DYD) from pedigree BLUP in the full data set on EBV from pedigree-based BLUP (PED), and genomic EBV from single-step (ss)SNPBLUP based on groups of genotyped animals in the truncated data set for the valida- tion bulls (n = 762). Journal of Dairy Science Vol. 106 No. 7, 2023 Predictive Abilities of Validation Bulls and Cows PED = pedigree-based BLUP; SY = ssSNPBLUP consisting of sires with classified daughters and young animals; CY = ssSNPBLUP consisting of cows with records and young animals; SCY = ssSNPBLUP consisting of sires with classified daughters, cows with records, and young animals; NOUPG and UPG = without unknown-parent group (UGP) and with UPG; 10, 20, and 30 = assumed proportion of polygenic residual variance = 0.10, 0.20, and 0.30; sta = stature; cwi = chest width; bde = body depth; ang = angularity; ran = rump angle; rls = rear leg side view; fan = foot angle; fua = fore udder attachment; ruh = rear udder height; ruw = rear udder width; usu = udder support; ude = udder depth; ftp = front teat placement. 0.56 (0.29–0.75) for SCY. Correlations and reliabilities showed similar trends, with SCY being the highest and CY the lowest among the groups. across-country evaluation (MACE). In the multistep genomic evaluation implemented in Japan, the refer- ence population included approximately 6,000 foreign genotyped bulls with MACE EBV. The inclusion of MACE EBV into ssSNPBLUP can increase the reli- ability of GEBV for young animals. Alkhoder et al. (2022) considered the use of bull MACE EBV for the ssSNPBLUP in German Holsteins and this may have resulted in higher correlations than the ssGBLUP of Tsuruta et al. (2021), which used only US Holsteins national data. Although the number of genotyped cows in the trun- cated data set was only 6,897, the use of genotyped cows with records slightly improved the predictive abil- ity of GEBV for young animals compared with the use of only genotyped bulls with daughters. Only bulls with daughters in Japan were included in the analyses. In practice, foreign bulls with in- ternational EBV are included according to multiple Journal of Dairy Science Vol. 106 No. 7, 2023 4857 Biases for Validation Bulls and Cows Regression coefficients of Y on EBV and GEBV Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP Figure 4. Regression coefficients of adjusted phenotypes (Yadj) from pedigree-based BLUP in the full data set on EBV from pedigree-based BLUP (PED), and genomic EBV from single-step (ss)SNPBLUP based on groups of genotyped animals in the truncated data set for the valida- tion cows (n = 22,594). Predictive Abilities of Validation Bulls and Cows PED = pedigree-based BLUP; SY = ssSNPBLUP consisting of sires with classified daughters and young animals; CY = ssSNPBLUP consisting of cows with records and young animals; SCY = ssSNPBLUP consisting of sires with classified daughters, cows with records, and young animals; NOUPG and UPG = without unknown-parent group (UGP) and with UPG; 10, 20, and 30 = assumed proportion of polygenic residual variance = 0.10, 0.20, and 0.30; sta = stature; cwi = chest width; bde = body depth; ang = angularity; ran = rump angle; rls = rear leg side view; fan = foot angle; fua = fore udder attachment; ruh = rear udder height; ruw = rear udder width; usu = udder support; ude = udder depth; ftp = front teat placement. Predictive Abilities of Validation Bulls and Cows PED = pedigree-based BLUP; SY = ssSNPBLUP consisting of sires with classified daughters and young animals; CY = ssSNPBLUP consisting of cows with records and young animals; SCY = ssSNPBLUP consisting of sires with classified daughters, cows with records, and young animals; NOUPG and UPG = without unknown-parent group (UGP) and with UPG; 10, 20, and 30 = assumed proportion of polygenic residual variance = 0.10, 0.20, and 0.30; sta = stature; cwi = chest width; bde = body depth; ang = angularity; ran = rump angle; rls = rear leg side view; fan = foot angle; fua = fore udder attachment; ruh = rear udder height; ruw = rear udder width; usu = udder support; ude = udder depth; ftp = front teat placement. 4857 Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP Figure 4. Regression coefficients of adjusted phenotypes (Yadj) from pedigree-based BLUP in the full data set on EBV from pedigree-based BLUP (PED), and genomic EBV from single-step (ss)SNPBLUP based on groups of genotyped animals in the truncated data set for the valida- tion cows (n = 22,594). PED = pedigree-based BLUP; SY = ssSNPBLUP consisting of sires with classified daughters and young animals; CY = ssSNPBLUP consisting of cows with records and young animals; SCY = ssSNPBLUP consisting of sires with classified daughters, cows with records, and young animals; NOUPG and UPG = without unknown-parent group (UGP) and with UPG; 10, 20, and 30 = assumed proportion of polygenic residual variance = 0.10, 0.20, and 0.30; sta = stature; cwi = chest width; bde = body depth; ang = angularity; ran = rump angle; rls = rear leg side view; fan = foot angle; fua = fore udder attachment; ruh = rear udder height; ruw = rear udder width; usu = udder support; ude = udder depth; ftp = front teat placement. Figure 4. Regression coefficients of adjusted phenotypes (Yadj) from pedigree-based BLUP in the full data set on EBV from pedigree-based BLUP (PED), and genomic EBV from single-step (ss)SNPBLUP based on groups of genotyped animals in the truncated data set for the valida- tion cows (n = 22,594). Journal of Dairy Science Vol. 106 No. 7, 2023 CONCLUSIONS // / / ( ) Liu, Z., M. E. Goddard, B. J. Hayes, F. Reinhardt, and R. Reents. 2016. Technical note: Equivalent genomic models with a residual polygenic effect. J. Dairy Sci. 99:2016–2025. https://doi.org/10 .3168/jds.2015-10394. In a national evaluation of the type traits of Japanese Holstein populations, the ssSNPBLUP model, includ- ing UPG, was implemented. The predictive ability of the ssSNPBLUP model was influenced by the group of genotyped animals, and biases were reduced with the increased residual polygenic variance. Thus, when implementing the ssSNPBLUP model for routine ge- nomic evaluation, it is necessary to appropriately set the parameter of residual polygenic variance for each trait to control the overdispersion of GEBV in young animals. The use of UPG did not significantly affect the predictive ability and overdispersion of GEBV in young animals, but differences in the definition of UPG may affect GEBV of individual animals. A further verifica- tion of the optimal UPG definition for ssSNPBLUP should be performed in the future. / Liu, Z., M. E. Goddard, F. Reinhardt, and R. Reents. 2014. A single- step genomic model with direct estimation of marker effects. J. Dairy Sci. 97:5833–5850. https://doi.org/10.3168/jds.2014-7924. Masuda, Y., I. Misztal, A. Legarra, S. Tsuruta, D. A. L. Lourenco, B. O. Fragomeni, and I. Aguilar. 2017. Technical note: Avoiding the direct inversion of the numerator relationship matrix for geno- typed animals in single-step genomic best linear unbiased predic- tion solved with the preconditioned conjugate gradient. J. Anim. Sci. 95:49–52. https://doi.org/10.2527/jas.2016.0699. Masuda, Y., P. M. VanRaden, I. Misztal, and T. J. Lawlor. 2018. Dif- fering genetic trend estimates from traditional and genomic evalu- ations of genotyped animals as evidence of preselection bias in US Holsteins. J. Dairy Sci. 101:5194–5206. https://doi.org/10.3168/ jds.2017-13310. Misztal, I., A. Legarra, and I. Aguilar. 2014. Using recursion to com- pute the inverse of the genomic relationship matrix. J. Dairy Sci. 97:3943–3952. https://doi.org/10.3168/jds.2013-7752. Misztal, I., Z. Vitezica, A. Legarra, I. Aguilar, and A. Swan. 2013. Un- known-parent groups in single-step genomic evaluation. J. Anim. Breed. Genet. 130:252–258. https://doi.org/10.1111/jbg.12025. // / / Osawa, T., and J. Saburi. 2018. Study of single-step SNP BLUP in a Japanese Holstein population. Volume 1, page 131 in Proceedings of the World Congress on Genetics Applied to Livestock Produc- tion, Auckland, New Zealand. Electronic Poster Session—Methods and Tools—Models and Computing Strategies. ACKNOWLEDGMENTS The authors thank the staff at the National Livestock Breeding Center (Nishigo-mura, Fukushima, Japan) and Hokkaido Holstein Agricultural Cooperation (Sap- poro, Hokkaido, Japan) for their support. No human or animal subjects were used, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board. The authors have not stated any conflicts of interest. Patry, C., and V. Ducrocq. 2011. Evidence of biases in genetic evalu- ations due to genomic preselection in dairy cattle. J. Dairy Sci. 94:1011–1020. https://doi.org/10.3168/jds.2010-3804. p // g/ /j Quaas, R. L. 1988. Additive genetic model with groups and rela- tionships. J. Dairy Sci. 71:91–98. https://doi.org/10.1016/S0022 -0302(88)79986-5. Quaas, R. L., and E. J. Pollak. 1981. Modified equations for sire mod- els with groups. J. Dairy Sci. 64:1868–1872. https://doi.org/10 .3168/jds.S0022-0302(81)82778-6. Strabel, T., T. Jankowski, and J. Jamrozik. 2006. Adjustments for heterogeneous herd-year variances in a random regression model for genetic evaluations of Polish Black-and-White cattle. J. Appl. Genet. 47:125–130. https://doi.org/10.1007/BF03194611. Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP Osawa et al.: SNP BLUP MODEL WITH UNKNOWN-PARENT GROUP of genomic recursions in single-step genomic best linear unbiased predictor (BLUP) with a large number of genotypes. J. Dairy Sci. 98:4090–4094. https://doi.org/10.3168/jds.2014-9125. of genomic recursions in single-step genomic best linear unbiased predictor (BLUP) with a large number of genotypes. J. Dairy Sci. 98:4090–4094. https://doi.org/10.3168/jds.2014-9125. eter of residual polygenic variance reduced the overdis- persion of the GEBV for all traits. In the ssSNPBLUP model, it is important to set an appropriate parameter of residual polygenic variance according to the trait to reduce the overdispersion of GEBV in young animals. Along with developing genomic prediction methods, it is also necessary to increase the number of “reference animals,” using reliable genetic evaluations or external information such as MACE EBV of foreign bulls to reduce bias in genomic predictions for Japanese Hol- steins. // / / Hsu, W. L., D. J. Garrick, and R. L. Fernando. 2017. The accuracy and bias of single-step genomic prediction for populations under selection. G3 (Bethesda) 7:2685–2694. https://doi.org/10.1534/g3 .117.043596. Konstantinov, K. V., and M. E. Goddard. 2020. Application of multi- variate single-step SNP best linear unbiased predictor model and revised SNP list for genomic evaluation of dairy cattle in Austra- lia. J. Dairy Sci. 103:8305–8316. https://doi.org/10.3168/jds.2020 -18242. Konstantinov, K. V., and M. E. Goddard. 2020. Application of multi- variate single-step SNP best linear unbiased predictor model and revised SNP list for genomic evaluation of dairy cattle in Austra- li J D i S i 103 8305 8316 htt //d i /10 3168/jd 2020 lia. J. Dairy Sci. 103:8305–8316. https://doi.org/10.3168/jds.2020 -18242. Lidauer, M., I. Strandén, E. A. Mäntysaari, J. Poso, and A. Kettunen. 1999. Solving large test-day models by iteration on data and pre- conditioned conjugate gradient. J. Dairy Sci. 82:2788–2796. https: //doi.org/10.3168/jds.S0022-0302(99)75536-0. Biases for Validation Bulls and Cows Regression coefficients of Yadj on EBV and GEBV from PED and ssSNPBLUP for the validation cows are shown in Figure 4. Similar to the validation bulls, the regression coefficients in ssSNPBLUP approached 1.0 as the parameter of residual polygenic variance increased. The inclusion of UPG in the model and genotyped groups of animals also showed no significant differences in the regression coefficients. Regression coefficients of DYD on GEBV and EBV from PED and ssSNPBLUP for the validation bulls are shown in Figure 3. The regression coefficients in PED were closest to 1.0, but most traits in ssSNPBLUP had regression coefficients less than 1.0, implying an overdispersion in the predictions. However, the regres- sion coefficients approached 1.0 as the parameter of residual polygenic variance increased. The regression coefficients were mostly similar, regardless of the use of UPG across the groups of genotyped animals. This study included several traits for which increas- ing the parameter of residual polygenic variance to 0.3 did not result in regression coefficients for validation bulls greater than 0.9. However, increasing the param- Journal of Dairy Science Vol. 106 No. 7, 2023 4858 REFERENCES net. 47:125–130. https://doi.org/10.1007/BF03194611. Strandén, I., K. Matilainen, G. P. Aamand, and E. A. Mäntysaari. 2017. Solving efficiently large single-step genomic best linear un- biased prediction models. J. Anim. Breed. Genet. 134:264–274. https://doi.org/10.1111/jbg.12257. Aguilar, I., I. Misztal, D. L. Johnson, A. Legarra, S. Tsuruta, and T. J. Lawlor. 2010. Hot topic: A unified approach to utilize phenotypic, full pedigree, and genomic information for genetic evaluation of Holstein final score. J. Dairy Sci. 93:743–752. https://doi.org/10 .3168/jds.2009-2730. Taskinen, M., E. A. Mäntysaari, and I. Strandén. 2017. Single-step SNP-BLUP with on-the-fly imputed genotypes and residual poly- genic effects. Genet. Sel. Evol. 49:36. https://doi.org/10.1186/ s12711-017-0310-9. Alkhoder, H., Z. Liu, D. Segelke, and R. Reents. 2022. 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https://openalex.org/W2785756464	http://medlib.mef.hr/3285/1/brangan_s_et_al_rep_3285.pdf	English	null	Health literacy of hospital patients using a linguistically validated Croatian version of the Newest Vital Sign screening test (NVS-HR)	PloS one	2,018	cc-by	8,351	RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Introduction Various tests of health literacy (HL) have been developed over the past years as a result of the finding that poor HL may be a stronger predictor of a person’s health than age, income, employment status, education level, and race [1]. Additionally, HL has been shown to impact patients’ knowledge, health behaviours, health outcomes, and medical costs [2]. A systematic review on low health literacy and health outcomes from 2011 [3] showed that low health liter- acy is consistently associated with more hospitalizations; greater use of emergency care; lower receipt of mammography screening and influenza vaccine; poorer ability to demonstrate tak- ing medications appropriately; poorer ability to interpret labels and health messages; and, Data Availability Statement: All relevant data are within the paper. Editor: Peter J. Schulz, Università della Svizzera italiana, SWITZERLAND Editor: Peter J. Schulz, Università della Svizzera italiana, SWITZERLAND Received: August 19, 2017 Accepted: January 22, 2018 Published: February 15, 2018 Copyright: © 2018 Brangan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Sanja Brangan1, Martina Ivanisˇić2, Goranka Rafaj3, Gill Rowlands4 1 Department of Educational Technology, Andrija Sˇ tampar School of Public Health, School of Medicine, University of Zagreb, Zagreb, Croatia, 2 Department of Anesthesiology, Reanimatology, and Intensive Care, University Hospital Center Osijek, Osijek, Croatia, 3 Study in Nursing, Technical College Bjelovar, Bjelovar, Croatia, 4 Institute of Health and Society, Newcastle University, Newcastle upon Tyne, United Kingdom skusec@snz.hr Abstract a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 The Newest Vital Sign (NVS) is a simple, quick and accurate screening test for health liter- acy (HL). It has been validated for different languages but, to date, not for the Croatian lan- guage. The aim of this study was to develop a linguistically validated Croatian version of the NVS and to use it at a later stage in a pilot study of health literacy assessment of hospital patients in Croatia. A full linguistic validation procedure was applied, including forward and backward translation, expert panel review, cognitive interview with 10 respondents from general population, and full involvement in the procedure of one of the screening test devel- opers, the lead author of the NVS-UK version. HL testing on 100 hospital patients (55% women, median age 63.5 years) revealed 58% of patients had less than adequate HL level (scores less than 4), and mean NVS total score was 3.34. A positive significant association was observed between HL and educational level (p = 0.002). A high percentage of patients (92%) did not object to being tested for HL by their primary care physician or in hospital, and 99% of patients would recommend HL testing among patients in general. The respondents’ positive views on HL testing and mean completion time of 4 minutes indicate that the Croa- tian version of the NVS (NVS-HR) could be recommended for use in both clinical and research settings in Croatia. Health literacy of hospital patients using a linguistically validated Croatian version of the Newest Vital Sign screening test (NVS-HR) Sanja Brangan1*, Martina Ivanisˇić2, Goranka Rafaj3, Gill Rowlands4 OPEN ACCESS Citation: Brangan S, Ivanisˇić M, Rafaj G, Rowlands G (2018) Health literacy of hospital patients using a linguistically validated Croatian version of the Newest Vital Sign screening test (NVS-HR). PLoS ONE 13(2): e0193079. https://doi.org/10.1371/ journal.pone.0193079 Data Availability Statement: All relevant data are within the paper. Funding: The authors received no specific funding for this work. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 1 / 14 Health literacy with Croatian NVS among elderly persons, poorer overall health status and higher mortality rates. Data on the US population [2] show that nearly half of the adult population has difficulty understanding and using health information. In Europe, a study on HL in eight EU countries [4] showed that peo- ple with low HL have a higher prevalence of long-term conditions, which in turn are more likely to be limiting, and that low HL level is significantly associated with worse self-assessed health. Research on HL in Croatia is still in its infancy, with no studies published at the national level [5] and previous published studies either using a nonvalidated instrument [6] or focusing mostly on readability, comprehension or informed consent issues [5,7–11]. There are multiple definitions of health literacy. One of the most commonly used is ’the personal, cognitive and social skills which determine the ability of individuals to gain access to, understand and use information to promote and maintain good health’ [12]. This definition includes three ’levels’ of HL: basic or functional; communicative or interactive; and critical HL [12]. Although the levels are not presented as hierarchial, it is reasonable to assume that basic functional HL and numeracy skills are important in forming the basis for more complex com- petencies [13]. The Newest Vital Sign (NVS) is a test of functional health literacy, assessing reading com- prehension and numeracy skills. It was first developed for US population in 2005 [13], and the UK version (NVS-UK) was developed in 2013 [14], with adaptations for the European context. The NVS is a 6-item test based on the ability to read and apply information from an ice cream nutrition label; it takes three minutes to administer; and the scoring enables identification of three groups of patients: likely marginal/inadequate HL, likely limited HL, and likely adequate HL [13,15,16]. The NVS (US version) scores correlate with the scores of the widely used but lengthier validated instrument for HL assessment, the Test of Functional Health Literacy in Adults–TOFHLA [17], which served as the reference standard. Data Availability Statement: All relevant data are within the paper. In clinical settings it is recom- mended that the NVS scores are recorded in the patient’s chart, so that future communication can be tailored to ensure patient understanding [16]. Ever since the original NVS was developed by Weiss et al. [13] for the US population, both in English and Spanish, there have been translations/adaptations of the NVS for other lan- guages. Review of the literature published in English on details of translation procedures lists the following language versions of the NVS: Turkish [18], Dutch [19,20], Portuguese [21], Ara- bic [22,23], Japanese [24], Italian [25], Malay [26], and Brazilian Portuguese [27]. The report on HL survey in eight EU countries [4] does not describe translation procuredure of the NVS for Bulgarian, German, Greek, and Polish versions although data on HL results are included for those countries as well; however, a German language version is briefly described by Berens et al. [28]. A Hungarian NVS version is described in a paper in Hungarian language [29]. The usually adopted but complex procedure of translation/cultural adaptation–termed also linguistic validation, of patient-reported outcome (PRO) instruments is described by Acquadro et al. [30]. This includes initial engagement with the instrument developer, forward and back- ward translation, review of the reconciled version with the developer of the original instrument, cognitive interviews, and expert panel review. To the best of our knowledge, based on literature review, there is no test of functional health literacy developed in or translated into Croatian lan- guage that has undergone full linguistic validation procedure. The aim of this study was to develop a linguistically validated Croatian version of the NVS health literacy test and to use it at a later stage in a pilot study of health literacy assessment of hospital patients in Croatia. Materials and methods The cognitive inter- view results were again reviewed by 3 out of 5 experts and the developer of the NVS-UK. The final version (NVS-HR) was reached when there were no more suggestions for modifications in the linguistic validation step. followed the procedure as described by Acquadro et al. [30] for PRO instruments. The NVS-UK version was translated into Croatian by two independent professional translators, native speakers of Croatian, who were given an instruction to take into account the Croatian style of ice cream nutrition labels found in the market, which are in line with the EU and Croa- tian regulations [31], but at the same time to keep the Croatian translation as close as possible to the source text of the NVS-UK, and to check the NVS-US for further assistance, if needed. One forward translator was also provided with the Italian translation of the NVS-UK and the Spanish translation of the NVS-US for further assistance, since they were also fluent in these two languages. A panel of five experts covering the fields of linguistics, health literacy, public health, medicine (neurology), nutrition, and nursing, reviewed the two forward translations taking into account the same instructions as given for the forward translation step and pre- pared a reconciled version by agreeing on the best translation option for the entire NVS test. The reconciled version was backtranslated by a professional translator, native speaker of English and fluent in Croatian. The backtranslated version was reviewed by a panel of 3 out of 5 experts, as well as by the developer of the NVS-UK. Cognitive interviews were done face-to- face by a trained interviewer with 10 respondents recruited from general population in an urban area by a medical nurse, with the inclusion criteria of both genders, varying age and occupation, lower educational level (primary and secondary school, that is, up to 12 years of schooling). Respondents were given a hard copy of the NVS ice cream nutrition label to read to themselves, then answered the NVS questions read out loud by the interviewer, and after- wards discussed the instrument with the interviewer focusing on: general impression (length and difficulty of the instrument); questions about which parts/words/phrases they did not understand; what each NVS item meant to them in their own words, with examples; how they reached the answers to NVS questions; and suggestions for modifications. Materials and methods The Croatian version of the NVS instrument (NVS-HR) is based on the NVS-UK version, which adapted the NVS-US for the European context. The linguistic validation process 2 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 Health literacy with Croatian NVS followed the procedure as described by Acquadro et al. [30] for PRO instruments. The NVS-UK version was translated into Croatian by two independent professional translators, native speakers of Croatian, who were given an instruction to take into account the Croatian style of ice cream nutrition labels found in the market, which are in line with the EU and Croa- tian regulations [31], but at the same time to keep the Croatian translation as close as possible to the source text of the NVS-UK, and to check the NVS-US for further assistance, if needed. One forward translator was also provided with the Italian translation of the NVS-UK and the Spanish translation of the NVS-US for further assistance, since they were also fluent in these two languages. A panel of five experts covering the fields of linguistics, health literacy, public health, medicine (neurology), nutrition, and nursing, reviewed the two forward translations taking into account the same instructions as given for the forward translation step and pre- pared a reconciled version by agreeing on the best translation option for the entire NVS test. The reconciled version was backtranslated by a professional translator, native speaker of English and fluent in Croatian. The backtranslated version was reviewed by a panel of 3 out of 5 experts, as well as by the developer of the NVS-UK. Cognitive interviews were done face-to- face by a trained interviewer with 10 respondents recruited from general population in an urban area by a medical nurse, with the inclusion criteria of both genders, varying age and occupation, lower educational level (primary and secondary school, that is, up to 12 years of schooling). Respondents were given a hard copy of the NVS ice cream nutrition label to read to themselves, then answered the NVS questions read out loud by the interviewer, and after- wards discussed the instrument with the interviewer focusing on: general impression (length and difficulty of the instrument); questions about which parts/words/phrases they did not understand; what each NVS item meant to them in their own words, with examples; how they reached the answers to NVS questions; and suggestions for modifications. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 Materials and methods The cognitive inter- view results were again reviewed by 3 out of 5 experts and the developer of the NVS-UK. The final version (NVS-HR) was reached when there were no more suggestions for modifications in the linguistic validation step. The NVS-HR was then used to assess HL within a pilot study involving a convenience sam- ple of 100 hospital patients in an urban area of eastern Croatia, a part of the country that was heavily affected by war in the 1990s, with a high unemployment rate (12.1%, July 2016) [32], and increasing emigration of young people. This part of the study was performed in the largest hospital of Osijek-Baranja County, the University Hospital Center Osijek, with the reported total bed capacity of 986 for stationary patients in 2016 [33]. Exclusion criteria were: patients under 18 years of age and patients from departments that provide care to mental patients since no mental capacity assessments were planned. The hospital departments included in the study, with the respective bed capacity as reported by the hospital nurse, were: Surgery (185), Internal Medicine (150), ENT (26), Orthopedics (40). Taking into account exclusion criteria and expecting occasional objective barriers to NVS testing, such as postoperative pain, high per- centage of patients without glasses [10], the approachable realistic number in each department was estimated as 50, 50, 20 and 10 patients, respectively. Expecting a relatively high response rate, we set the target number of 100 patients in the sample for this pilot study. The study was approved by the hospital ethics committee, Committee for Ethical and Pro- fession Issues of Medical Nurses and Technicians, University Hospital Center Osijek, and each patient signed an informed consent form. All patients from the four hospital departments were approached consecutively until the target number of 100 was reached. The testing was done in January 2017, by a trained interviewer, a medical nurse employed in the hospital. Data were collected on patient sociodemographic characteristics and their overall opinions on HL testing, with questions constructed for the purpose of the study. Each patient was given a lami- nated copy of the NVS-HR ice cream nutrition label, and the NVS-HR questions were read PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 3 / 14 Health literacy with Croatian NVS out loud to the patient. No use of a calculator or any supporting materials was allowed. The NVS-HR testing completion time was recorded for each patient. Stage 1 –Linguistic validation of the NVS Stage 1 –Linguistic validation of the NVS The backtranslation of the nutrition label as worded in the Croatian reconciled version is given in Fig 1. The backtranslator’s second option for some words is presented in square brackets. Analysis of consideration points, made by the expert panel upon backtranslation of the rec- onciled version, resulted in keeping the NVS text in Croatian unmodified since reflecting well the document in English and complying with the standards used in Croatian nutrition labels. The only problem arose with Question 2 (Q2) wording “how many ice creams maximum are you allowed to eat?”, which could be interpreted as ‘how many containers/packages’, so it was changed to: “what maximum amount of ice cream may you eat?”. Cognitive interviews with 10 respondents from general population revealed that the respon- dents considered the NVS test short enough and not complicated, although requiring some concentration and mathematical skills. Some respondents asked for some questions to be repeated, which was done, and one asked for a calculator, which was not allowed. Demo- graphic characteristics and NVS scores for 10 respondents in the cognitive interview step are given in Table 1. The NVS nutrition label was well understood, although some respondents found the fat sub- types confusing, suggested one word only for calories, or did not understand whether ‘guar gum’ was related to rubber gloves and therefore to allergic reaction mentioned in the test. The NVS test questions raised a few concerns. Some respondents miscalculated Question 1 either by confusing ‘portion’ with ‘package’ or not knowing where to find the correct values, and one respondent mentioned not necessarily reacting to peanuts. The interviewer also noted that for some respondents it was difficult to calculate when hearing a question just once so they miscal- culated, or they focused on numbers only or some words only forgetting e.g. a difference between serving size and package. The expert panel and the developer agreed that modifications suggested by respondents were not needed since they were either irrelevant or would actually change the discriminatory power of the test, except for one wording: ‘guar gum’ (Croatian: ‘guar guma’) may be closely related to ‘rubber gloves’ (Croatian: ‘gumene rukavice’–made of gum), which made some respondents think ‘if allergic to rubber gloves, it’s not safe to eat this ice cream’. Therefore, this confusing wording of ‘guar gum’ was modified to ‘guar mixture’. Materials and methods Reflecting the scoring in current versions of the NVS, the NVS-HR total scores (0–6) were interpreted as follows: 4 = adequate HL; 2–3 = intermediate HL; 0–1 = low HL. Question 7, since considered a subquestion of Question 6 also in the NVS-UK, when answered correctly, was interpreted as ’Question 6 answered correctly’. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS version 22.0; Chicago, IL, USA) software. Collected data are described by frequencies and percentages. We used means, standard deviation (SD), and median values to summarize the respondents’ characteristics and performance on the test. For categorical variables, Fisher’s exact test was used to compare the groups. Values of p < 0.05 were considered statistically significant. https://doi.org/10.1371/journal.pone.0193079.t001 Stage 2 –Health literacy testing among hospital patients A total of 144 consecutive patients from the four hospital departments were considered for HL testing using the NVS-HR, to reach the target sample size of 100 participants. For the 44 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 4 / 14 Health literacy with Croatian NVS Fig 1. NVS nutrition label backtranslated from Croatian. patients not included in the sample, since no informed consent could have been obtained, the following reasons were given: not having reading glasses (15); problems with eyesight (2); not having a hearing aid (1); severe medical condition–poor general health (8), mental illness (3), Table 1. Demographic characteristics, completion time, and NVS scores of 10 respondents in cognitive interview step. No. Age Gender Educational level (total years of schooling) Occupation Residence Completion time (min.) NVS score 1 46 F Secondary school (11) unemployed shoe maker urban 4.31 6 2 38 F Primary school (8) cleaner urban 9.05 3 3 32 F Secondary school (11) hair dresser rural 2.25 6 4 18 F Secondary school (12) unemployed trade worker rural 6.37 5 5 21 F Secondary school (12) secretary urban 8.28 5 6 55 M Secondary school (11) self-employed in farming business rural 8.45 6 7 50 M Secondary school (12) retired army officer rural 3.48 5 8 43 M Secondary school (12) construction worker urban 4.15 5 9 24 M Secondary school (12) shop assistant urban 2.45 6 10 68 M Primary school (4) retired worker in agriculture rural 7.32 5 Demographic characteristics, completion time, and NVS scores of 10 respondents in cognitive interview step. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 5 / 14 Health literacy with Croatian NVS postoperative pain (7); illiterate patients (2); refusal to participate–because of no personal gain from the study (1), no clear purpose of study results (2), not stating a reason (3). The final sam- ple included 49 patients from Internal Medicine department, 34 from Surgery, 10 from ENT, and 7 from Orthopedics. Of the total 100 patients who participated in the study, 55% were women, 44% over 65 years of age (median age 63.5 years, range 18–84), 59% retired, 58% with secondary level edu- cation (11 or 12 years of schooling), 67% with very low or no income, 53% with self-reported chronic condition, and 69% with BMI 25 (32% overweight, 37% obese). Patient sociodemo- graphic and baseline characteristics are given in Table 2. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 Stage 2 –Health literacy testing among hospital patients Mean completion time was 4.2 minutes (SD = 1.2 minutes), with range 1.9–8.8 minutes. Mean NVS total score was 3.34 (SD = 1.3), with most patients (31%) scoring 3 points. The NVS total scores with percentage of patients per score are shown in Fig 2. Percentage of patients who gave correct answer to each of the 6 NVS questions is given in Table 3, with backtranslation of each NVS-HR question. Q7, as a subquestion of Q6, when Table 2. Patient sociodemographic and baseline characteristics (n = 100). Variable % Gender Male 45 Female 55 Residence Urban 64 Rural 36 Age (years) < 45 15 45–65 41 > 65 44 Education Without or incomplete primary school 8 Primary school 22 Secondary school 58 College or more 12 Employment status Student / training 3 Unemployed 5 Housewife 8 Self-employed 2 Retired 59 Employed (full time) 23 Monthly income [34] No income 3 < 3000 HRK 64 3000–6000 HRK 24 6000–12000 HRK 9 BMI Underweight (<18.5) 3 Normal weight (18.5–24.99) 28 Overweight (25–29.99) 32 Obesity (30) 37 Self-reported chronic condition No chronic condition 47 Diabetes 21 Gastritis 5 Other 27 HRK C ti k (EUR 1 HRK 7 6 J 2017) able 2. Patient sociodemographic and baseline characteristics (n = 100). Table 2. Patient sociodemographic and baseline characteristics (n = 100). PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 6 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 Health literacy with Croatian NVS Fig 2. NVS total score with percentage of patients. https://doi org/10 1371/journal pone 0193079 g002 Fig 2. NVS total score with percentage of patients. https://doi.org/10.1371/journal.pone.0193079.g002 https://doi.org/10.1371/journal.pone.0193079.g002 answered correctly, was interpreted as ’Q6 answered correctly’. The easiest question was Q5 and the most difficult was Q1 (only 24% correct), where most patients, 59 out of 76 who answered incorrectly, replied incorrectly ’250 kcal’. Adequate HL level was found in 42% of patients; 51% had intermediate HL level, and 7% had low HL level. HL levels by sociodemographic and baseline characteristics are shown in Table 4. A positive significant association was observed between HL level and educational level, showing that patients with higher education scored significantly higher on the NVS test. Namely, no patient with high educational level had low HL, and 91.7% of those patients had adequate HL. Statistically significant differences were observed in employment status as well Table 3. Percentage of correct answers to each NVS-HR question. NVS question % of correct answers Q1. How many calories (kcal) will you consume if you eat the entire package? 24 Q2. If you are advised not to eat more than 60 grams of carbohydrates for dessert, what maximum amount of ice cream may you eat? 33 Q3. Imagine your doctor advising you to reduce the amount of saturated fat in your diet. You normally consume 42 g of saturated fat daily, which includes one serving of ice cream. If you stop consuming ice cream, how many grams of saturated fat will you be consuming every day? 47 Q4. If you normally consume 2500 calories every day through food, what percentage of your daily intake of calories (kcal) will you consume if you eat one serving of ice cream? 74 Q5. Imagine that you are allergic to the following substances: penicillin, peanuts, rubber gloves and bee stings. Is it safe for you to eat this ice cream? 80 Q6. [If the answer to Question 5 is "no"]: Why not? 76 (Q7. [If the answer to Question 6 is "Because I could have an allergic reaction", ask:] Why would you have an allergic reaction?) https://doi.org/10.1371/journal.pone.0193079.t003 Table 3. Percentage of correct answers to each NVS-HR question. 7 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 Health literacy with Croatian NVS Table 4. Health literacy levels by sociodemographic and baseline characteristics (n = 100). Characteristic Health literacy level (%; n) P-value Low Intermediate Adequate Age group (years) P = 0.130 < 45 0.0% (0) 33.3% (5) 66.7% (10) 45–65 12.2% (5) 46.3% (19) 41.5% (17) > 65 4.5% (2) 61.4% (27) 34.1% (15) Gender P = 0.269 Male 11.1% (5) 53.3% (24) 35.6% (16) Female 3.6% (2) 49.1% (27) 47.3% (26) Educational level P = 0.002 Low (no education; incomplete or complete primary school) 10.0% (3) 66.7% (20) 23.3% (7) Medium (secondary school) 6.9% (4) 51.7% (30) 41.4% (24) High (college or above) 0.0% (0) 8.3% (1) 91.7% (11) Residence P = 0.167 Urban 3.1% (2) 53.1% (34) 43.8% (28) Rural 13.9% (5) 47.2% (17) 38.9% (14) Employment status P = 0.036 Student / training 0.0% (0) 0.0% (0) 100.0% (3) Unemployed 20.0% (1) 20.0% (1) 60.0% (3) Housewife 0.0% (0) 62.5% (5) 37.5% (3) Self-employed 50.0% (1) 0.0% (0) 50.0% (1) Retired 8.5% (5) 59.3% (35) 32.2% (19) Employed (full time) 0.0% (0) 43.5% (10) 56.5% (13) Monthly income P = 0.390 No income 0.0% (0) 100.0% (3) 0.0% (0) < 3000 HRK 7.8% (5) 53.1% (34) 39.1% (25) 3000–6000 HRK 4.2% (1) 37.5% (9) 58.3% (14) 6000–12000 HRK 11.1% (1) 55.6% (5) 33.3% (3) BMI P = 0.583 Underweight (<18.5) 33.3% (1) 33.3% (1) 33.3% (1) Normal weight (18.5–24.99) 3.6% (1) 57.1% (16) 39.3% (11) Overweight (25–29.99) 9.4% (3) 53.1% (17) 37.5% (12) Obesity (30) 5.4% (2) 45.9% (17) 48.6% (18) Self-reported chronic condition P = 0.901 No chronic condition 6.4% (3) 46.8% (22) 46.8% (22) Diabetes 9.5% (2) 52.4% (11) 38.1% (8) Gastritis 0.0% (0) 80.0% (4) 20.0% (1) Other 7.4% (2) 51.9% (14) 40.7% (11) https://doi.org/10.1371/journal.pone.0193079.t004 le 4. Health literacy levels by sociodemographic and baseline characteristics (n = 100). but the number of patients in many analyzed groups was too low to draw any firm and clear conclusions. Statistically significant associations between HL levels and other sociodemo- graphic or baseline characteristics were not found. Patients also replied to several questions on HL testing in general. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 Discussion [30] for translation/cultural adaptation of PRO Cultural issues with the NVS test related to unfamiliarity with nutrition labels in the respon- dents’ daily activities have been described [19,26] and also observed in this study with hospital patients. When asked about reading ice cream or other food labels, 89% of patients mentioned Cultural issues with the NVS test related to unfamiliarity with nutrition labels in the respon- dents’ daily activities have been described [19,26] and also observed in this study with hospital patients. When asked about reading ice cream or other food labels, 89% of patients mentioned never reading ice cream nutrition labels, and 62% mentioned never or rarely reading food labels. However, we feel that a test based on understanding and use of a nutrition label remains a valid screening tool for HL as it directly relates to an activity required for healthy eating, and tests the abilities to navigate through text, find the relevant information, and draw conclusions leading to potential health-related decision-making. Also, the NVS allows clinicians and health administrators to rapidly assess HL in their patients–within 3 minutes [13], in 3–6 minutes [21], or around 4 minutes [22] as found in this study. Some language versions did require lon- ger mean completion times– 6.28 [18] and 10.8 [23], but HL testing in Iraq was self-adminis- tered, allowing the participants to read the entire text on their own, which perhaps yielded such long completion times. Although a study on the use of the NVS has indicated to a limited practicality of this test in the elderly population with the mean completion time of 11 minutes [37], 25% of the participants over the age of 65 in this Croatian study completed the test within 4 minutes, and only three required more than 6 minutes. Although different methodologies have been used for development of the different language versions, and the sample size and profiles have varied in the NVS validation studies, results of HL testing may be compared internationally, but to a certain degree and with caution. HL test- ing with NVS-HR on 100 hospital patients identified 42% of patients with adequate HL, 51% with intermediate HL, and 7% with low HL (mean total score 3.34). A very similar mean score of 3.4 was observed with both NVS-US English version [13] and the NVS-UK [14], and slightly lower (3.2) in Kuwait [22] and Iraq (3.02) [23]. A high percentage of patients (92%) did not object to being tested for HL by their primary care physician or in hos- pital; three patients said they would feel shame, two patients said it would be a waste of the physician’s time, and three gave no reason for their objection. Also, 99% of patients would rec- ommend HL testing among patients in general, and 98% were of the opinion HL testing results would help physicians and medical nurses in their communication with patients. When asked PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 8 / 14 Health literacy with Croatian NVS about reading ice cream or other food labels, 89% mentioned never reading ice cream nutri- tion labels, and 62% mentioned never or rarely reading food labels. Discussion The development of robust HL tests is lengthy and complex, as reflected in the description of the development of the original NVS (US version) [13]. The cultural and linguistic adaptations for another country and/or language need to undergo a rigorous procedure for the test to be acceptable and useful [19,20]. This implies the process of cultural adaptation as well as transla- tion where needed. The Croatian version of the NVS underwent full linguistic validation pro- cedure as described by Acquadro et al. [30] for translation/cultural adaptation of PRO instruments, before it was used for HL testing of 100 hospital patients. Since the rigorous pro- cedure included forward/backward translations by professional translators experienced in the procedure, along with pre-testing on general population, expert reviews, and full involvement of the lead author of the NVS-UK version, only minor modifications were required to reach the final text version. The procedures adopted for the NVS versions in other countries have varied from a Delphi technique [14,20], full [19,21,23,27] or partial linguistic validation [22,24,25], to forward and backward translation only [18,26]. When the step of cognitive inter- views is excluded, valuable information may be missing on why some NVS questions result in incorrect answers. Cognitive interviews in this study revealed why some respondents miscal- culated in Question 1 (Q1)–confusing ’portion’ with ’package’, which was done by 59 hospital patients tested for HL as they replied incorrectly ’250 kcal’. Q1 was identified as the most diffi- cult; the same, along with Q3, was found in a study among Canadian immigrants [35], and Q3 alone, considered very long and complex [20] was the most difficult in some other language versions [18,36] that at the same time found Q5 the easiest, identical to this Croatian study. The development of robust HL tests is lengthy and complex, as reflected in the description of the development of the original NVS (US version) [13]. The cultural and linguistic adaptations for another country and/or language need to undergo a rigorous procedure for the test to be acceptable and useful [19,20]. This implies the process of cultural adaptation as well as transla- tion where needed. The Croatian version of the NVS underwent full linguistic validation pro- cedure as described by Acquadro et al. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 Discussion On the other hand, very low mean scores were observed in Spanish [13], Dutch [19], Turkish [18], and Japanese [24] study groups– 1.6, 1.8, PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 9 / 14 Health literacy with Croatian NVS 2.6, and 2.1, respectively. Also, the NVS-HR mean score was much lower than with NVS-D [20] and NVS-PT [21] of 4.2 and 4.4, respectively. However, the NVS-D testing [20] was partly done online on respondents who had access to and were familiar with the use of the Internet, and were allowed to read the NVS questions and use a calculator, whereas the NVS-PT [21] testing was done mostly on students. Additionally, the NVS-HR mean score is similar to the average EU score of 3.5, as reported in a study on HL among 8 EU countries [4], where ade- quate HL level was found in 55.3% of the total study population. Interestingly, with the NVS-BR [27], adequate HL was found in 48.7% of adult pharmacy clients compared to 33.5% of public school teachers. The lower HL levels among teachers compared to the general popu- lation are explained by the fact that the teachers may have not paid enough attention to the NVS since they were given another lengthy instrument to complete first, leading the authors to note that the NVS scores might be impacted by the number of items in multiple-scale surveys. g p y p y HL testing with NVS-HR showed a significant positive association between educational level and HL, reflecting finding from a systematic review [38], the NVS-UK [14], a HL survey of 8 EU countries for the general sample [4], the NVS-D [19,20], and the Turkish study [18]. Whilst the associations between HL and education level appear to be consistent across coun- tries, it is important to note the international variations in associations between other socio- economic and demographic determinants of health and HL. For instance, no statistical associ- ation between HL and gender was found with the NVS-HR, and neither in a systematic review of US studies [38] or some NVS language versions where it was measured and reported [4,13,23,27]. On the other hand, the Turkish study [18] found the lowest NVS scores in female participants whereas the NVS-D scores [20] were significantly higher in the female study pop- ulation. Discussion As to age, both of those studies found older participants to have lower NVS scores, the same as the Portuguese [21] and the Brazilian Portuguese study [27], but not with the NVS-HR. It is likely that these differences reflect differences in national education and health systems, and national culture and context [4,39], meaning that an understanding of HL within different countries requires local measurement and exploration of the associations with socio- economic and demographic factors, and cannot be imputed from other national settings [39]. Weiss et al. [13] cautioned that patients with probable marginal or inadequate literacy (NVS scores below 2) cannot be reliably identified by clinicians if asked only about their edu- cational level, as education does not always predict literacy. Although HL testing with NVS-HR showed significant positive association between educational level and HL level, edu- cational level alone would not identify each patient with limited literacy–for example, 6.9% of those with medium educational level had low HL, and 23.3% with low educational level had adequate HL level, so an objective HL test applied in clinical practice, especially when it takes around 4 minutes, should be considered a better and more precise predictor. Weiss et al. con- sider the NVS test as ’a quick and accurate screening test for limited literacy’ suitable for pri- mary health care settings [13]. Authors of the NVS-UK version consider this instrument a simple and accurate predictor of health literacy skills, which can be administered by both clini- cal and non-clinical personnel, and it has a potentially valuable role in improving clinical prac- tice and patient communication [14]. Furthermore, some studies have shown that healthcare providers, including physicians and nurses, overestimate their patients’ health literacy levels, but also that they have limited knowledge and skill related to health literacy assessment. For example, one NVS testing showed that 63% of patients had a high likelihood of limited HL and 22% of patients had a high likelihood of adequate HL, whereas nurses reported 19% and 68%, respectively [40]. The potential use of a quick, sensitive and specific measure of HL in clinical practice has been recognized; however, there has been anxiety about the routine use in clinical practice due to concerns about the impact of stigma on the patients [41], and hence on the doctor-patient relationship. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 Discussion This has resulted in suggestions that a better approach is one of PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 10 / 14 Health literacy with Croatian NVS ’universal precautions’, where the focus is on developing a health service accessible to all, regardless of HL capacities [42]. Whilst this is a commendable approach, it is likely that finan- cial and time constraints may hamper or preclude such an approach. Our finding that patients in this Croatian study had no objections (92%) to HL testing, with as many as 99% recom- mending HL testing among patients, and 98% being of the opinion that HL testing results would help physicians and medical nurses in their communication with patients, is supported by Ryan et al. [43]. This brings an additional option to health care providers in resource-con- strained settings such as Croatia. ’universal precautions’, where the focus is on developing a health service accessible to all, regardless of HL capacities [42]. Whilst this is a commendable approach, it is likely that finan- cial and time constraints may hamper or preclude such an approach. Our finding that patients in this Croatian study had no objections (92%) to HL testing, with as many as 99% recom- mending HL testing among patients, and 98% being of the opinion that HL testing results would help physicians and medical nurses in their communication with patients, is supported by Ryan et al. [43]. This brings an additional option to health care providers in resource-con- strained settings such as Croatia. Among the limitations of this study, we would include a relatively small sample size (n = 100), which in turn yielded a relatively high percentage of the participants in retirement (59%), not formally employed (75%), with low income (67%), being overweight or obese (69%), and even of the age 45 years (85%). However, this was a convenience sample where the patients were approached consecutively in the four hospital departments, so it reflected the actual situation both in the hospital and the area. Also, this pilot study was limited to a single hospital center but it is the only one in this eastern county and among the five largest hospitals in Croatia. Selection of the hospital setting for participant recruitment may be justified by the fact that a large hospital center would admit patients from different urban and rural smaller primary care units in the county. Conclusion This study developed the Croatian version of the NVS health literacy screening tool, based on the UK version since adapted for the European context, and following a full linguistic valida- tion procedure. HL testing with NVS-HR on 100 hospital patients identified 42% of patients with adequate HL, found the mean total NVS score of 3.34, and statistically positive association of HL with educational level. These findings are in line with the results of other studies describ- ing the development of the NVS for other languages. Results also showed a short average com- pletion time of the NVS-HR of 4 minutes, and the exceptionally high percentage (99%) of study participants who would recomment HL testing among patients in general. Considering the fact that the NVS is considered a quick and simple but also an accurate screening test for health literacy, we recommend the NVS-HR for HL testing in clinical settings in Croatia. As in other countries, the NVS-HR is also likely to be a useful tool for researchers investigating the impact of health literacy on health and illness. Discussion On the other hand, any generalizations of the findings for the general population should be made carefully considering the selection of participants and the small sample size. Finally, data on ethnicity were not collected, although ethnicity plays an important role in HL testing worldwide [3,4], due to the fact that 90% of the population both in Croatia and the city of Osijek declare themselves as Croats [44]. Acknowledgments The authors would like to thank all study participants, the translators and expert panel mem- bers for their contribution to this study. References 1. Ad Hoc Committee on Health Literacy for the American Council on Scientific Affairs, American Medical Association. Health literacy: report of the council on scientific affairs. JAMA. 1999; 281(6):552–7. PMID: 10022112 2. Institute of Medicine. Health Literacy: A Prescription to End Confusion. Nielsen-Bohlman L, Panzer A, Kindig DA, editors. Washington, DC: National Academies Press; 2004. 3. Berkman ND, Sheridan SL, Donahue KE, Halpern DJ, Crotty K. Low health literacy and health out- comes: an updated systematic review. Ann Intern Med. 2011; 155:97–107. https://doi.org/10.7326/ 0003-4819-155-2-201107190-00005 PMID: 21768583 4. HLS-EU Consortium: Comparative report on health literacy in eight EU member states. The European Health Literacy Project 2009–2012. Vienna: Ludwig Boltzmann Institute for Health Promotion Research, 2012. (Second extended and revised version, 2014) Available from: http://lbihpr.lbg.ac.at. w8.netz-werk.com/sites/files/lbihpr/attachments/neu_rev_hls-eu_report_2015_05_13_lit.pdf 5. Vučemilo L, Borovečki A. Readability and content assessment of informed consent forms for medical procedure sin Croatia. PLoS ONE 2015; 10(9):e0138017. https://doi.org/10.1371/journal.pone. 0138017 PMID: 26376183 6. Dukić N, Blecich A, Cerović Lj. Economic implications of insufficient health literacy. Ekonomska istrazˇi- vanja–Economic Research 2013; 26(Suppl 1):117–32. 7. Brangan S. Development of SMOG-Cro readability formula for healthcare communication and patient education. Coll Antropol 2015; 39(1):11–20. PMID: 26040062 8. Brangan S. Quantitative assessment of text difficulty in Croatian language. Rasprave: Časopis Instituta za hrvatski jezik i jezikoslovlje 2014; 40(1):35–58. 9. Vučemilo L, C´ urković M, Milosˇević M, Mustajbegović J, Borovečki A. Are physician-patient communica- tion practices slowly changing in Croatia?–a cross-sectional questionnaire study. Croat Med J 2013; 54 (2):185–91. https://doi.org/10.3325/cmj.2013.54.185 PMID: 23630146 10. Kusec S, Oreskovic S, Skegro M, Korolija D, Busic Z, Horzic M. Improving comprehension of informed consent. Patient Educ Couns. 2006; 60(3):294–300. https://doi.org/10.1016/j.pec.2005.10.009 PMID: 16427762 11. Kusec S, Mastilica M, Pavlekovic G, Kovacic L. Readability of patient information on diabetes on the Croatian Web sites. Stud Health Technol Inform 2002; 90:128–32. PMID: 15460674 12. Nutbeam D. Health literacy as a public health goal: a challenge for contemporary health education and communication strategies into the 21st century. Health Promot Int. 2000; 15(3):259–67. 13. Weiss BD, Mays MZ, Martz W, Castro KM, DeWalt DA, Pignone MP, et al. Quick assessment of literacy in primary care: the newest vital sign. Ann Fam Med. 2005; 3:514–22. https://doi.org/10.1370/afm.405 PMID: 16338915 14. Rowlands G, Khazaezadeh N, Oteng-Ntim E, Seed P, Barr S,Weiss BD. Development and validation of a measure of health literacy in the UK: the newest vital sign. BMC Public Health. 2013; 13:116. https:// doi.org/10.1186/1471-2458-13-116 PMID: 23391329 15. Project administration: Sanja Brangan. Project administration: Sanja Brangan. Supervision: Sanja Brangan, Goranka Rafaj, Gill Rowlands. Visualization: Sanja Brangan, Martina Ivanisˇić, Goranka Rafaj, Gill Rowlands. Writing – review & editing: Sanja Brangan, Martina Ivanisˇić, Goranka Rafaj, Gill Rowlands. Author Contributions Conceptualization: Sanja Brangan, Martina Ivanisˇić, Goranka Rafaj, Gill Rowlands. Data curation: Sanja Brangan, Martina Ivanisˇić. Formal analysis: Sanja Brangan. Investigation: Martina Ivanisˇić. Conceptualization: Sanja Brangan, Martina Ivanisˇić, Goranka Rafaj, Gill Rowlands. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193079 February 15, 2018 11 / 14 Health literacy with Croatian NVS Project administration: Sanja Brangan. Resources: Martina Ivanisˇić, Gill Rowlands. Supervision: Sanja Brangan, Goranka Rafaj, Gill Rowlands. Visualization: Sanja Brangan, Martina Ivanisˇić, Goranka Rafaj, Gill Rowlands. Writing – original draft: Sanja Brangan. Writing – review & editing: Sanja Brangan, Martina Ivanisˇić, Goranka Rafaj, Gill Rowlands. 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https://openalex.org/W2043235398	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2568845/pdf/mlab-96-04-370.pdf	English	null	Teaching teens to “Get Net Smart for Good Health”: comparing interventions for an Internet training program	Journal of the Medical Library Association	2,008	cc-by	4,467	METHODS The project involved a combination of structured workshops and hands-on activities, held at local high schools with 10th and 11th graders, from year 1 (2005) to year 3 (2007). Classes of students enrolled in a health sciences vocational curriculum were selected by their teachers to participate in the program based on their interest in pursuing careers in science and health care. Workshops included a pretest and posttest, structured instruction, a health-related website evaluation exer- cise, and a satisfaction survey. Workshops were modified in year 2 to include a role-playing compo- nent, and an evaluation of the role-playing exercise was added to the program in year 3. The project was approved by the MUSC Institutional Review Board. p Given this challenge, a team of outreach specialists at the Medical University of South Carolina (MUSC) Library in Charleston, South Carolina, experimented for three years with ways to teach high school students to critically evaluate health websites. The team’s goal was to develop and compare interven- tions for teaching students how to evaluate health websites and transfer what they had learned to new situations. Using the results discussed here, the team developed an outreach program, ‘‘Get Net Smart for Good Health,’’ that engages students in active learning through a hands-on computer workshop and a role-playing activity called ‘‘Cyber Court.’’ A program description and portable document format (PDF) file of the ‘‘Get Net Smart for Good Health’’ curriculum are available online [7]. This paper describes program development and evaluation re- sults for 2005 to 2007. Workshops The workshops took place at four different schools over three years, with no students repeating a workshop. The average size of participating high school classes was twelve students, and classes were composed of all tenth graders, all eleventh graders, or a mixture of tenth and eleventh graders. Workshops were held at MUSC with the exception of workshops for the tenth grade classes from school A in year 2 and the tenth grade classes from school D in year 3, due to shortages of funds for transportation costs. * Supported by the National Library of Medicine under contract no. N01-LM-1-3522 and by the Medical University of South Carolina Project EXPORT Center on Metabolic Syndrome and Minority Health (5 P60 MD000267), funded by the National Center on Minority Health and Health Disparities. Lillian D. Trettin, PhD; Janice C. May, MPA; Nancy C. McKeehan, MSLS Lillian D. Trettin, PhD; Janice C. May, MPA; Nancy C. McKeehan, MSLS See end of article for author’s affiliations. DOI: 10.3163/1536-5050.96.4.013 INTRODUCTION With their casual acceptance of information on the Internet, high school students are a tough audience for librarians. A detailed study of 12 middle and high school students in 2003 revealed that 77% of the students’ searches for answers to health questions led them no farther than a search engine’s first links [1]. Another study based on two dozen focus groups (157 teens) reported that students acknowledged lacking the skill to evaluate Internet sites [2]. An anecdotal report from National Public Radio suggested that, even in college, students forgot to verify sources unless asked to do so—and that faculty rarely asked [3]. These habits can become entrenched by adulthood, and as librarians, educators, and computer systems analysts agree, most Internet users are guilty of taking information on the Internet at face value, even if they know what steps to take to evaluate content [4–6]. * Supported by the National Library of Medicine under contract no. N01-LM-1-3522 and by the Medical University of South Carolina Project EXPORT Center on Metabolic Syndrome and Minority Health (5 P60 MD000267), funded by the National Center on Minority Health and Health Disparities. { Based on a poster presented at MLA ’07, the 107th Annual Meeting of the Medical Library Association; Philadelphia PA; May 23, 2007. Supplemental Appendixes A and B and Tables 2 and 3 are available with the online version of this journal. Teaching teens to ‘‘Get Net Smart for Good Health’’: comparing interventions for an Internet training program{ ‘‘Get Net Smart for Good Health’’ is part of a larger outreach effort aimed at residents of South Carolina, a state with one of the lowest high school completion rates and literacy rates in the nation [8]. In 2002, the MUSC Library created a Website for laypeople called Hands on Health—South Carolina with initial funding from the Duke Endowment [9]. Collaborators in the site’s development included the South Carolina State Library, the University of South Carolina School of Medicine Library, and the South Carolina Hospital Association. Hands on Health—South Carolina is a gateway to reputable sites like MedlinePlus, with additional content written for readers with low literacy skills. The site is currently being translated into Spanish. The site is tailored to address the needs and interests of South Carolina residents with special features like ‘‘Go Local-SC,’’ which directs readers to local services, many of them free or low cost. Further information about the National Library of Medicine’s Go Local initiatives is available at the National Library of Medicine Internet site [10]. The MUSC team uses Hands on Health—South Carolina and MedlinePlus extensively in the ‘‘Get Net Smart’’ program, as well as in workshops for other target audiences. Participants at North Charleston High School (NCHS) (school A in Table 1) to ensure that the program met state curricular standards. NCHS is an inner-city school, serving a 90% low-income, minority student popula- tion. Nearly all the students were African American. One class at each of 2 suburban high schools (schools B and C) with 40% minority students also attended the workshop in year 1. In year 2, students taking the workshop attended 1 school serving 90% low-income, minority students (school A) and 1 school with a 40% minority constituency (school B). In year 3, student participants attended an urban magnet school serving primarily low-income minority students (school D). credibility site was 24–30, and the ideal average score for an objectionable or low credibility site was 6–12. Students also filled out a satisfaction survey assessing their perceptions of the workshop. During the year 1 evaluation process, a higher than expected change in test scores, combined with the poor comprehension students exhibited with the web exer- cise, indicated that the pretest/posttest instrument needed redesign. The team decided to replace the short-answer format with a multiple-choice format and introduced a revised instrument in year 2. While the revision included new items, the team calculated multiyear evaluation results using only the two items that were used every year. Following the revision process, the team held the workshop with students at schools A and B in year 2. The pretest/posttest (Appendix A online), website exercise (Appendix B online), and satisfaction survey were again administered. That year, the team experimented with an additional role-playing activity to add an element of active learning to complement the largely didactic workshop [11]. This activity, ‘‘Cyber Court,’’ entailed small groups of three or four students preparing a hypothetical courtroom scene to dramatize whether a particular health site was reliable enough to be considered good evidence. Students had to say, in their own words, why a particular website was or was not reliable and why that mattered, given a set of circumstances. As teachers felt that eleventh grade students would be better equipped to take on the role- playing exercise, only eleventh grade students partici- pated in the exercise. J Med Libr Assoc 96(4) October 2008 Participants S il During year 1, MUSC staff developed the first workshop with the help of a health sciences teacher Supplemental Appendixes A and B and Tables 2 and 3 are available with the online version of this journal. J Med Libr Assoc 96(4) October 2008 370 Brief communications: Trettin et al. Table 1 Comparison of students’ pretest and posttest evaluations from each class over three years High school Grade level Number of students Pretest proportion of students giving correct answers % (n) Posttest proportion of students giving correct answers % (n) Percentage point difference in improvement % Year 1 A 10 15 7 (1) 53 (8) 46 B 10 and 11 15 20 (3) 53 (8) 33* C 10 16 12.5 (2) 69 (11) 56.5* Year 2 A (class 1) 10 11 64 (7) 91 (10) 27 A (class 2) 10 7 29 (2) 71 (5) 42 A (class 3) 10 9 22 (2) 44 (4) 22 A (class 4) 11 5 40 (2) 80 (4) 40 B 10 and 11 12 50 (6) 75 (9) 25 Year 3 D (class 1) 10 16 31 (5) 87.5 (14) 56.5 D (class 2) 10 16 12.5 (2) 56 (9) 43.5 D (classes 3 & 4{) 11 20 40 (8) 85 (17) 45 * Trial pretest/posttest, revised for years 2–3. { Classes combined for workshop. Table 1 Comparison of students’ pretest and posttest evaluations from each class over three years Table 1 Comparison of students’ pretest and posttest evaluations from each class over three years Table 1 Comparison of students’ pretest and posttest evaluations from each class over three years * Trial pretest/posttest, revised for years 2–3. { Classes combined for workshop. at North Charleston High School (NCHS) (school A in Table 1) to ensure that the program met state curricular standards. NCHS is an inner-city school, serving a 90% low-income, minority student popula- tion. Nearly all the students were African American. One class at each of 2 suburban high schools (schools B and C) with 40% minority students also attended the workshop in year 1. In year 2, students taking the workshop attended 1 school serving 90% low-income, minority students (school A) and 1 school with a 40% minority constituency (school B). In year 3, student participants attended an urban magnet school serving primarily low-income minority students (school D). Year 3 In year 3, 52 students in 4 classes attended the workshop. Student interest in workshop presentations averaged 3.7 (scale51–5), with 11th graders demon- strating more interest (4.0) than 10th graders (3.4). Less than 50% of the students (15/52) answered all 3 questions correctly on the pretest. On the posttest, however, increases for all classes were over 100%, and 77% of all students (40/52) answered all questions correctly on the posttest. While the workshops took place at a new school in year 3 (school D), the learning context was similar to that of NCHS (school A in years 1 and 2): the teachers and the health sciences curriculum were comparable, and the students were 10th or 11th graders and 91% (20/22) African American. However, because students attending school D, a magnet school, expressed career interest in technological vocational training, they may have been more highly motivated to learn Internet evaluation techniques. p p ( ) Team members asked students to rank sites according to the 6 credibility factors noted earlier using a score of 1 (very bad) to 5 (very good) for each one. According to year 1 results, the students tended to rate low-credibility sites highly. They generally ranked high-credibility sites with an appropriate number of points, but the range in points awarded to each site in each category was also high (10– 14 points), indicating a lack of consistency in the class (Table 2 online). Based on these results, the MUSC team streamlined the workshop to make sure that key issues were reinforced and extraneous content dropped. A multiple-choice format replaced a short- answer format for the pretest/posttest. In year 3, the team also introduced an evaluation for the ‘‘Cyber Court’’ role-playing exercise, in which students defended or disputed the case for using a particular website as evidence in a hypothetical court case. As noted, this exercise was only implemented in 11th grade classes (n522 students). Most (n516/22) of the students preferred the combination of work- shop and role-playing activity to either one alone (Table 3 online). This finding was particularly true of students who reported greater understanding, more enjoyment, and higher confidence in their ability to evaluate sites. No students reported preferring the workshop without role playing. Twenty-seven per- cent (6/22) reported preferring role playing without the workshop. RESULTS The website exercise in year 2 consisted of assessing one rather than two sites, allowing students more time to evaluate, and students did a better job of assigning appropriate values to Internet-based health sites. No apparent differences in performance emerged between urban classes (with a high propor- tion of minority students) and suburban classes. The team observed that students seemed to perform best with access to a well-equipped computer lab and sufficient class time. A two-hour workshop with time for a break seemed optimum [13]. Year 1 Year 1 results indicated that the 46 participating students were not very engaged. Satisfaction indica- tors showed low interest: class averages of students’ responses to the workshop ranged from 2.75 to 3.7 on a Likert scale of 1 to 5, with a response of 1 indicating ‘‘not interested’’ and a response of 5 indicating ‘‘very interested.’’ Students’ answers on the website exercise indicated confusion about how to evaluate sites. Students wrote comments like ‘‘Get students more involved’’ and ‘‘Give students more time to explore on their own.’’ Some students evaluated sites only according to what they found interesting. A few made astute observations, such as: ‘‘This was not a good site. It was flashy but no updated information date was posted’’ [12]. But in general, students made cursory evaluations and mistook quantity of informa- tion for quality. Results from the pretest and posttest indicated improvement, but because the high rate of improvement failed to coincide with signs of com- prehension on the website exercise, and, even with improvement, no more than 69% (11/16) of any 1 class answered questions correctly, the team decided to substantially revise both the workshop and the pretest/posttest (Tables 1 and 2 online). Year 2 Interest among students rose from year 1 to year 2, with the average satisfaction score for the 44 students who participated in year 2 rising to 4.0 (scale51–5). Pretest/posttest data indicated that in addition to enjoying themselves more, year 2 students also were better able to evaluate websites after participating in the more focused workshop. Year 2 students were drawn from 5 classes. In 3 of those classes, less than 50% of the students (6/21, including almost all the 11th graders) answered the 2 pretest questions correctly. On the posttest, all but 1 class showed a rise in accuracy, with the result that 73% of all the students (32/44) from that year answered all ques- tions correctly on the posttest. One class had relatively high pretest scores (64%, or 7/11) but only modest improvement (a rise of 27 percentage points, from 7 to 10 of 11 students answering correctly). Curriculum At each school, the team conducted a hands-on Internet workshop using the ‘‘Get Net Smart’’ curriculum. Workshops were preceded by brief pretests related to website evaluation (Appendix A online). The curriculum included guidelines on how to evaluate consumer health websites, recommenda- tions of reputable sites, a site evaluation exercise, and a role-playing activity. Students learned to consider six credibility factors: site sponsorship, accuracy of content, author qualifications, editorial policy, privacy protection, and contact availability (Appendix B online). After demonstrating how to evaluate con- sumer health sites, the presenters allowed students a little time to explore the sites on their own. Students also took a posttest identical to the pretest. p p At the end of the workshop, the trainers asked all students to complete an exercise in which they ranked 2 different websites using each of the 6 credibility factors listed above with a score of 1 (very bad) to 5 (very good). Team members assigned each student 2 websites to review from a list of a dozen sites the team put together to reflect typically reputable and objec- tionable consumer health sites. While the sites differed in content, their adherence or lack of adherence to the credibility factors was comparable. The ideal average score for a reputable or high p In year 3, the MUSC team held the workshop with and without the role-playing activity at a technology magnet school (school D) with a 90% minority population. The team added a process evaluation of the role-playing activity to the other instruments to see if students found it beneficial. This evaluation consisted of a survey with both Likert-scale and short- answer components (Appendix C online). The team combined student self-reporting with pretest/posttest results and instructor observations of student behav- ior in the preliminary evaluation reported here. 371 Brief communications: Trettin et al. Year 3 But 73% (n516/22) said they preferred the combination of workshop and role-playing activ- ity because it increased their confidence in their ability to evaluate sites. REFERENCES g y g In year 3, students with low pretest scores improved more dramatically than students in year 2 did. In both years, eleventh graders showed less improvement than tenth graders, perhaps indicating that sophomores might be more receptive to training than juniors who were one year closer to leaving high school. Taking time to question students about how they use the Internet outside the classroom and teachers about classroom dynamics could improve effectiveness. Finally, year 1 results, which indicated low engage- ment with and understanding of the workshop content, emphasized the importance of planning for a test phase in the development of a new program. 1. Hansen D, Derry HA, Resnick PJ, Richardson CR. Ado- lescents searching for health information on the Internet: an observational study. J Med Internet Res. 2003 Oct;5(4):e25. 1. Hansen D, Derry HA, Resnick PJ, Richardson CR. Ado- lescents searching for health information on the Internet: an observational study. J Med Internet Res. 2003 Oct;5(4):e25. 2. Gray NJ, Klein JD, Noyce PR, Sesselberg TS, Cantrill JA. The Internet: a window on adolescent health literacy. J Adolesc Health. 2005 Sep;37(3):243. 1. Hansen D, Derry HA, Resnick PJ, Richardson CR. Ado- lescents searching for health information on the Internet: an observational study. J Med Internet Res. 2003 Oct;5(4):e25. 2. Gray NJ, Klein JD, Noyce PR, Sesselberg TS, Cantrill JA. The Internet: a window on adolescent health literacy. J Adolesc Health. 2005 Sep;37(3):243. 3. Cornish A. Testing computer literacy [Internet]. Morning Edition National Public Radio; 25 Apr 2005 [cited 11 Sep 2007]. ,http://www.npr.org/templates/story/story.php? storyId54618058.. 4. Ivanitskaya L, O’Boyle I, Casey A. Health information literacy and competencies of information age students: results from the interactive online Research Readiness Self- Assessment (RRSA). J Med Internet Res. 2006 Apr;8(2):e6. 5. Eysenbach G, Kohler C. How do consumers search for and appraise health information on the world wide web? qualitative study using focus groups, usability tests, and in- depth interviews. BMJ. 2002 Mar 9;324(7337):573–7. 6. Fogg BJ. How do people evaluate a web site’s credibility? results from a large study. Consumer Reports Web Watch [Internet]. Stanford University Persuasive Technolo- gy Lab; 29 Oct 2002 [cited 11 Sep 2007]. ,http://www .consumerwebwatch.org/dynamic/web-credibility-reports- evaluate-abstract.cfm.. y ( ) 2. Gray NJ, Klein JD, Noyce PR, Sesselberg TS, Cantrill JA. The Internet: a window on adolescent health literacy. J Adolesc Health. 2005 Sep;37(3):243. p 3. Cornish A. Testing computer literacy [Internet]. DISCUSSION AND CONCLUSION This study had several limitations. Because the MUSC team relied on a small, non-randomized convenience sample of students, the results cannot be considered representative of the state’s high school students. The school populations may have had unidentified differ- ences, despite similarities among schools of the same type. For example, only one of the two urban schools was a vocational magnet school. Some change in level J Med Libr Assoc 96(4) October 2008 372 Brief communications: Trettin et al. which students teach Internet evaluation skills to family members and others in their community. of performance could also be attributed to revisions in the program and the pretest and posttest after year 1. Improvements in years 2 and 3 could attest also to trainers’ improvements in delivery. Some findings in year 3 are based primarily on student self-reporting and trainer observation, and hence they require validation with more rigorous methods. y y Teenagers will probably continue to take the Internet for granted, but there are positive signs that they can be encouraged to use it safely. This study found that role-playing reinforces students’ ability to critically evaluate sites, particularly if it is preceded by intensive and focused skill-building activities for which adequate class time is allowed. Additionally, the study suggests that such training earlier in students’ high school careers is more effective. Librarians can play a key role in guiding adolescents’ interactions with web-based health information through programs such as ‘‘Get Net Smart.’’ Despite these limitations, year 2 and 3 results suggested that ethnicity and type of school had no discernible bearing on students’ performance and that an extra year of instruction and maturation did not improve students’ abilities to critically evaluate web- sites. The variability of results suggests that individual students’ skills influenced performance, and classroom dynamics (level of collegiality) and time of day (early morning or immediately before lunch) might also have. J Med Libr Assoc 96(4) October 2008 REFERENCES Morning Edition National Public Radio; 25 Apr 2005 [cited 11 Sep 2007]. ,http://www.npr.org/templates/story/story.php? storyId54618058.. 4. Ivanitskaya L, O’Boyle I, Casey A. Health information literacy and competencies of information age students: results from the interactive online Research Readiness Self- Assessment (RRSA). J Med Internet Res. 2006 Apr;8(2):e6. This study also contributes to the limited amount of literature related to how adolescents and students of the Millennial generation (the first generation raised with access to the Internet) evaluate Internet health sites by examining average students with variable computer skills [14]. Existing literature typically focuses on self-perceptions and close observation of students who have self-selected based on interest in using the Internet [1, 2]. The combination of workshop and role-playing activity in ‘‘Get Net Smart’’ provides the opportunity to test and observe both individual and group uses of the Internet by students who are selected for their interest in health careers, not the Internet, per se. Additional research with teens could target specific interests (such as those of teens who are HIV/AIDS positive) [15] and could incorporate digital advances, such as gaming in virtual environments, to which these students readily respond [16, 17]. 5. Eysenbach G, Kohler C. How do consumers search for and appraise health information on the world wide web? qualitative study using focus groups, usability tests, and in- depth interviews. BMJ. 2002 Mar 9;324(7337):573–7. p 6. Fogg BJ. How do people evaluate a web site’s credibility? results from a large study. Consumer Reports Web Watch [Internet]. Stanford University Persuasive Technolo- gy Lab; 29 Oct 2002 [cited 11 Sep 2007]. ,http://www .consumerwebwatch.org/dynamic/web-credibility-reports- evaluate-abstract.cfm.. 7. EXPORT Center on Metabolic Syndrome (H3O)and Minority Health. Outreach: get net smart for good health [Internet]. Charleston, SC: Medical University of South Carolina for National Center on Minority Health and Health Disparities; 2006 [cited 6 Dec 2007]. ,http://export.musc .edu/outreach/get_net_smart.html.. 7. EXPORT Center on Metabolic Syndrome (H3O)and Minority Health. Outreach: get net smart for good health [Internet]. Charleston, SC: Medical University of South Carolina for National Center on Minority Health and Health Disparities; 2006 [cited 6 Dec 2007]. ,http://export.musc .edu/outreach/get_net_smart.html.. 8. United Health Foundation. Snap shot: South Carolina. America’s health rankings: a call to action for people and their communities. 2006 ed. Minnetonka, MN: United Health Foundation; 2006. p. 72. 9. Medical University of South Carolina Library. Hands on health: South Carolina [Internet]. Charleston, SC: Medical University of South Carolina; 2007 [cited 6 Dec 2007]. ,http://www.hohsc.org.. 10. REFERENCES 2008 Summer;15(1/2), Pullman, WA: Washington State University Research Exchange; 2007 [cited 26 Mar 2007]. ,http://hdl.handle.net/2376/1121.. 14. Johnson C, Lindsay E, Walter S. Learning more about how they think: information literacy instruction in a campus-wide critical thinking project. Col Undergrad Libr [Internet]. 2008 Summer;15(1/2), Pullman, WA: Washington g State University Research Exchange; 2007 [cited 26 Mar 2007]. ,http://hdl.handle.net/2376/1121.. p 15. Flicker S, Goldberg E, Read S, Veinot T, McClellan A, Saulnier P, Skinner H. HIV-positive youth’s perspectives on the Internet and eHealth. J Med Internet Res. 2004 Jul– Sep;6(3):e32. p 16. Nelson B, Ketelhut D. Scientific inquiry in educational multi-user virtual environments. Educ Psych Review. 2007;19(3):265–83. 17. Sweeney R. Reinventing library buildings and services for the millennial generation. Libr Adm Manag. 2005 Fall;19(4):165–75. 18. Partridge H, Hallam G. Educating the millennial generation for evidence based information practice. Libr Hi Tech. 2006;24(3):400–19. REFERENCES National Library of Medicine. About Go Local [Internet]. Washington, DC: The Library; 2007 [cited 6 Dec 2007]. ,http:// www.nlm.nih.gov/medlineplus/golocal/about.html.. 11. Francis B, Kelly J. Active learning: its role in health sciences libraries. Med Ref Serv Q. 1997;16(1):25–37. 12. McKeehan N. Hands-on health education for South Carolinians: quarterly report. Charleston SC: November 2005. Submitted to Network of National Libraries of Medicine, National Institutes of Medicine. NLM contract no. N01-LM-1-3522. g 8. United Health Foundation. Snap shot: South Carolina. America’s health rankings: a call to action for people and their communities. 2006 ed. Minnetonka, MN: United Health Foundation; 2006. p. 72. y p For librarians, there are several potential benefits to using a program like ‘‘Get Net Smart’’ with small groups of students. Librarians can become hidden by the impersonal nature of communication in the digital age. Outreach activities allow librarians to forge more personal connections with a generation learning to think differently about information and resources [17, 18]. Students can gain a better appreciation of the range of services librarians can provide. Also, the potential exists for expanded community involvement when librarians enter the classroom: they can partic- ipate in or advise parent–teacher association meet- ings, school health fairs, after-school programs, adult education classes, and service-learning projects in p 9. Medical University of South Carolina Library. Hands on health: South Carolina [Internet]. Charleston, SC: Medical University of South Carolina; 2007 [cited 6 Dec 2007]. ,http://www.hohsc.org.. p g 10. National Library of Medicine. About Go Local [Internet]. Washington, DC: The Library; 2007 [cited 6 Dec 2007]. ,http:// www.nlm.nih.gov/medlineplus/golocal/about.html.. 11. Francis B, Kelly J. Active learning: its role in health sciences libraries. Med Ref Serv Q. 1997;16(1):25–37. 11. Francis B, Kelly J. Active learning: its role in health sciences libraries. Med Ref Serv Q. 1997;16(1):25–37. 12. McKeehan N. Hands-on health education for South Carolinians: quarterly report. Charleston SC: November 2005. Submitted to Network of National Libraries of Medicine, National Institutes of Medicine. NLM contract no. N01-LM-1-3522. 373 Brief communications: Ralston et al. 13. Trettin L. Get Net smart for good health: teaching high school students to evaluate Internet-based health sites, an internal program evaluation. Charleston SC: May 2006. Submitted to National Network of Libraries of Medicine. NLM contract no. N01-LM-1-3522. 14. Johnson C, Lindsay E, Walter S. Learning more about how they think: information literacy instruction in a campus-wide critical thinking project. Col Undergrad Libr [Internet]. J Med Libr Assoc 96(4) October 2008 Received December 2007; accepted April 2008 AUTHORS’ AFFILIATIONS Lillian D. Trettin, PhD, trettinl@musc.edu, Assistant Professor; Janice C. May, MPA, mayjc@musc.edu, Program Coordinator; Nancy C. McKeehan, MSLS, mckeehan@musc.edu, Associate Library Director; Medical University of South Carolina Library, 17 Ashley Avenue, P.O. Box 250403, Charleston, SC 29425 374
https://openalex.org/W1969598286	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0045787&type=printable	English	null	Modelling Stream-Fish Functional Traits in Reference Conditions: Regional and Local Environmental Correlates	PloS one	2,012	cc-by	11,018	Introduction and Esselman and Allan [9] for Mesoamerican streams suggested that landscape-scale factors had stronger relative influence on fish assemblages. In Europe, Ferreira et al. [12] for Western Iberia and Pont et al. [13] for France reported that both watershed and reach-scale factors were equally important in structuring fish species. Although all these studies have contributed greatly to our understanding of fish-environment relations across multiple spatial scales at the taxonomic level, little attention has been paid to the factors that determine the functional organization of stream-fish assemblages in reference conditions (but see [7]). Recently, Logez et al. [14] tested the functional structures of some European fish assemblages in least disturbed conditions, but they only used five environmental variables. We are unaware of any studies in European undegraded streams evaluating the roles of a large set of local and regional environmental factors in determining the functional organization of fish assemblages. Biomonitoring and stream restoration are important tools to re- establish the health of river ecosystems with the emphasis placed on the restoration of ecological processes and ecosystem function- ality [1]. Therefore, identifying the primary ‘natural’ environ- mental gradients that control biological assemblages at the functional level should provide information to help river manage- ment and predict the consequences of anthropogenic stressors. The structure of any local biological assemblage is a function of several biotic and abiotic factors operating on multiple spatial and temporal scales [2,3]. In fact, many studies (e.g., [4–10]) have shown that both local factors, such as channel morphology and fish cover, and regional factors (many of which are correlated with one another and influence local conditions), such as climate, and both large-scale land use and water transfer, can contribute to the structure of fish assemblages. The question of which spatial scale reveals the strongest relationships among environmental variables and fish assemblages has attracted considerable interest, although those conclusions may depend on the biological unit employed (species/functional groups) [8], the degree of anthropogenic landscape disturbance [7,11], or the geo-climatic heterogeneity of the studied area [9]. Of the studies that directly addressed this question in least disturbed regions, Wang et al. [7] reported stronger relations at a local scale in north-central United States, The functional perspective has been recently emphasized in stream-fish ecology, in which the assemblage traits are the primary focus [8,15–17]. Abstract Identifying the environmental gradients that control the functional structure of biological assemblages in reference conditions is fundamental to help river management and predict the consequences of anthropogenic stressors. Fish metrics (density of ecological guilds, and species richness) from 117 least disturbed stream reaches in several western Iberia river basins were modelled with generalized linear models in order to investigate the importance of regional- and local-scale abiotic gradients to variation in functional structure of fish assemblages. Functional patterns were primarily associated with regional features, such as catchment elevation and slope, rainfall, and drainage area. Spatial variations of fish guilds were thus associated with broad geographic gradients, showing (1) pronounced latitudinal patterns, affected mainly by climatic factors and topography, or (2) at the basin level, strong upstream-downstream patterns related to stream position in the longitudinal gradient. Maximum native species richness was observed in midsize streams in accordance with the river continuum concept. The findings of our study emphasized the need to use a multi-scale approach in order to fully assess the factors that govern the functional organization of biotic assemblages in ‘natural’ streams, as well as to improve biomonitoring and restoration of fluvial ecosystems. Editor: Carlos Garcia de Leaniz, Swansea University, United Kingdom ditor: Carlos Garcia de Leaniz, Swansea University, United Kingdom Received May 8, 2012; Accepted August 23, 2012; Published September 24, 2012 Copyright: 2012 Oliveira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was financially supported by the National Forest Authority (AFN) (http://www.afn.min-agricultura.pt) under project AQUARIPORT, and post- doctoral grants from the Foundation for Science and Technology (FCT) (http://www.fct.pt) (JMO - SFRH/BPD/44624/2008; PS - SFRH/BPD/39067/2007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jmoliveira@utad.pt Joa˜o M. Oliveira1,2*, Pedro Segurado2, Jose´ M. Santos2, Amı´lcar Teixeira3, Maria T. Ferreira2, Rui V. Cortes1 1 Centro de Investigac¸a˜o e de Tecnologias Agro-Ambientais e Biolo´gicas (CITAB), Universidade de Tra´s-os-Montes e Alto Douro, Vila Real, Portugal, 2 Centro de Estudos Florestais (CEF), Instituto Superior de Agronomia, Universidade Te´cnica de Lisboa, Lisboa, Portugal, 3 Centro de Investigac¸a˜o de Montanha (CIMO), Escola Superior Agra´ria, Instituto Polite´cnico de Braganc¸a, Braganc¸a, Portugal September 2012 \| Volume 7 \| Issue 9 \| e45787 Fish Functional Traits in Reference Conditions The climate reflects both the elevation and the proximity of the Atlantic Ocean, ranging from temperate oceanic on the northwest of the study area, to Mediterranean in most of the country [36]. Temperatures increase southwards and mean annual precipitation is generally higher in the northern area. The rainfall pattern exhibits strong intra-and inter-annual variability, with a discharge regime that is typical of Mediterranean systems [37]. Therefore, high floods occur from autumn to late-winter, with a gradual decline in flow that eventually dries out during late spring and summer in the most arid areas. In riverine systems fish assemblages may reveal significant variations along broad geographic gradients, suggesting adaptation to spatial (i.e. latitudinal and longitudinal) changes in abiotic variables. For example, Marsh-Matthews and Matthews [26], in a study of streams from Iowa to Texas, reported that variations in fish assemblage composition reflected the overall north-south arrangement of drainages sampled. Ferreira et al. [27] described latitudinal variations of Portuguese fish assemblages, based on climatic and topographic factors, and Pusey and Kennard [28] also found a strong north-south gradient in structure of fish assemblages of Northern Queensland. Several works have addressed the phenomenon of longitudinal variation of both fish species richness and fish functional groups (e.g., [9,15,25,29–33]). These studies have demonstrated that variables that represent longitudinal position in the catchment, including local habitat characteristics (e.g., depth, width, slope, discharge) and its regional drivers (e.g., climatic variables, drainage area) correlate strongly to fish assemblage variation. For example, McGarvey and Hughes [32] and McGarvey and Ward [33] described longitudinal patterns in both fish guilds and species richness as a function of river discharge across large geographic areas. These patterns are usually attributed to concepts that relate the gradient in physical factors that occur along river systems, to changes in assemblage structure and function. The landscape varies greatly across the study area. The northern and central part of the country part is dominated by narrow and steep valleys, mostly forested with English oak (Quercus robur) and Pyrenean oak (Quercus pyrenaica). Riparian forests are dense and typically dominated by the common alder (Alnus glutinosa) and the narrow-leafed ash (Fraxinus angustifolia), frequently found in association with willows (Salix spp.) and poplars (Populus spp.). Pines (Pinus spp.) and Eucalyptus plantations are also characteristic of this area, which also supports a patchy pattern of orchards, vineyards, olive groves and irrigated crops. Fish Functional Traits in Reference Conditions Fish Functional Traits in Reference Conditions complex. The inland area is dominated by the pre-Mesozoic Hesperic Massif – a geological unit that includes granites, schists and quartzites with various degrees of metamorphism – whereas the coast is dominated by tertiary layers under quaternary deposits, but including a few calcareous areas; in the southern region, below Tagus river, the flat platforms of the Hesperic Massif predominate in extensive areas and further south, geology is mainly composed of sedimentary deposits (e.g., sandstones, limestones, conglomerates and marls), mainly with a calcareous background. Thus, species traits can be used to examine relations between the fundamental ecological function of fish assemblages and the environmental variables in natural conditions [19,20]. The functional guild approach is the cornerstone for the development of multi-metric indices, such as the Indices of Biotic Integrity, to assess the biological condition of aquatic systems across large landscapes [21–22]. The use of guilds has several advantages: (1) species in a guild are more likely than an entire assemblage or taxonomic group to respond to a common subset of environmental variables [23]; (2) density of individual species may be more fluctuating via biotic and abiotic factors than density of whole guilds [24]; (3) since the use of the guild concept is largely less geographically constrained than the taxonomic level, it may be more useful for comparing the organization of fish assemblages across large geographic scales [25]. Thus, species traits can be used to examine relations between the fundamental ecological function of fish assemblages and the environmental variables in natural conditions [19,20]. The functional guild approach is the cornerstone for the development of multi-metric indices, such as the Indices of Biotic Integrity, to assess the biological condition of aquatic systems across large landscapes [21–22]. The use of guilds has several advantages: (1) species in a guild are more likely than an entire assemblage or taxonomic group to respond to a common subset of environmental variables [23]; (2) density of individual species may be more fluctuating via biotic and abiotic factors than density of whole guilds [24]; (3) since the use of the guild concept is largely less geographically constrained than the taxonomic level, it may be more useful for comparing the organization of fish assemblages across large geographic scales [25]. Ethics Statement We selected data from fish samples of Portuguese streams collected by a consortium of Portuguese national universities 1996–2006. From a collection of more than 500 sites, only least disturbed sites were considered (‘reference data set’ or RD). The identification followed the EU-FAME project approach [16,42], in which human disturbance is ranked semi-quantitatively using all available field data and GIS information. In our study we considered fifteen disturbance variables at catchment, segment and reach scales (Table S1), that were scored to the degree they deviated from minimally disturbed conditions (from 1 for no deviation, to 5 for highly degraded). The RD included sites with a classification of 1 and 2 on, at least, 80% of the 15 variables, All animal work (from sampling to handling) was conducted in accordance to relevant national and international guidelines to minimise discomfort to individuals [34,35]. The necessary permits for fish sampling were obtained from the National Forest Authority. Fish Functional Traits in Reference Conditions The southern area is more homogenous and largely characterized by agriculture, Mediterranean shrublands and evergreen oak (Quercus suber and Quercus ilex) woodlands. Meander valleys and small floodplains are characteristic of the middle and lower reaches of the main rivers. Most streams in this region have narrow riparian forests, primarily dominated by willows and ashes, which, in the driest areas, are replaced by shrublands mainly composed by the oleander (Nerium oleander) and the Ibero-African shrubby spurge (Flueggea tinctoria). The biogeographic isolation of the Iberian Peninsula by the Atlantic Ocean and the Pyrenees allowed the development of a characteristic fish fauna, similar to other European Mediterranean peninsulas [38–39], characterized by a low number of families, with most of the species belonging to the family Cyprinidae, a high degree of diversification at the species level, and the greatest European percentage of endemics [40]. There is also a long history of alien fish introductions representing a total of twenty three species, with more than half of the introductions occurring after the beginning of 20th century, mainly for fishery and ornamental purposes [41]. In this study we use a combination of multivariate statistical techniques to further our objectives: (1) to identify the regional- and local-scale environmental gradients explaining the functional structure of western Iberian fish assemblages at least disturbed sites (reference conditions); and (2) to assess the relative importance of these abiotic gradients on the abundance of fish guilds and fish species richness. We also hypothesized that the generic biological patterns would be associated with broad geographic gradients, with variations of the functional organization of fish assemblages either with latitude or along the streams. Introduction This can be achieved by distinguishing functional groups (or guilds): groups of species that exploit the same class of environmental resources in a similar way [18]. Species can be grouped into guilds on the basis of many different life-history traits (e.g., reproduction, feeding, habitat), reflecting morphological, physiological, and behavioural adaptations to their environment. 1 September 2012 \| Volume 7 \| Issue 9 \| e45787 PLOS ONE \| www.plosone.org Study Area The study area is located on the west side of the Iberian Peninsula and includes all the hydrological network of Portuguese catchments (Figure 1), with most of the rivers running in a NE–SW direction towards the Atlantic coast. Geology of the country is September 2012 \| Volume 7 \| Issue 9 \| e45787 PLOS ONE \| www.plosone.org 2 Fish Functional Traits in Reference Conditions PLOS ONE \| www.plosone.org 3 September 20 September 2012 \| Volume 7 \| Issue 9 \| e45787 September 2012 \| Volume 7 \| Issue 9 \| e45787 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 3 3 Fish Functional Traits in Reference Conditions Figure 1. Location of the sampling sites. The elevation and the main river basins are also shown (a - Minho; b - Lima; c - Ca´vado; d - Ave; e - Douro; f - Vouga; g - Mondego; h - Lis; i - Extremadura coastal basins; j - Tagus; k - Sado; l - Mira; m - Algarve costal basins; n - Guadiana). doi:10.1371/journal.pone.0045787.g001 Figure 1. Location of the sampling sites. The elevation and the main river basins are also shown (a - Minho; b - Lima; c - Ca´vado; d - Ave; e - Douro; f - Vouga; g - Mondego; h - Lis; i - Extremadura coastal basins; j - Tagus; k - Sado; l - Mira; m - Algarve costal basins; n - Guadiana). doi:10.1371/journal.pone.0045787.g001 stream flows were lower (but still had full connectivity between habitats), thus ensuring a higher fishing efficiency, while at the same time, we avoided the extreme-flow events that typically occur during the rainy season from causing bias in fish sampling or in the measurement of local habitat variables [43]. Electrofishing distances followed CEN standards for assessing fish species composition and abundance for a given site (point 3.3.2 in [34]). This distance was at least 20 times the mean wetted width of the channel to encompass complete sets of the characteristic stream form (e.g., riffles, pools, runs); minimum sampled length was 100 m, and maximum sampled length was 600 m. The entire widths of wadeable streams were fished by walking slowly upstream and using one anode for every 5 m of stream width. Study Area The majority of sites were sampled by wading, but a few rivers with mean depths exceeding 0.8 m were electrofished by boat moving downstream, again sampling all habitat types, but focusing on the margins. Although capture efficiency estimates, assumed to be constant across sites, were not available for the study area, previous works indicated that this sampling effort was sufficient to ensure accurate characterization of fish species composition and allowing a maximum classification of 3 on 20% of those variables, with the exception of the ‘alien fish abundance’, which must be included in class 1 (no aliens) or 2 (,15% aliens). Thus, a median pressure classification of three was allowed for no more than three abiotic variables. This allowance was based on the assumption that minor deviations from excellent/good conditions do not signifi- cantly alter biological patterns at the assemblage level [27]. In addition, only sites spaced out 2 km from each other were retained for analyses to reduce problems of spatial autocorrelation while maintaining a sufficiently large sample size [43]. This yielded a total of 117 stream reaches (mean stream width = 5.6 m; min 1.0 m-max 30.0 m) from all main Portuguese catchments (Minho, Lima, Ca´vado, Ave, Douro, Vouga, Mondego, Lis, Extremadura coastal basins, Tagus, Sado, Guadiana and Algarve costal basins) (Figure 1), that cover the range of natural conditions across the country (Table 1). Fish Metrics To test whether the functional structure of fish assemblages responded to environmental gradients, we considered ten metrics related to the total density of fish guilds grouped into five ecological functions, following Noble et al. [23]: 1) overall tolerance guilds, based on the ability of species to survive and reproduce in a wide range of natural environmental conditions (non-tolerant (NOTO), and tolerant (TOLE)); 2) trophic guilds, based on food items in the diet of adult individuals (invertivores (INVE) and omnivorous (OMNI)); 3) feeding habitat guilds, based on the preferred habitat to live and feed (benthic (BENT) and water column (WACO)); 4) reproduction guilds, based on spawning substrate (lithophilic (LITH), phytolithophilic (PHLI), and polyphilic (POLY)); 5) migratory behaviour guild (potamo- dromous species (POTA)). The fish species were generally assigned for these guilds based on Fame Consortium [45,46] with a few modifications supported by more recent grey or published data, completed by expert judgment when necessary (Table 2). These assemblage traits represent important aspects of the biology and ecology of fish species [14,23], and could be considered of great interest to environmental assessment and management [19]. Following Logez et al. [14], we also considered number of native species (NATI) and total density of native individuals (DENS) as metrics. Table 2. Catch (median and 1st-3rd quartiles; individuals per 1000m2), frequency of occurrence (FO) (%), index of relative dominance (IRD), and functional guilds for fish taxonomic groups collected in Portugal. Table 2. Catch (median and 1st-3rd quartiles; individuals per 1000m2), frequency of occurrence (FO) (%), index of relative dominance (IRD), and functional guilds for fish taxonomic groups collected in Portugal. Table 2. Catch (median and 1st-3rd quartiles; individuals per 1000m2), frequency of occurrence (FO) (%), index of relative dominance (IRD), and functional guilds for fish taxonomic groups collected in Portugal. Species Catch FO IRD Guilds Acondrostoma oligolepis 68 (32–106) 17.1 195.2 TOLE, OMNI, WACO, PHLI Anguilla anguilla 10 (5–22) 30.8 75.1 TOLE, INVE, BENT Cobitis paludica 7 (2–21) 24.8 42.7 TOLE, INVE, BENT, POLI Iberochondrostoma almacai 13 (11–17) 4.3 1.2 NOTO, OMNI, PHLI Iberochondrostoma lemmingii 11 (5–19) 10.3 8.5 NOTO, OMNI, PHLI Iberochondrostoma lusitanicum 11 (7–15) 5.1 0.8 TOLE, OMNI, PHLI Luciobarbus bocagei 20 (8–70) 29.1 226.0 TOLE, OMNI, BENT, LITH, POTA Luciobarbus spp. 54 (7–199) 10.3 62.2 TOLE, OMNI, BENT, LITH, POTA Lampetra spp. Fish Functional Traits in Reference Conditions Fish Functional Traits in Reference Conditions abundance in the studied streams [27,43–44]. Fish were identified and measured in the field and returned alive to the water. Because juveniles of the four Luciobarbus species (L. comizo/microcephalus/ sclateri/steindachneri) present in the southern Portuguese basins can not be reliably identified to species, we grouped them as Luciobarbus spp. For the same reason we grouped Lampetra fluviatilis and L. planeri. Environmental Data For each site, environmental data were obtained at regional and local scales (Table 1). Following Hoeinghaus et al. [8], we considered regional factors as those associated with processes occurring over an area larger than the in-stream scale. Regional environmental variables included: (1) catchment area, catchment shape index (perimeter-area ratio), stream order, catchment mean elevation, catchment elevation range, catchment mean slope, catchment slope range, mean flow accumulation and drainage (stream) density (all these variables were partially derived from a Digital Terrain Model, with approximately 90 m resolution, from the NASA Shuttle Radar Topographic Mission, that is available from the CGIAR-CSI SRTM 90m Database: http://srtm.csi. cgiar.org/, accessed 2012 January 16); (2) mean annual precipi- tation, mean summer precipitation, and summer ombrothermic index [47] (these climatic variables were determined from 30 seconds (600–800 metres) resolution maps of monthly precipita- tion and monthly temperature that are available in the WorldClim website: http://www.worldclim.org/, accessed 2012 January 16). Regional variables were computed in a GIS environment using ArcGIS 9.2 (ESRI Inc., Redlands, CA, USA). Hydrology function from the ArcGIS Spatial Analyst package was used to produce the variables related to the river catchment network. Local environ- mental variables included: (1) elevation, channel slope, and channel sinuosity (stream length/valley length) (all derived from a Digital Terrain Model); (2) mean annual temperature and mean July temperature (both determined from the climate models described above); (3) water temperature and conductivity (both quantified at mid-channel, using a multiparameter meter); (4) dominant substrate (1 = silt, ,0.02 cm; 2 = sand, 0.02–0.2 cm; 3 = gravel, 0.2–1.6 cm; 4 = pebble, 1.6–6.4 cm; 5 = cobble, 6.4– 26.0 cm; 6 = boulder, .26.0 cm; 7 = bedrock), mean wetted width, mean depth, and maximum depth (all collected from 3 to 10 cross-sectional transects depending on distance fished (i.e. Only fish taxa presented at least at five sites are presented. The IRD for each group was calculated by multiplying the percent frequency of occurrence by the percent of relative abundance of that group. Guild abbreviations are defined in Methods. doi:10 1371/journal pone 0045787 t002 Fish Sampling During spring–summer base flow, sites were electrofished (DC, 300–700 V, or pulsed DC, 400–1000 V) once. In this period n and range of environmental variables measured at 117 least disturbed sites in Portugal. ble 1. Median and range of environmental variables measured at 117 least disturbed sites in Portugal. Table 1. Median and range of environmental variables measured at 117 least disturbed sites in Portug Table 1. Median and range of environmental variables measured at 117 least disturbed sites in Portugal. Environmental variables Median (range) Regional variables Catchment area (km2) 38.00 (4.40–1340.00) Catchment shape index 1.08 (0.20–4.88) Stream order 2 (1–6) Catchment mean elevation (m) 599.00 (75.00–1256.00) Catchment elevation range (m) 612.00 (111.00–1421.00) Catchment mean slope (%) 0.57 (0.08–2.11) Catchment slope range (%) 1.75 (0.09–5.87) Mean flow accumulation 82.00 (21.00–487.00) Drainage density (km/km2) 0.94 (0.62–1.33) Mean annual precipitation (mm) 1107.00 (556.00–1612.00) Mean summer precipitation (mm) 25.00 (6.00–49.00) Summer ombrothermic index 2.10 (0.59–4.75) Local variables Elevation (m) 247.00 (6.00–952.00) Channel slope (%) 1.04 (0.01–16.40) Channel sinuosity 1.45 (1.11–2.22) Mean annual temperature (uC) 13.54 (8.81–16.97) Mean July temperature (uC) 21.60 (17.70–24.60) Conductivity (mS/cm) 63.00 (10.00–769.00) Water temperature (uC) 17.80 (7.80–29.80) Mean width (m) 5.60 (1.00–30.00) Mean depth (m) 0.40 (0.13–1.00) Maximum depth (m) 1.00 (0.20–3.00) Mean width-depth ratio 14.00 (2.86–70.67) Dominant substrate (class) 5 (2–7) Woody cover (class) 3 (1–5) Macrophyte cover (class) 2 (1–5) Shading (class) 3 (1–5) doi:10.1371/journal.pone.0045787.t001 September 2012 \| Volume 7 \| Issue 9 \| e45787 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 4 045787.t002 PLOS ONE \| www.plosone.org 5 September 2012 \| Volume 7 \| Issue 9 \| e45787 Statistical Analyses To reduce multicollinearity among environmental variables, principal component analysis (PCA), using Varimax rotation to ease interpretability [48], was performed separately on regional and local level variable groups. The reduced set of principal components, that still retained enough environmental variation, was used as the predictor gradients set in posterior analyses. To investigate the relative influence of regional and local environmental gradients on the considered fish metrics, General- ized Linear Models (GLM; [49]) were adjusted. For functional guilds and DENS (density-based metrics), GLM were based on Negative Binomial error distribution with log as the link function. For NATI, GLM based on Poisson error distribution with log as the link function was used. The quadratic terms of all predictors were tested for inclusion in models, in order to account for unimodal responses of fish metrics. PCA on instream local variables generated two components with eigenvalues .1, which accounted for 40.0% of the total variation. Local gradient 1 (L1) also represented a latitudinal variation, and was positively related to elevation, channel slope, and dominant substrate, and negatively related to mean annual temperature and conductivity, representing a shift from southern low-gradient and warmer stream reaches, with finer substrates and more productive waters, to northern higher gradient and cooler reaches, with coarser substrata and lower conductivity. L2 exhibited a habitat-size longitudinal gradient, and was positively Model selection procedure was based on the Information Theoretic Approach (ITA) [50], using the Akaike Information Criteria (AIC) as a measure of information loss of each candidate model, with the best fitting model having the lowest AIC. The difference between the AIC of each candidate model and the AIC of the top-ranked model was computed (Di), in order to identify those models with larger support. Akaike weights (wi) were also computed for each candidate model, representing the probability of each model being selected as the best fitting model if the data were collected again under identical circumstances. Parameters of the final model were computed as the average of the parameters of models weighted by the respective wi. Only models with Di #2, which are typically those considered to have substantial support, were considered in model averaging [50]. The relative importance of each variable was assessed using the probability of each variable to be included in the best approximating models. This was estimated by summing the wi of all candidate models where the variable was included. Table 3. Statistical Analyses Loadings of regional and local variables on the first two principal components (PC) extracted by PCA and the eigenvalues and proportions of variance accounted for by each axis for the 117 sites in Portugal. PCR axes PCL axes Regional variables R1 R2 Local variables L1 L2 Catchment area 20.84 Elevation 0.67 Catchment shape index 0.75 Channel slope 0.60 Stream order 20.80 Mean annual temperature 20.80 Catchment mean elevation 0.75 Conductivity 20.72 Catchment elevation range 0.78 Mean width 0.61 Catchment mean slope 0.77 Mean depth 0.93 Mean flow accumulation 20.89 Maximum depth 0.84 Mean annual precipitation 0.86 Dominant substrate 0.71 Mean summer precipitation 0.90 Ombrothermic index 0.94 eigenvalue 5.3 3.1 eigenvalue 3.7 2.3 variance (%) 44.2 26.2 variance (%) 24.6 15.4 Marked loadings are $ \|0.60\|. doi:10.1371/journal.pone.0045787.t003 All statistical analyses were performed using functions and routines implemented in R software version 2.12 [51]. Fish Metrics 21(16–104) 4.3 5.5 NOTO, BENT, LITH Pseudochondrostoma duriense 21 (8–51) 26.5 114.1 NOTO, OMNI, BENT, LITH, POTA Pseudochondrostoma polylepis 42 (30–67) 12.0 29.7 NOTO, OMNI, BENT, LITH, POTA Salmo trutta 35 (17–81) 53.8 587.0 NOTO, INVE, WACO, LITH, POTA Squalius alburnoides 93 (54–183) 32.5 525.8 NOTO, INVE, WACO, LITH Squalius aradensis 157 (45–353) 9.4 91.0 NOTO, INVE, WACO, LITH Squalius carolitertii 25 (9–53) 35.0 242.3 NOTO, INVE, WACO, LITH Squalius pyrenaicus 70 (29–219) 32.5 634.1 NOTO, INVE, WACO, LITH Aliens 9 (4–18) 13.0 0.7 Only fish taxa presented at least at five sites are presented. The IRD for each group was calculated by multiplying the percent frequency of occurrence by the percent of relative abundance of that group. Guild abbreviations are defined in Methods. doi:10.1371/journal.pone.0045787.t002 September 2012 \| Volume 7 \| Issue 9 \| e45787 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Fish Functional Traits in Reference Conditions cyprinid S. aradensis dominated the southern small Mediterranean- type drainages. about one transect per 30 m of electrofishing distance)); (5) woody cover (woody debris and submerged roots), macrophyte cover, and overhanging tree shading (all visually assessed and scored on a grade scale for cover with the following classes (1 = ,5%; 2 = 5– 25%; 3 = 25–50%; 4 = 50–75%; 5 = .75%). Environmental Gradients Principal component analysis (PCA) performed on large-scale regional variables yielded two components with eigenvalues .1, which accounted for 70.4% of the total variation (Table 3). Principal component 1 (regional gradient 1, R1) was positively loaded on catchment mean elevation, catchment elevation range, catchment mean slope, mean annual precipitation, mean summer precipitation, and summer ombrothermic index, and therefore described a south-north latitudinal gradient, mainly related to climate and topography, with increasing values for catchments with higher mean annual and summer precipitation, lower aridity, and both higher elevation and terrain relief. R2 showed marked negative loadings on catchment area, stream order, and mean flow accumulation, and was positively loaded with catchment shape index, therefore describing an upstream-downstream longitudinal gradient, with decreasing values for sites representing higher drainage areas and higher volumes of water discharged from the upper catchment. Model Averaging For the metrics with significant estimates of GLM coefficients, the number of models with substantial support (i.e. with Di #2) ranged from 1 (POLY) to 7 (INVE), with cumulative Akaike weights varying from 0.49 (DENS) to 0.99 (POLY), and variability explained by the top-ranked models ranging from 21% (OMNI) to 56% (NATI) (Table 4). All coefficients of models for POTA showed no statistical support, with 95% confidence intervals overlapping zero. For all the analyzed metrics, models including regional environmental variables (R1 and R2) were more likely to be selected as more informative (Figure 2). Furthermore, regional variables showed higher support for all metrics following calculation of unconditional 95% confidence intervals (Table 4). Thus, R1 had a unimodal effect for the five metrics where it showed a larger support and higher relative importance (NOTO, INVE, WACO, LITH, and DENS), while R2 displayed a unimodal effect for NATI, and had a negative linear response for TOLE, OMNI, and BENT. On the other hand, L1 exhibited a large support and a substantial relative importance for the metrics NATI, TOLE, and OMNI, where it showed a negative linear effect. L2 displayed the weakest support for all metrics according to the 95% confidence intervals and had the lowest relative importance. INVE was primarily influenced by R1, showing a unimodal response to this gradient. The wi of the best models was 0.77 and the top-ranked model had a R-square of 0.31. OMNI, on the other hand, was negatively affected by both R2 and L1. The best models had a wi of 0.83 and the R-square of the top-ranked model was 0.21. BENT displayed a negative response to R2 and, hence, tends to be positively correlated with downstream reaches. The wi of the best models was 0.72 and the top-ranked model had a R-square of 0.28. Conversely, WACO was primarily influenced by R1, showing a unimodal relationship along this gradient. The best models had a wi of 0.62 and the R-square of the top-ranked model was 0.37. LITH also exhibited a unimodal response to R1. The wi of the best models was 0.73 and the top-ranked model had a R-square of 0.33. POLY was negatively responsive to R1, although the response showed poor statistical support (only one model). The cumulative wi was near 1 and the R-square of the model was 0.32. Fish Functional Traits in Reference Conditions loaded on mean width, mean depth and maximum depth, with increasing values for larger and deeper downstream sites. conditions (L1), which means that density of tolerant fishes tend to be higher at downstream sites representing lowland streams with gentle slopes, highly productive and warm waters, and finer substrates. The best models had a wi of 0.73 and the R-square of the top-ranked model was 0.30. Model Averaging NATI was primarily influenced by R2, with a unimodal response, and by L1, with a linear negative response, meaning that a maximum richness is attained in middle-order reaches with low levels of altitude, slope, and substrate, and with warm and productive waters. The wi of the best models was 0.66 and the top- ranked model had a R-square of 0.56. DENS displayed a unimodal response to R1. The best models had a wi of 0.49 and the R-square of the top-ranked model was 0.36. Effects of Environmental Gradients on Metrics NOTO was primarily influenced by the regional-level gradient related to the latitudinal variation of climate and topography (R1), showing a unimodal response (Table 4). Thus, we expect density of non-tolerants to be higher in northern and central midelevation streams, with medium catchment slopes and more stable flows. The wi of the best models was 0.66 and the top-ranked model had a R-square of 0.28. TOLE was negatively affected by both the regional-level gradient related to the longitudinal stream contin- uum (R2) and the latitudinal variation of in-stream habitat Fish Assemblages We collected 29 species of fish from 9 families, representing a total of 17 822 individuals. About 82% of this total were endemic cyprinids. The aliens were represented in 15 sites (13%) and contributed only 1% of the total abundance (in eleven of these sites the relative abundance was less than 10%). Species richness ranged from 1 to 7 with a median of 3. The catch for the sampling sites ranged from 13 to 1623 and had a median of 179 individuals per 1000 m2. As expected, fish species with large distributions in Portugal were the most common (Table 2), mainly Salmo trutta, species from the genus Squalius, Luciobarbus bocagei and Anguilla anguilla. Conversely, most of the rare species in our study are endemic to small catchments or have restricted distribution in larger basins. Fish assemblages in the northern and central regions were dominated by A. oligolepis, P. duriense, L. bocagei, S. carolitertii, and S. trutta in the basins north of Tagus basin, and by P. polylepis, L. bocagei, S. pyrenaicus, and S. alburnoides in the Tagus basin. In the Guadiana basin three fish groups exhibited clear dominance: Luciobarbus spp., C. paludica, and S. pyrenaicus/alburnoides. The small September 2012 \| Volume 7 \| Issue 9 \| e45787 PLOS ONE \| www.plosone.org 6 Fish Functional Traits in Reference Conditions Fish Functional Traits in Reference Conditions Metric N Swi R2 R1 R12 R2 R22 L1 L12 L2 NOTO 4 0.66 0.28 20.42 (20.70, 20.15) 20.21 (20.37, 20.06) 0.10 (20.17, 0.36) 20.05 (20.20, 0.10) TOLE 2 0.73 0.30 20.14 (20.62, 0.34) 20.66 (21.11, 20.20) 21.52 (22.12, 20.91) INVE 7 0.77 0.31 20.45 (20.70, 20.20) 20.20 (20.36, 20.04) 0.07 (20.12, 0.27) 0.05 (20.13, 0.24) 20.07 (20.25, 0.12) OMNI 3 0.83 0.21 20.17 (20.70, 0.36) 20.87 (21.34, 20.40) 21.01 (21.63, 20.38) 20.05 (20.27, 0.16) BENT 3 0.72 0.28 20.42 (21.01, 0.18) 20.93 (21.31, 20.56) 20.21 (20.78, 0.36) WACO 3 0.62 0.37 20.46 (20.64, 20.27) 20.26 (20.41, 20.11) 0.12 (20.09, 0.33) 20.02 (20.09, 0.06) LITH 4 0.73 0.33 20.50 (20.79, 20.21) 20.23 (20.39, 20.08) 20.08 (20.27, 0.12) 0.13 (20.18, 0.44) POLY 1 0.99 0.32 22.11 POTA 5 0.69 0.09 0.01 (20.15, 0.16) 20.35 (20.72, 0.01) 0.21 (20.25, 0.66) 0.01 (20.06, 0.09) NATI 2 0.66 0.56 20.34 (20.48, 20.20) 20.08 (20.14, 20.02) 20.16 (20.26, 20.05) 0.01 (20.04, 0.07) DENS 4 0.49 0.36 20.31 (20.43, 20.19) 20.16 (20.29, 20.03) 20.04 (20.16, 0.08) 20.11 (20.23, 0.02) For each response variable (fish metric), the table provides the cumulative Akaike weight (Swi), calculated by summing the Akaike weights of the N best approximating models (Di #2), Nagelkerke r-square of the top-ranked mod g g y We found that the density-based metrics strongly influenced by the regional gradient R1 displayed unimodal relationships, indicating lower fish densities in the northern reaches that had higher catchment relief and slope, and strong runoff events, and higher densities at some intermediate levels. This result is concordant with the findings of Oliveira et al. [44] in the least disturbed and forested areas of northern and central Portugal, who found more and larger individuals in midelevation streams with gentler slopes. Possibly, lower gradient sites, with intermediate flow levels, tend to show higher diversity of available habitats for fish. Contrariwise, in the mountainous upper catchments the environ- mental conditions can be restrictive, corresponding to smaller, cooler and fast-flowing habitats that could limit both fish abundance and richness [60–61]. Additionally, it is known that altitude influences species occurrence through water temperature [62]. For example, as long as temperatures remain cold enough for trout reproduction and growth, increased temperatures that also increase metabolism and food supply are likely to increase trout production [63–64]. Fish Functional Traits in Reference Conditions For each response variable (fish metric), the table provides the cumulative Akaike weight (Swi), calculated by summing the Akaike weights of the N best approximating models (Di #2), Nagelkerke r-square of the top-ranked model (R2), and model-averaged estimates of GLM coefficients (unconditional 95% confidence intervals in parentheses) for the gradients included in the model (and quadratic term). Coefficients where the 95% confidence interval does not overlap zero are represented in bold. 2 0.66 0.56 20.34 (20.48, 20.20) 20.08 (20.14, 20.02) 20.16 (20.26, 20.05) 0.01 (20.04, 0.07) 4 0.49 0.36 20.31 (20.43, 20.19) 20.16 (20.29, 20.03) 20.04 (20.16, 0.08) 20.11 (20.23, 0.02) ponse variable (fish metric), the table provides the cumulative Akaike weight (Swi), calculated by summing the Akaike weights of the N best approximating models (Di #2), Nagelkerke r-square of the top-ranked model odel-averaged estimates of GLM coefficients (unconditional 95% confidence intervals in parentheses) for the gradients included in the model (and quadratic term). Coefficients where the 95% confidence interval does zero are represented in bold. rainfall (and, consequently, run-off), and drainage area. Over thirty years ago, the importance of landscape-stream interactions was clearly stated by Hynes [52] ‘in every respect the valley rules the stream’. The regional patterns operate on large temporal and spatial scales constraining the dynamics of smaller units, and, consequently, the resulting physical patterns will strongly influence the biological assemblages [53–55]. In fact, although stream fish assemblages are influenced by factors that occur at multiple scales, large scale measurements may be the best predictors of assemblage structure in studies like ours dealing with broad geographical areas [56]. Consequently, our prediction, that functional organization of fish assemblages would be associated with broad latitudinal and longitudinal gradients, was supported by our findings. Most guild metrics were related to strong north-south gradients (R1 or L1), corresponding partly to changes in climate and topography, with precipitation, altitude and slope increasing, and temperature and aridity decreasing, from south to north. This division broadly corresponds to different forms of ecosystem functioning, ranging from the colder, steeper and higher-altitude northern streams to the warmwater, and lowland southern systems. Thus, given the large geographical extent of the present study, the pronounced latitudinal gradient in fish assemblage variation was to be expected. Ferreira et al. [27] suggested a similar organisation of fluvial systems in Portugal but their work has been hampered by lack of representative network of reference sites. Fish Functional Traits in Reference Conditions On the other hand, the upstream-downstream gradient (R2) also greatly influenced local assemblages at the basin level. In fact, interactions between the catchment landscapes and aquatic organisms can change predictability with longitudinal position [56]. Although studies dealing with longitudinal variation of fish assemblages in Iberian streams [57–59] are limited, and do not only consider least disturbed reaches, they also provided evidences of assemblage changes along these lotic systems. Table 4. Summary results of model selection for the relations between fish metrics and environmental gradients reflected in each of the two components extracted from principa component analyses of regional (R) and local (L) variables. Metric N Swi R2 R1 R12 R2 R22 L1 L12 L2 NOTO 4 0.66 0.28 20.42 (20.70, 20.15) 20.21 (20.37, 20.06) 0.10 (20.17, 0.36) 20.05 (20.20, 0.10) TOLE 2 0.73 0.30 20.14 (20.62, 0.34) 20.66 (21.11, 20.20) 21.52 (22.12, 20.91) INVE 7 0.77 0.31 20.45 (20.70, 20.20) 20.20 (20.36, 20.04) 0.07 (20.12, 0.27) 0.05 (20.13, 0.24) 20.07 (20.25, 0.12) OMNI 3 0.83 0.21 20.17 (20.70, 0.36) 20.87 (21.34, 20.40) 21.01 (21.63, 20.38) 20.05 (20.27, 0.16) BENT 3 0.72 0.28 20.42 (21.01, 0.18) 20.93 (21.31, 20.56) 20.21 (20.78, 0.36) WACO 3 0.62 0.37 20.46 (20.64, 20.27) 20.26 (20.41, 20.11) 0.12 (20.09, 0.33) 20.02 (20.09, 0.06) LITH 4 0.73 0.33 20.50 (20.79, 20.21) 20.23 (20.39, 20.08) 20.08 (20.27, 0.12) 0.13 (20.18, 0.44) POLY 1 0.99 0.32 22.11 POTA 5 0.69 0.09 0.01 (20.15, 0.16) 20.35 (20.72, 0.01) 0.21 (20.25, 0.66) 0.01 (20.06, 0.09) NATI 2 0.66 0.56 20.34 (20.48, 20.20) 20.08 (20.14, 20.02) 20.16 (20.26, 20.05) 0.01 (20.04, 0.07) DENS 4 0.49 0.36 20.31 (20.43, 20.19) 20.16 (20.29, 20.03) 20.04 (20.16, 0.08) 20.11 (20.23, 0.02) For each response variable (fish metric), the table provides the cumulative Akaike weight (Swi), calculated by summing the Akaike weights of the N best approximating models (Di #2), Nagelkerke r-square of the top-ranked mode (R2), and model-averaged estimates of GLM coefficients (unconditional 95% confidence intervals in parentheses) for the gradients included in the model (and quadratic term). Coefficients where the 95% confidence interval doe not overlap zero are represented in bold Table 4. Summary results of model selection for the relations between fish metrics and environmental gradients reflected in each of the two components extracted from principa component analyses of regional (R) and local (L) variables. Discussion Our study revealed that generic patterns of the fish life-history traits in Iberian least disturbed streams were primarily associated with regional features, such as catchment elevation and slope, Figure 2. Relative importance of each variable given by the probability of the variable to be included in the best models as measured by the Akaike weights. The relative importance of each variable corresponds to the sum of its weight with the weight of its quadratic term with a maximum value of 1. Metrics abbreviations as follows: NOTO - density of non-tolerant individuals, TOLE - density of tolerant individuals, INVE - density of invertivores individuals, OMNI - density of omnivorous individuals, BENT - density of benthic individuals, WACO - density of water column individuals, LITH - density of lithophilic individuals, POLY - density of polyphilic individuals, POTA - density of potamodromous individuals, NATI - number of native species, DENS - total density of native individuals. doi:10.1371/journal.pone.0045787.g002 Figure 2. Relative importance of each variable given by the probability of the variable to be included in the best models as measured by the Akaike weights. The relative importance of each variable corresponds to the sum of its weight with the weight of its quadratic term with a maximum value of 1. Metrics abbreviations as follows: NOTO - density of non-tolerant individuals, TOLE - density of tolerant individuals, INVE - density of invertivores individuals, OMNI - density of omnivorous individuals, BENT - density of benthic individuals, WACO - density of water column individuals, LITH - density of lithophilic individuals, POLY - density of polyphilic individuals, POTA - density of potamodromous individuals, NATI - number of native species, DENS - total density of native individuals. doi:10.1371/journal.pone.0045787.g002 September 2012 \| Volume 7 \| Issue 9 \| e45787 PLOS ONE \| www.plosone.org 7 7 Fish Functional Traits in Reference Conditions September 2012 \| Volume 7 \| Issue 9 \| e45787 Fish Functional Traits in Reference Conditions Reinforcing this hypothesis is the fact that our dominant benthic species (endemic cyprinids) have mouths in a inferior position that allow them to feed very effectively on the riverbed [83]. At the broadest scale, climate and topography strongly contribute to the general hydrological characteristics, temperature regime, substrate type and local slope [77]. This is particularly evident in a significant area of the northern region, where the topography and climate described earlier support permanent and coarse-bottomed streams, with low-conductive waters and dense riparian vegetation. Particularly, relief makes a major contribution to the erosive force acting on streambed, and substrate size tends to be generally larger in the steeper gradients [53]. Changes in substrates influence the dominant reproduction guild [60], and thus higher density of lithophils was generally associated with the northern and central midelevation streams. Conversely, and despite the poor statistical performance of the model, polyphilics appear to be associated with the southern areas having lower catchment relief and slope (and, consequently, finer substrates), and lower annual and summer rainfall. Since this guild is largely represented by C. paludica, we may conclude that this species exhibited spatial patterns that are consistent with other studies in Mediterranean streams [12,78]. In the northern region, streams generally maintain more stable flows during periods of low rainfall, comparatively to the southern intermittent streams. Such more stable hydrological conditions would favour species having narrower water and habitat flexibility [79], and thus higher density of non-tolerants was expected. We also found higher A valid understanding of patterns and processes in ‘natural’ conditions is an essential prerequisite for viable ecosystem management [88]. The findings of our study emphasized the need to use a multi-scale approach to fully assess the factors that govern the functional organization of biotic assemblages in reference conditions, as well as to improve biomonitoring and restoration of fluvial ecosystems. The hierarchical models postulate that the units of streams are constrained by larger scales, from biota to local habitat to basin. To identify the spatial scale that explain most of the variation in biotic composition is to find the scale at which the most important physical/chemical processes constraining the assemblages can be found [89]. Thus, the effectiveness of actions that strive to improve river health rely on the ability to distinguish between and understand the multiscale environmental gradients. Fish Functional Traits in Reference Conditions densities of invertivores in these northern and central streams densely shaded by deciduous vegetation. These systems receive large amounts of allochthonous inputs of food provided by the riparian vegetation [80–82], and thus fish assemblages may include high densities of invertivores species [67]. In particular, terrestrial invertebrates can comprise more than 50% of energy intake by stream fishes and are often a preferred prey of juvenile salmonids [82]. Like the typically carnivorous S. trutta, the endemic invertivores cyprinids have a mouth in a superior position, allowing them to feed more effectively on terrestrial invertebrates, and thus these prey may significantly contribute to their diet [83–84]. Moreover, alders – the dominant riparian trees in the northern region – provide much larger inputs of invertebrates to streams than southern dominant trees as ashes or oleanders [80]. Since most of our water column species are active swimmers that feed on drifting and surface prey, we expected that this guild also had a similar association with the latitudinal gradient R1. However, these conclusions should be tested in the more general context of the links between the carbon fluxes in the Iberian stream ecosystems and the functional structure of fish assemblages [85]. Our results show strong longitudinal variability for some guild metrics, such as increasing of omnivorous, tolerant and benthic individuals with stream size. The RCC suggests that in the downstream reaches the lower trophic contribution of the riparian vegetation and the higher quantities of particulate organic matter, tend to increase the proportion of generalist feeders [86]. This prediction was also confirmed for European streams [14,29,87], and for fish assemblages of four continents [25]. We expected a similar response for the density of tolerants, since both metrics are strongly associated in Mediterranean streams, increasing along the longitu- dinal gradient [14]. Additionally, the present study also provides support that local habitat conditions (i.e. gradient L1) may be of great importance in determining the density of omnivorous and tolerant individuals. In fact, for downstream sites with similar drainage areas, we found higher proportion of these two guilds in the southern stream reaches with gentler slopes, warmer temper- atures, finer substrates and higher conductivities. But levels of sedimentation also increase with organic matter (even in low disturbed rivers), and thus the density of species which are adapted to substrate may increase along the upstream-downstream gradient, contrary to the ones that feed on invertebrates in the water column. Fish Functional Traits in Reference Conditions systems. Although other river ecology concepts have been developed over the last three decades, the RCC still adequately describes river zones, as the ones dealt with in this work, where longitudinal processes predominate [68]. In fact, this concept still offers stream ecologists a powerful integrative framework for research (e.g., [25,31,69]). In our study the upper stream reaches were characterized by S. trutta alone or in association with a few reophilic species. A decrease in the number of species towards headwater streams is a general pattern of the river continuum hypothesis, and as we pointed out can be largely explained by the cold temperatures and the high channel slopes that filter out other species [70–71]. Contrary to the typical increase in species with stream size (e.g., [25,32–33,72–74]), we did not find higher species richness in the larger streams of our study (20–30 m). In fact, our results seems to agree once more with the RCC which predicts maximum biotic diversity in midsize streams in response to habitat diversity and the number of niches available to different autecological types [67]. A parabolic relationship between species richness and stream order in near-natural systems was also found by Aarts and Nienhuis [31] in France. Anthropogenic disturbances have impacted streams worldwide and this can explain divergences from the predictions of the RCC, which was developed for unperturbed fluvial systems. However, even in our study, which only considered reference stream reaches, the non-linear response of species richness with measures of stream size should be interpreted in the light of the contemporary fish assemblages, because of the river impoundment that has taken place in Portugal in the last sixty years. In particular, diadromous migrations have been obstructed by large dams in some river basins, and thus relatively more diverse fish assemblages were likely to occur in the past. Independently of stream size, local variables (L1) also explained an important component of variation in species richness, suggesting that processes at the reach scale may contribute to spatial heterogeneity within the stream continuum. In fact, although the RCC has been an effective framework for understanding the structure and function of assemblages along river systems, longitudinal relationships may be clouded by local factors [75]. For example, McNeely [76] have reported an unusual pattern in species diversity along a pristine stream, with habitat diversity overriding the typical upstream-downstream gradients. Fish Functional Traits in Reference Conditions Lower productive waters, that usually characterize the upper reaches, also have lesser abundance and diversity of invertebrate fauna [65–66], constraining the food supply of invertivores species. On the other hand, and given the unimodal responses of these metrics, they also decreased at the extreme opposite of the R1 gradient, i.e., in the southern stream reaches with lower catchment relief and slope, and more hydrological variability (as discussed below). We also found a strong unimodal response for the longitudinal component of the metric ‘number of native species’. One of the most cited riverine ecosystem theories, the river continuum concept (RCC) [67], explicitly predicts changes in fish assemblages along the longitudinal abiotic gradients that occur in the river September 2012 \| Volume 7 \| Issue 9 \| e45787 8 Fish Functional Traits in Reference Conditions September 2012 \| Volume 7 \| Issue 9 \| e45787 References Esselman PC, Allan JD (2010) Relative influences of catchment- and reach-scale abiotic factors on freshwater fish communities in rivers of northeastern Mesoamerica. Ecol Freshw Fish 19: 439–454. 32. 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Fish Functional Traits in Reference Conditions In spite of these perspectives, most stream restoration efforts often concentrate on a discrete river reach or segment involving localized interventions even when larger-scale processes may be paramount [90–91]. However, the results of our study should be viewed with caution, as they represent an ecological snapshot. Temporal variation, which could September 2012 \| Volume 7 \| Issue 9 \| e45787 September 2012 \| Volume 7 \| Issue 9 \| e45787 PLOS ONE \| www.plosone.org 9 Fish Functional Traits in Reference Conditions Fish Functional Traits in Reference Conditions not be detected by our single sampling at each site, may play a role in understanding the spatial distribution of fish guilds. Future studies should consider monitoring fish assemblages at appropriate temporal scales, to provide additional insights for understanding the environmental correlates of the functional structure of Iberian fish assemblages and their implication for river management. Acknowledgments The authors thank Paulo Pinheiro, Rui Rivaes, Raul Arenas, Ana Geraldes and Angelo Saraiva for their assistance in the field, and Paulo Pinheiro for his assistance with GIS data processing. We thank two anonymous reviewers for critical manuscript reviews. Supporting Information Conceived and designed the experiments: JMO JMS MTF RVC. Analyzed the data: JMO PS JMS. Wrote the paper: JMO PS JMS. Collected the data: JMO JMS AT. Significant manuscript editing: AT MTF RVC. Conceived and designed the experiments: JMO JMS MTF RVC. Analyzed the data: JMO PS JMS. Wrote the paper: JMO PS JMS. Collected the data: JMO JMS AT. Significant manuscript editing: AT MTF RVC. 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https://openalex.org/W3033686643	https://peerj.com/articles/9300.pdf	English	null	The effect of journal guidelines on the reporting of antibody validation	PeerJ	2,020	cc-by	13,750	How to cite this article Hoek JM, Hepkema WM, Halffman W. 2020. The effect of journal guidelines on the reporting of antibody valida- tion. PeerJ 8:e9300 http://doi.org/10.7717/peerj.9300 The effect of journal guidelines on the reporting of antibody validation 1 Department of Psychology, University of Groningen, Groningen, The Netherlands 2 Institute for Science in Society, Radboud University Nijmegen, Nijmegen, The Netherlands ABSTRACT Background. Despite the widespread use of antibodies as a research tool, problems with specificity, lot-to-lot consistency and sensitivity commonly occur and may be important contributing factors to the ‘replication crisis’ in biomedical research. This makes the validation of antibodies and accurate reporting of this validation in the scientific literature extremely important. Therefore, some journals now require authors to comply with antibody reporting guidelines. Methods. We used a quasi-experimental approach to assess the effectiveness of such journal guidelines in improving antibody reporting in the scientific literature. In a sample of 120 publications, we compared the reporting of antibody validation and identification information in two journals with guidelines (Nature and the Journal of Comparative Neurology) with two journals without guidelines (Science and Neuroscience), before and after the introduction of these guidelines. Results. Our results suggest that the implementation of antibody reporting guidelines might have some influence on the reporting of antibody validation information. The percentage of validated antibodies per article slightly increased from 39% to 57% in journals with guidelines, whereas this percentage decreased from 23% to 14% in journals without guidelines. Furthermore, the reporting of validation information of all primary antibodies increased by 23 percentage points in the journals with guidelines (OR = 2.80, 95% CI = 0.96-INF; adjusted p = 1, one-tailed), compared to a decrease of 13 percentage points in journals without guidelines. Fortunately, the guidelines seem to be more effective in improving the reporting of antibody identification information. The reporting of identification information of all primary antibodies used in a study increased by 58 percentage points (OR = 17.8, 95% CI = 4.8-INF; adjusted p = 0.0003, one-tailed) in journals with guidelines. This percentage also slightly increased in journals without guidelines (by 18 percentage points), suggesting an overall increased awareness of the importance of antibody identifiability. Moreover, this suggests that reporting guidelines mostly have an influence on the reporting of information that is relatively easy to provide. A small increase in the reporting of validation by referencing the scientific literature or the manufacturer’s data also indicates this. Results. Our results suggest that the implementation of antibody reporting guidelines might have some influence on the reporting of antibody validation information. The percentage of validated antibodies per article slightly increased from 39% to 57% in journals with guidelines, whereas this percentage decreased from 23% to 14% in journals without guidelines. INTRODUCTION Antibodies are veritable workhorses in biomedical research. Used to label specific molecules or antigens (generally proteins), they allow researchers to map biomolecular processes in the cell. Through techniques such as western blotting (WB), immunohistochemistry, or ELISA, the use of antibodies has become widespread in biological research. However, binding antibodies to antigens is intricate and full of complications. Antibodies may lack specificity and their affinity for specific antigens may vary. Furthermore, their affinity may vary with experimental conditions (Baker, 2015b), for example when pH levels or reagents denature proteins, altering protein folding and thereby the epitopes to which antibodies bind. Among tens of thousands of antibodies on offer (largely from commercial suppliers), researchers need to identify precisely which antibodies are most suitable for their antigens of interest, under their precise experimental circumstances. To aid this selection, extensive support tools have been developed, such as the Antibodypedia, or the Antibodyregistry, which support the identification of antibodies with research resource IDs (RRIDs) (Bandrowski et al., 2016). Nevertheless, even with the help of such resources, the practice of antibody use remains complex. While researchers may identify the correct antibody for their specific research purposes in principle, verifying that the correct antibodies are used in practice is a different matter. Antibodies may vary from batch to batch, suppliers may not always be able to guarantee relevant quality standards, or earlier mistakes may be obfuscated by relying on locally established routines, such as habits, experimental skills, and techniques passed on in a laboratory. If identification and validation information about antibodies is not reported accurately, the possibility of experimental replication is jeopardised, and subsequent research may be built on errors. In turn, this may lead to wasted research, missed opportunities for medical innovation, or even patient safety risks. Taken together, the costs involved may be considerable. In fact, some commentators suggest problems with antibody validation, or lack of validation information, may be an important contributing factor to the ‘replication crisis’ in biomedical research (Freedman, Cockburn & Simcoe, 2015). For over a decade, various researchers have expressed concerns about insufficient antibody validation in biomedical research (Baker, 2015a; Bordeaux et al., 2010; Saper & Sawchenko, 2003). The challenges are considerable. One study validating over 5,000 commercial antibodies for the Human Protein Atlas (HPA) showed that half of these antibodies were not suitable for the specific immunohistochemistry application in the HPA. ABSTRACT Furthermore, the reporting of validation information of all primary antibodies increased by 23 percentage points in the journals with guidelines (OR = 2.80, 95% CI = 0.96-INF; adjusted p = 1, one-tailed), compared to a decrease of 13 percentage points in journals without guidelines. Fortunately, the guidelines seem to be more effective in improving the reporting of antibody identification information. Submitted 15 January 2020 Accepted 14 May 2020 Published 3 June 2020 Corresponding author Joyce M. Hoek, j.m.hoek@rug.nl Academic editor Jeffrey Stuart Additional Information and Declarations can be found on page 22 DOI 10.7717/peerj.9300 Copyright 2020 Hoek et al. Distributed under Creative Commons CC-BY 4.0 Submitted 15 January 2020 Accepted 14 May 2020 Published 3 June 2020 Corresponding author Joyce M. Hoek, j.m.hoek@rug.nl Academic editor Jeffrey Stuart Additional Information and Declarations can be found on page 22 DOI 10.7717/peerj.9300 C i ht p g p g y The reporting of identification information of all primary antibodies used in a study increased by 58 percentage points (OR = 17.8, 95% CI = 4.8-INF; adjusted p = 0.0003, one-tailed) in journals with guidelines. This percentage also slightly increased in journals without guidelines (by 18 percentage points), suggesting an overall increased awareness of the importance of antibody identifiability. Moreover, this suggests that reporting guidelines mostly have an influence on the reporting of information that is relatively easy to provide. A small increase in the reporting of validation by referencing the scientific literature or the manufacturer’s data also indicates this. Conclusion. Combined with the results of previous studies on journal guidelines, our study suggests that the effect of journal antibody guidelines on validation practices by themselves may be limited, since they mostly seem to improve antibody identification instead of actual experimental validation. These guidelines, therefore, may require ad- ditional measures to ensure effective implementation. However, due to the explorative nature of our study and our small sample size, we must remain cautious towards other factors that might have played a role in the observed change in antibody reporting behaviour. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Subjects Cell Biology, Molecular Biology, Science Policy Keywords Antibodies, Validity, Journal guidelines, Replication crisis INTRODUCTION The researchers concluded that every application of antibodies requires application-specific validation (Berglund et al., 2008). Advocates of tighter validation have suggested techniques and principles to ensure correct antibodies are used and to improve reporting of antibody validation information in publications, although there is no universal standard as yet (GBSI, 2016). At the very Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 2/26 least, these advocates suggest that researchers should report information that establishes the identity of antibodies used, via reference to RRIDs or supplier identifiers, including catalogue and batch number (Vasilevsky et al., 2013). While many researchers rely on the literature to establish application-specific validity of antibodies, actual validation testing avoids repetition of older mistakes. This testing should verify not just the identity, avoiding supply-line errors (such as misidentification during personal exchange, or transport via suppliers), but also the antibody’s specificity for the target protein and sensitivity in the specific application. Relatively simple checks include staining a western blot to check whether antibodies recognise antigens of the correct molecular weight, omitting the primary antiserum, and performing pre-adsorption controls (Saper & Sawchenko, 2003). While these methods might be useful as a first indicator of an antibody’s specificity, they are not very stringent, and antibodies might still be found to be nonspecific upon more thorough validation (Andersson et al., 2017; Bordeaux et al., 2010; Hewitt et al., 2014; O’Hurley et al., 2014). The International Working Group of Antibody Validation (IWGAV) proposed a very stringent validation procedure in 2016, with five ‘pillars’ for application-specific validation. These include genetic strategies (testing the antibody in conditions when the protein is not expressed), orthogonal strategies (comparing results for varying amounts of target protein identified by other means), independent antibody strategies (comparing results with alternative antibodies), expression of tagged proteins (using affinity tags or fluorescent proteins), and immunocapture followed by mass spectrometry. The IWGAV advises researchers to carry out at least one of these five methods in order to validate whether an antibody is truly specific to the application at hand. Furthermore, the IWGAV advises suppliers to also use at least one of these five pillars for validation, including validation for each new batch of antibodies, and to provide specific information on optimal use (Uhlén et al., 2016). Several initiatives encourage researchers to improve antibody validation. INTRODUCTION Apart from calls to action in editorials, the development of databases, and improved validation by suppliers, some journals have also stepped up to the plate. The first and most vocal initiative was taken by the Journal of Comparative Neurology (JCN), introducing explicit requirements for antibody validation in its author instructions in 2003 (Saper, 2005; Saper & Sawchenko, 2003). Several journals followed suit and now require various levels of antibody validation information: some require only identification information; others require documentation or actual experimental proof of application-specific validity (Steve et al., 2018; Gore, 2013; Nature, 2013). In essence, these journal initiatives constitute rule-based or ‘regulatory’ policy, attempting to change researchers’ behaviour through regulation. These vary from insistent but voluntary guidelines, through checklists that attempt to ‘nudge’ authors into compliance (Babic et al., 2019), or actual rules at penalty of editorial rejection. In policy sciences, the track record of rule-based behaviour modification is mixed, at best. Rules require policing, which is expensive, and rules tend to fail if they lack community support, for example if they are perceived as meaningless or ineffectual. In matters of Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 3/26 research integrity, rules that blatantly diverge from actual practice have been shown to induce cynicism among researchers, decreasing willingness to comply (Clair, 2015). Although journals have long been considered powerful ‘gatekeepers of science’ (Crane, 1967), and journals are generally expected to maintain standards in research communities, their power to do so is not absolute. Journals are also dependent on research communities for submitted articles and willingness of reviewer cooperation, compounded by the hard economics of publishing in which volume is often the backbone of the business model (Larivière, Haustein & Mongeon, 2015). Hence, the possibility of journals to raise standards may be limited by what the research community is willing to maintain. From this perspective, it is interesting to investigate the effect of journal antibody guidelines on the presence and quality of reported validation information. For these purposes, we chose to compare antibody validation information in journals with guidelines and in equivalent journals without guidelines, i.e., a control group. Our hypothesis was that antibody validation information would improve in the journals that introduced antibody validation guidelines. Before we turn to the details of our research method, we want to discuss some key methodological assumptions in validation testing, which touch upon quite fundamental issues of philosophy of science. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 INTRODUCTION First, we want to make an important distinction between antibody reliability and antibody validity. Antibodies may reliably produce the same results if experimental conditions are reproduced exactly, but if these conditions are based on misconceptions, they could simply reliably reproduce systematic error. A striking example of how the reproduction of error might lead research astray, comes from the recent evaluation of antibodies against oestrogen receptor beta in breast cancer research. Andersson et al. (2017) discovered that the antibodies most cited in the literature had systematically stained the wrong proteins in tissue samples and that the target protein was not even present in breast cancer tissue, affecting two decades of research. Hence, validation would ideally constitute the experimental confirmation under different conditions, providing more robust replication, rather than identical reproduction, as recent philosophy of science has argued (Leonelli, 2018). In the case of antibodies, the principles of the ‘five pillars’ offer such variation in experimental conditions. In contrast, strictly speaking, reference to exact reproduction of antibodies as used in the literature only constitutes proof of reliability, although it is frequently presented as proof of validity. Second, while exact reproduction can be considered important evidence for antibody reliability in specific applications, in the practice of antibody research this evidence is not absolute: it always remains possible to question whether the reproduced experiment was indeed identical or performed equally expertly. Lack of exact reproduction raises the possibility that the repeat experiment failed to reproduce the exact original conditions and materials, or lacked some quality in the performer, such as particular experimental skills (Collins, 1985). In the case of antibodies, this is particularly pertinent in the case of polyclonal antibody batches that have run out, for example as source animal populations are terminated. Moreover, whether an application of antibodies constitutes an exact reproduction may be difficult to establish based on concise information provided in databases or even publications. Lack of reproduction therefore does not automatically Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 4/26 Figure 1 Overview of the research strategy. Samples of 15 articles were taken from journals that did (1 and 2) and that did not (3 and 4) introduce antibody reporting guidelines. These samples were taken from a timeframe before (August 2002–August 2003) and after (June 2017–June 2018) the introduction of guidelines by journal 1 and 2. INTRODUCTION An increase in antibody validation reporting was expected between the sam- ples connected with a black arrow, while no difference was expected between the samples connected with a dashed grey arrow. Full-size DOI: 10.7717/peerj.9300/fig-1 Figure 1 Overview of the research strategy. Samples of 15 articles were taken from journals that did (1 and 2) and that did not (3 and 4) introduce antibody reporting guidelines. These samples were taken from a timeframe before (August 2002–August 2003) and after (June 2017–June 2018) the introduction of guidelines by journal 1 and 2. An increase in antibody validation reporting was expected between the sam- ples connected with a black arrow, while no difference was expected between the samples connected with a dashed grey arrow. Full-size DOI: 10.7717/peerj.9300/fig-1 disqualify the reliability of the original experiment and exact reproduction may be hard to assess in practice. In scoring the quality of validation information, we should therefore pay attention to information that establishes the identity of antibodies, to evidence for antibodies’ reliability in the specific intended purposes, and to evidence that experimentally confirms the antibodies’ adequacy to identify the intended antigens with precision. While only the latter constitutes proper validation, information about identity and reliability is a precondition of such higher levels of validation. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 1Editorial policies at JCN have recently changed. The Antibody Characterization section in force at the time of this study is no longer a requirement. Research strategy In this exploratory study, we used a quasi-experimental study design to assess whether journal antibody validation guidelines can improve antibody validation reporting (see Fig. 1). We selected two journals that had adopted such guidelines and two journals with a comparable impact factor and research area, but without guidelines. We took random samples of 15 articles from each journal from a one-year timeframe before and after the two journals implemented antibody reporting guidelines. We used the study performed by Han et al. (2017) as an indication of the sample size and did not perform a power calculation beforehand. Since the first selected journal with guidelines created awareness of the antibody problem in August 2003, the ‘before guidelines’ samples consisted of articles that were published between August 2002 and August 2003. The ‘after guidelines’ samples consisted of articles that were published between June 2017 and June 2018, because the second journal with guidelines implemented its most stringent guidelines in June 2017. We then used an antibody validation coding table to assess the antibody validation information provided by each of the articles. We evaluated a total of 120 articles. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 5/26 Journal selection Intervention group The Journal of Comparative Neurology (JCN) and Nature were selected as the two journals with antibody validation guidelines. With regard to antibodies, JCN requires authors to add an ‘Antibody Characterization’ section to the methods section of a paper.1 This section should include complete identification information of the used antibodies and proof of their validity. Authors can report their own validation or describe and cite evidence from the literature. The guidelines further specify that just a casual reference to a supplier’s data sheet does not count as reliable evidence of specificity (information from Author Guidelines section of website Journal Comparative Neurology, retrieved on 04-05-2018). When authors want to submit a manuscript to JCN, they need to check a box that states that their publication provides full (validation) information on all antibodies used (article submission process tested on 09-05-2018). The journal Nature has been asking authors in the life sciences to fill out a reporting checklist since May 2013. Since June 2017, this checklist has been replaced by a reporting summary to be published as supplementary information alongside the article. While the previous checklist and current reporting summary are not uniquely intended for antibodies, these documents do ask for exhaustive identification and validation information on antibodies. Furthermore, filling out this reporting summary is a requirement for publication in Nature. The form needs to be uploaded at submission to prevent manuscript processing delays (article submission process tested on 09-05-2018). A more elaborate explanation of these journals’ guidelines can be found in Article S1. Control group The InCites Journal Citations Reports function of Web of Science (WoS) was used to find journals comparable to JCN and Nature. Comparable journals were chosen by selecting the first journal with a similar impact factor from the Cited Journal Data. Journals in this network are considered similar because they fit in the same research area and are part of the same citation network. In the case of JCN, the most similar journal is Neuroscience with a similar impact factor and research category of neurosciences. The journal Neuroscience does not have antibody validation guidelines. No specific information about antibodies can be found in the author guidelines for article submission on Neuroscience’s website. The website does provide information on resource materials in general. The source of all materials used should be provided and the location of each supplier should be provided on first use in the text (information from Guide for Authors section on website Neuroscience, retrieved 04-05-2018). However, at submission, authors are not asked to provide information on the materials they used or on antibodies specifically (article submission process tested on 09-05-2018). A comparable journal to Nature is Science, as they are both multidisciplinary journals with similar impact factors. Using the same method as before, Science is also the first journal with a comparable impact factor in the list of cited journals of Nature. While Science does state that antibodies should be validated, this instruction is cursory and hard to find. It is Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 6/26 written between brackets on the ‘Editorial Policies’ page of the Science website, rather than in author instructions. There is no further elaboration on what validation should consist of. Furthermore, the manuscript submission process of Science never asks authors for validation information on materials that are used in a study and never mentions antibodies. Hence, Science can be regarded as having very marginal, to no guidelines on antibody reporting when compared to Nature (information from Information for Authors/Editorial Policies section on website Science, retrieved 04-05-2018; article submission process tested on 09-05-2018). Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 Article selection WoS was used to search for articles published between 2002 and 2003 or between 2017 and 2018 in each of the four journals. These lists of articles were then randomly sorted by assigning them a random number and sorting these numbers from smallest to largest. The first 15 articles in these lists in the time span of August 2002–August 2003 and June 2017–June 2018 (according to the WoS publication date) that contained antibodies as a resource material were then selected for assessment, selecting a total of 120 articles. Using antibodies as a resource material means that they are used as a tool in a biological method such as western blotting or immunohistochemistry. Antibodies can also themselves be the subject of scientific research. In this case the article was only included in the sample if antibodies were additionally used as a resource material. To determine if an antibody was used in a study, first the title of the article was used to determine if the article was published in a field that could use antibodies (e.g., life sciences and not physics). Next, the full text of the article was visually screened and screened for the word ‘anti’ to see if antibodies as a resource material were mentioned in the article. If this word did not occur in the full text in relation to antibodies, it was assumed that no antibodies had been used. In case of doubt, supplementary information was used to determine if an article should be included in the sample. g Coding protocol All articles and their supplementary information were then scanned for antibody (validation) information, and this information was collected in a coding table. The coding table was carefully constructed by evaluating the literature for different antibody validation methods, by consulting researchers working with antibodies, and by checking the table on a set of testing articles. Information was collected per article, not per antibody. If multiple antibodies were validated by multiple methods of validation, all methods were noted for this paper. In case of doubt about the answer to a question, the decision was always made to rule in favour of the article. For example, the highest number of validated antibodies would be written down in case of doubt about how many antibodies were validated. The coding table consisted mostly of ‘yes’ and ‘no’ questions such as: ‘are all primary antibodies validated?’ or ‘is validation carried out by positive control?’ When a particular question was not relevant for the evaluated article (for example, the questions about type of validation when no antibodies were validated), this question was evaluated as ‘not Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 7/26 Figure 2 Example of explicit antibody validation. To test the specificity of the antibody, a comparison was made between the western blots provided by the supplier of the antibody and western blots carried out by the authors of the paper. According to the supplier, two bands should be observed: one between 25 and 37 kDa and one between 50 and 75 kDa. According to the authors, the additional band at >250 kDa is likely caused by six connexin protein subunits that form a connexin hemi-channel. The authors explicitly mention antibody validation as the purpose of this experiment: ‘‘We tested the antibody’s specificity using a western blot of Gulf toadfish whole brain homogenates.’’ Figure from Rosner et al. (2018). Full-size DOI: 10.7717/peerj.9300/fig-2 Figure 2 Example of explicit antibody validation. To test the specificity of the antibody, a comparison was made between the western blots provided by the supplier of the antibody and western blots carried out by the authors of the paper. According to the supplier, two bands should be observed: one between 25 and 37 kDa and one between 50 and 75 kDa. According to the authors, the additional band at >250 kDa is likely caused by six connexin protein subunits that form a connexin hemi-channel. g Coding protocol The authors explicitly mention antibody validation as the purpose of this experiment: ‘‘We tested the antibody’s specificity using a western blot of Gulf toadfish whole brain homogenates.’’ Figure from Rosner et al. (2018). Full-size DOI: 10.7717/peerj.9300/fig-2 e DOI: 10.7717/peerj.9300/fig-2 applicable’. When it was impossible to answer a question for a certain article, this question was coded ‘unclear’. The answer ‘unclear’ was classified as missing in data analysis. We wanted to know the percentage of articles that report validation or information on all primary antibodies from the sample of all articles that use antibodies. Therefore, for the questions ‘are all primary antibodies validated?’ and ‘is the basic information of all primary antibodies complete?’, the answer ‘not applicable’ was coded as ‘no’ to calculate the percentage of articles that contain this information in all articles with antibodies. For all other questions about validation type, the answer ‘not applicable’ was classified as missing. We made this choice because we wanted to know the prevalence of these validation types in articles with validated antibodies, instead of their prevalence in all articles using antibodies. A full overview of the coding protocol the questions in the coding table and the different A full overview of the coding protocol, the questions in the coding table and the different antibody validation methods is provided in Article S2. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 Implicit and explicit validation Because we assessed antibody validation information by reading the publication, our coding made no distinction between explicit or purposeful validation and implicit or ‘accidental’ validation, as the intention to validate cannot always be inferred from the text. Whether explicitly presented as validation or not, in both cases the antibody (and thus the article it was used in) was coded as validated. However, for the interpretation of our findings, it is important to understand this distinction between implicit and explicit validation. Explicit validation means that the article provides information with the explicit purpose to attest antibody validity. In this case, experiments are presented with the purpose of validating the antibody, and/or the words antibody validation are explicitly mentioned. An example from one of the evaluated articles would be the use of western blot with molecular weight markers to validate the antibody (see Fig. 2). Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 8/26 Figure 3 Example of implicit antibody validation. Atg7 defective cells were generated by inhibiting gene expression of the Atg7 gene with RNA interference. The cells in which Atg7 was silenced show no stain- ing with anti-ATG7, while wild type cells do. This type of validation was marked as validation by negative control (RNA interference) and by genetic strategies of the five pillars. From Abu-Remaileh et al. (2017). Reprinted with permission from AAAS. Full-size DOI: 10.7717/peerj.9300/fig-3 Figure 3 Example of implicit antibody validation. Atg7 defective cells were generated by inhibiting gene expression of the Atg7 gene with RNA interference. The cells in which Atg7 was silenced show no stain- ing with anti-ATG7, while wild type cells do. This type of validation was marked as validation by negative control (RNA interference) and by genetic strategies of the five pillars. From Abu-Remaileh et al. (2017). Reprinted with permission from AAAS. F ll i DOI 10 7717/ j 9300/fi 3 Figure 3 Example of implicit antibody validation. Atg7 defective cells were generated by inhibiting gene expression of the Atg7 gene with RNA interference. The cells in which Atg7 was silenced show no stain- ing with anti-ATG7, while wild type cells do. This type of validation was marked as validation by negative control (RNA interference) and by genetic strategies of the five pillars. From Abu-Remaileh et al. (2017). Reprinted with permission from AAAS. Implicit and explicit validation Full-size DOI: 10.7717/peerj.9300/fig-3 Full-size DOI: 10.7717/peerj.9300/fig-3 Implicit validation here means that from reading the article, claims about the validity of the antibody can be made, but no experiments have specifically been reported to establish the validity of the antibody. The words ‘antibody validation’ are not mentioned. An example from one of the evaluated articles would be the use of RNA interference as an antibody validation method (see Fig. 3). Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 Question by the authors of the paper’, ‘negative and positive control’ and ‘other types of validation’ illustrates this. These types of validation are often consequential to the methods used in an experiment. For example, antibodies are used to validate the silencing of a gene, and, at the same time, the silencing of this gene can be seen as a method to validate the antibody. This is a type of circular reasoning that only holds if either the antibody or the silencing has proven to be valid by other methods. Therefore, it is difficult to judge if the experiment validates the antibody or not. Because of the low agreement on these questions, they were not taken into further consideration during data analysis. by the authors of the paper’, ‘negative and positive control’ and ‘other types of validation’ illustrates this. These types of validation are often consequential to the methods used in an experiment. For example, antibodies are used to validate the silencing of a gene, and, at the same time, the silencing of this gene can be seen as a method to validate the antibody. This is a type of circular reasoning that only holds if either the antibody or the silencing has proven to be valid by other methods. Therefore, it is difficult to judge if the experiment validates the antibody or not. Because of the low agreement on these questions, they were not taken into further consideration during data analysis. On the other hand, the more explicit types of validation are easier to recognize and to judge by readers of the paper. This is illustrated by the high percentage of agreement between both raters and kappa value on the categories ‘validation by reference to supplier, the literature, or a database’, ‘molecular weight similar to target’, ‘spatial localization’, ‘pre-adsorption’ and ‘secondary antibody without primary’. Methodological reliability 88% 0.75 0.31–1.00 8 Reference to validation information in database? 100% IND / 8 Antibody validation carried out by authors of article? 63% −0.20 −0.49–0.088 8 If carried out by authors, which method? Molecular weight similar to target (in WB)? 83% 0.57 −0.12–1.00 6 Spatial localization? 100% 1.00 1.00–1.00 6 Pre-adsorption/blocking peptide? 100% 1.00 1.00–1.00 6 Using secondary antibody without primary? 100% IND / 6 One of the five pillars? 100% 1.00 1.00–1.00 6 Positive control? 67% 0.33 −0.23–0.90 6 Negative control? 67% IND / 6 Other validation method? 50% 0.00 −0.75–0.75 6 Question ICC 95% CI N How many antibodies were used? 0.995 0.986–0.998 15 How many antibodies were validated? 0.684 0.263–0.886 14 Percentage of validated antibodies? 0.663 0.226–0.878 14 Methodological reliability All 120 articles were evaluated by one of us (Joyce Hoek). An independent rater (Wytske Hepkema) then assessed a randomly selected sample of these articles to determine the interrater reliability. This sample consisted of 2 articles for each journal and time frame, resulting in 16 articles or 13% of the total sample. Since Wytske Hepkema only analysed a small percentage of the total sample, we only used her answers to estimate the interrater reliability, and we used the answers of Joyce Hoek for data analysis. The percentage of agreement between both raters (Joyce Hoek and Wytske Hepkema) was calculated using the agree function of the irr package (Gamer et al., 2012) in R. For nominal variables, Cohen’s kappa was calculated using the cohen.kappa function of the psych package (Revelle & Revelle, 2015) in R. For continuous variables, the intra-class correlation coefficient (ICC) was calculated using the icc function of the irr package in R. ICC estimates were calculated based on a single-measures, consistency, two-way mixed-effects model. Missing values were not taken into account in the calculation of kappa or the ICC. Cohen’s kappa was indeterminate in some cases because one or both raters answered ‘yes’ or ‘no’ for all evaluated articles. This is indicated by IND in the table. Table 1 shows the percentage agreement and Cohen’s kappa or intra-class correlation coefficient. The agreement between raters varied considerably between the different questions, from a kappa of 1 to a worse than chance kappa of −0.20. Antibody validation information is often not explicitly reported, as opposed to antibody identification information, which also had a higher interrater agreement. The low agreement on antibody validation questions thus illustrates the current problem of antibody validation reporting: it is very difficult for a reader to assess the validity of antibodies used in paper and therefore assess the validity of the performed experiments. A more standardized way of reporting could improve this. The large disagreement indicates that it is difficult to establish the validity of antibodies from reading the publication when antibody validation is not explicitly mentioned. The disagreement between the two raters when it comes to the categories ‘validation carried out Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 9/26 Table 1 Interrater reliability of article coding process. Question Percentage agreement Cohen’s kappa 95% CI N Basic information all primary antibodies complete? 100% 1.00 1.00–1.00 16 Any validation information present (at least one antibody)? Methodological reliability 75% 0.50 0.13–0.87 16 All primary antibodies validated? 94% IND / 16 If validated: Reference to validation by antibody supplier? 100% IND / 8 Reference to validation in the literature? 88% 0.75 0.31–1.00 8 Reference to validation information in database? 100% IND / 8 Antibody validation carried out by authors of article? 63% −0.20 −0.49–0.088 8 If carried out by authors, which method? Molecular weight similar to target (in WB)? 83% 0.57 −0.12–1.00 6 Spatial localization? 100% 1.00 1.00–1.00 6 Pre-adsorption/blocking peptide? 100% 1.00 1.00–1.00 6 Using secondary antibody without primary? 100% IND / 6 One of the five pillars? 100% 1.00 1.00–1.00 6 Positive control? 67% 0.33 −0.23–0.90 6 Negative control? 67% IND / 6 Other validation method? 50% 0.00 −0.75–0.75 6 Question ICC 95% CI N How many antibodies were used? 0.995 0.986–0.998 15 How many antibodies were validated? 0.684 0.263–0.886 14 Percentage of validated antibodies? 0.663 0.226–0.878 14 by the authors of the paper’, ‘negative and positive control’ and ‘other types of validation’ illustrates this. These types of validation are often consequential to the methods used in an experiment. For example, antibodies are used to validate the silencing of a gene, and, at the same time, the silencing of this gene can be seen as a method to validate the antibody. This is a type of circular reasoning that only holds if either the antibody or the silencing has proven to be valid by other methods. Therefore, it is difficult to judge if the experiment validates the antibody or not. Because of the low agreement on these questions, they were not taken into further consideration during data analysis. On the other hand, the more explicit types of validation are easier to recognize and to judge by readers of the paper. This is illustrated by the high percentage of agreement between both raters and kappa value on the categories ‘validation by reference to supplier, the literature, or a database’, ‘molecular weight similar to target’, ‘spatial localization’, Table 1 Interrater reliability of article coding process. Question Percentage agreement Cohen’s kappa 95% CI N Basic information all primary antibodies complete? 100% 1.00 1.00–1.00 16 Any validation information present (at least one antibody)? 75% 0.50 0.13–0.87 16 All primary antibodies validated? 94% IND / 16 If validated: Reference to validation by antibody supplier? 100% IND / 8 Reference to validation in the literature? RESULTS Percentage of validated antibodies per article Data analysis We compared the reporting of antibody validation information between the samples of journals with and without guidelines and between the samples of August 2002–August Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 10/26 2003 and June 2017–June 2018. We expected the proportion of articles reporting validation to be higher in 2017 than in 2003 in journals that implemented guidelines. Furthermore, the proportion of validation was expected to be larger in 2017 in the samples of journals that implemented guidelines compared to journals that did not implement guidelines. We measured the difference in validation reporting by comparing the percentage of validated antibodies per article and the proportion of articles reporting any validation information. We also decided to look at the proportion of articles reporting complete identification or validation information on all primary antibodies as a more robust measure of validation rigour. For the comparisons of proportions, one-tailed p-values were calculated, since these hypotheses are directional. When comparing the samples of journals without guidelines between 2003 and 2017, no increase in proportion was expected. Likewise, the samples of similar journals with and without guidelines taken at baseline in 2003 were not expected to be different. For this reason, two-tailed p-values were calculated in these cases. Because of the small sample size, we used Fisher’s exact test (fisher.test in R) to compare the two proportions (instead of the chi-square or z-test for two proportions). This function uses the conditional Maximum Likelihood Estimate as an estimate of the odds ratio. For one-tailed tests, the odds ratio goes to infinity. This is indicated by INF in the text. To account for multiple comparisons, we used the Holm-Bonferroni correction (p.adjust in R) to adjust all calculated p-values. Data analysis was carried out using R version 3.3.3. In line with recent debate about the value of p-values (Wasserstein, Schirm & Lazar, 2019) and because of the exploratory nature of our study, we decided to not only look at statistically significant (p < 0.05) results, but also take into account meaningful but non-significant differences. To us, a meaningful difference is a substantial increase in proportion of validated articles, accompanied by a large odds ratio. We think these differences are interesting and might be good targets to further investigate if this study were to be replicated. We provide our data and R code in Data S1 and S2 and Article S3. Our data can also be found at https://doi.org/10.17026/dans-xhk-74m4. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 Percentage of validated antibodies per article For each evaluated article, the percentage of validated antibodies per article was calculated from the total number of antibodies used and the number of validated antibodies. An overview of these numbers can be found in Table S1. Figure 4 provides a descriptive overview of the percentages of validated antibodies in each evaluated article as well as the mean percentage per journal. This figure shows that the mean percentage of antibodies that have been validated per article for each journal slightly increased in both journals with guidelines (JCN and Nature) between 2003 and 2017. Combined, these journals increased from an average of 39% to 57% of validated antibodies per article. This percentage decreased in the journals that did not introduce reporting guidelines. These journals went from an average of 23% to 14% of validated antibodies per article. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 11/26 Figure 4 Change in percentage of validated antibodies per article before (2003) and after (2017) two of the journals (JCN and Nature) implemented antibody reporting guidelines. Grey dots represent the percentage of validated antibodies per article, red bars indicate the mean percentage of validated antibod- ies per article for each journal. Sample size: JCN 2003 n = 15, 2017 n = 14; Neuroscience 2003 n = 14, 2017 n = 15; Nature 2003 n = 15, 2017 n = 14; Science 2003 n = 15, 2017 n = 15. Full-size DOI: 10.7717/peerj.9300/fig-4 Figure 4 Change in percentage of validated antibodies per article before (2003) and after (2017) two of the journals (JCN and Nature) implemented antibody reporting guidelines. Grey dots represent the percentage of validated antibodies per article, red bars indicate the mean percentage of validated antibod- ies per article for each journal. Sample size: JCN 2003 n = 15, 2017 n = 14; Neuroscience 2003 n = 14, 2017 n = 15; Nature 2003 n = 15, 2017 n = 14; Science 2003 n = 15, 2017 n = 15. Full-size DOI: 10.7717/peerj.9300/fig-4 Furthermore, there is a difference in reporting between similar journals with and without guidelines. This difference in percentage was already present before JCN and Nature introduced guidelines, but it increased after the introduction of these guidelines. The difference in average percentage of validated antibodies between JCN and Neuroscience was 20 percentage points before the introduction of guidelines and increased to 49 percentage points after the introduction of guidelines by JCN. Percentage of validated antibodies per article Between Nature and Science, the difference in average percentage was 12 percentage points, which increased to a difference of 38 percentage points after the introduction of guidelines by Nature. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 Validation of at least one antibody Next, we made comparisons about antibody validation at the article level. Instead of looking at the percentage of validated antibodies per article, we now compared the percentage of articles that reported the validation of at least one antibody (see Tables S2 and S3). The reporting of validation improved slightly, but insignificantly, in the journals that had introduced guidelines. In addition, after the introduction of guidelines, 90% of the articles that use antibodies in journals with guidelines reported validation of at least one antibody compared to 53% of articles in journals without guidelines (OR = 7.6, 95% CI = 2.11-INF; adjusted p = .09, one-tailed). However, since it might be quite simple to implicitly validate at least one antibody per article, we also chose to compare the percentage of articles Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 12/26 Figure 5 Change in percentage of articles that reported validation information on all primary anti- bodies used in the study before (2003) and after (2017) two of the journals (JCN and Nature) imple- mented antibody reporting guidelines. Sample size: with guidelines 2003 n = 30, 2017 n = 28; without guidelines 2003 n = 30, 2017 n = 30, total 2003 n = 60, 2017 n = 58, JCN 2003 n = 15, 2017 n = 14; Neu- roscience 2003 n = 15, 2017 n = 15; Nature 2003 n = 15, 2017 n = 14; Science 2003 n = 15, 2017 n = 15. P- values (one-tailed) were determined with Fisher’s exact test and adjusted using the Holm-Bonferroni cor- rection. Full-size DOI: 10.7717/peerj.9300/fig-5 reporting antibody (validation) information for all primary antibodies that they used as a more robust measure of antibody reporting quality. Figure 5 Change in percentage of articles that reported validation information on all primary anti- bodies used in the study before (2003) and after (2017) two of the journals (JCN and Nature) imple- mented antibody reporting guidelines. Sample size: with guidelines 2003 n = 30, 2017 n = 28; without guidelines 2003 n = 30, 2017 n = 30, total 2003 n = 60, 2017 n = 58, JCN 2003 n = 15, 2017 n = 14; Neu- roscience 2003 n = 15, 2017 n = 15; Nature 2003 n = 15, 2017 n = 14; Science 2003 n = 15, 2017 n = 15. Validation of at least one antibody P- values (one-tailed) were determined with Fisher’s exact test and adjusted using the Holm-Bonferroni cor- rection. Full-size DOI: 10.7717/peerj.9300/fig-5 reporting antibody (validation) information for all primary antibodies that they used as a more robust measure of antibody reporting quality. Validation of all primary antibodies antibodies after the introduction of guidelines, compared to 0% in journals that did not implement guidelines (Neuroscience + Science), a difference of 46 percentage points (OR = INF, 95% CI = 6.5-INF; adjusted p = .0007, one-tailed). While a difference in percentage was already present before the introduction of guidelines in 2003, this difference was much smaller (10 percentage points, OR = 1.96, 95% CI = 0.43–10.3; adjusted p = 1, two-tailed). At the journal level, there was a large difference of 64 percentage points (OR = INF, 95% CI = 5.1-INF, adjusted p = .01, one-tailed) in validation reporting between the journals JCN and Neuroscience after June 2017. Between Nature and Science there was a difference in reporting of 29 percentage points (OR = INF, 95% CI = 1.09-INF; adjusted p = 1, one-tailed) after Nature had introduced guidelines, a difference that was not present before. Validation of all primary antibodies First, we compared the percentage of articles that report validation information on all the primary antibodies used per study. This percentage increased between 2003 and 2017 in both journals (JCN + Nature) that implemented reporting guidelines (see Fig. 5 and Tables S4 and S5). On average, this percentage increased by 23 percentage points in the journals that had implemented guidelines (OR = 2.80, 95% CI 0.96-INF; adjusted p = 1, one-tailed). In contrast, the percentage of articles with validation information on all primary antibodies decreased in both journals without guidelines (Science + Neuroscience). Furthermore, there was no meaningful increase in the total sample (18% to 22%, OR = 1.28, 95% CI = 0.48–3.52; adjusted p = 1, two-tailed). At the journal level, there was an increase of 24 percentage points (OR = 2.61, 95% CI = 0.60-INF, adjusted p = 1, one-tailed) in articles that reported validation information on all primary antibodies in JCN and an increase of 22 percentage points in Nature (OR = 5.3, 95% CI = 0.59-INF; adjusted p = 1, one-tailed). By comparing the journals with guidelines to the journals without guidelines, we can see that the difference in reporting between these similar journals in our sample substantially increased after the introduction of guidelines. After June 2017, 46% of the articles in journals that did implement guidelines (JCN + Nature) reported validation of all primary Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 13/26 antibodies after the introduction of guidelines, compared to 0% in journals that did not implement guidelines (Neuroscience + Science), a difference of 46 percentage points (OR = INF, 95% CI = 6.5-INF; adjusted p = .0007, one-tailed). While a difference in percentage was already present before the introduction of guidelines in 2003, this difference was much smaller (10 percentage points, OR = 1.96, 95% CI = 0.43–10.3; adjusted p = 1, two-tailed). At the journal level, there was a large difference of 64 percentage points (OR = INF, 95% CI = 5.1-INF, adjusted p = .01, one-tailed) in validation reporting between the journals JCN and Neuroscience after June 2017. Between Nature and Science there was a difference in reporting of 29 percentage points (OR = INF, 95% CI = 1.09-INF; adjusted p = 1, one-tailed) after Nature had introduced guidelines, a difference that was not present before. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 Identiﬁcation of all primary antibodies In addition to looking at antibody validation, we also evaluated how well antibody identification information was reported for all primary antibodies used in a study. In addition to looking at antibody validation, we also evaluated how well antibody identification information was reported for all primary antibodies used in a study. Antibodies are considered identifiable if enough information is provided in the publication for readers to be able to track down the antibody and obtain it themselves. In order to do so, either an RRID or the name, supplier and catalogue number of a commercial antibody needs to be reported. For non-commercial antibodies either an RRID or the host-animal and immunogen used needs to be reported. Figure 6 and Tables S6 and S7 show the percentage of articles that reports identification information on all primary antibodies that were used in the article. The percentage of articles that reported identification information on all primary antibodies increased considerably between 2003 and 2017 in journals that implemented antibody guidelines. Overall, the percentage of articles that reported identification information on all primary antibodies increased with 58 percentage points from 10% to 68% (OR = 17.8 95% CI = 4.8-INF; adjusted p = .0003, one-tailed) in journals with guidelines (JCN + Nature). Of the separate journals, JCN showed the most improvement in reporting. After the implementation of guidelines, its percentage increased from 20% to 93% of articles that use antibodies (OR = 45.2, 95% CI = 5.5-INF; adjusted p = .003, one-tailed). In the journals Neuroscience and Science this percentage also increased, but the increase was much smaller than in the journals with guidelines. Overall, the percentage of articles reporting identification information on all primary antibodies increased with 18 percentage points from 10% to 28% (OR = 3.36, 95% CI = 0.70–22.1; adjusted p = 1, two-tailed) in journals without guidelines (Neuroscience + Science). Apart from that, the overall reporting of identification information improved over time. For all journals combined this percentage increased by 37 percentage points (OR = 7.9, 95% CI = 2.81–26.3; adjusted p = .0005, two-tailed). This suggests that other factors such as an increase in awareness, the availability of identifying information, or a changing view on the importance of antibody reporting by the scientific community might also play a role in how well antibody identification information is reported. Hoek et al. Identiﬁcation of all primary antibodies (2020), PeerJ, DOI 10.7717/peerj.9300 14/26 Figure 6 Change in percentage of articles that reported identification information on all primary an- tibodies used in the study before (2003) and after (2017) two of the journals (JCN and Nature) imple- mented antibody reporting guidelines. Sample size: with guidelines 2003 n = 30, 2017 n = 28; without guidelines 2003 n = 30, 2017 n = 29; total all journals 2003 n = 60, 2017 n = 57; JCN 2003 n = 15, 2017 n = 15; Neuroscience 2003 n = 15, 2017 n = 15; Nature 2003 n = 15, 2017 n = 13; Science 2003 n = 15, 2017 n = 14. P-values were determined with Fisher’s exact test and adjusted using the Holm-Bonferroni correction. 1two-tailed p-values, other p-values are one-tailed. Full-size DOI: 10.7717/peerj.9300/fig-6 Figure 6 Change in percentage of articles that reported identification information on all primary an- tibodies used in the study before (2003) and after (2017) two of the journals (JCN and Nature) imple- mented antibody reporting guidelines. Sample size: with guidelines 2003 n = 30, 2017 n = 28; without guidelines 2003 n = 30, 2017 n = 29; total all journals 2003 n = 60, 2017 n = 57; JCN 2003 n = 15, 2017 n = 15; Neuroscience 2003 n = 15, 2017 n = 15; Nature 2003 n = 15, 2017 n = 13; Science 2003 n = 15, 2017 n = 14. P-values were determined with Fisher’s exact test and adjusted using the Holm-Bonferroni correction. 1two-tailed p-values, other p-values are one-tailed. Full-size DOI: 10.7717/peerj.9300/fig-6 Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 Change in type of validation If an article reported validation of at least one antibody, the types of validation that were carried out in the article were further specified. An article can contain multiple types of antibody validation, since one antibody can be validated in multiple ways, or multiple antibodies can be validated by different methods. First, a distinction was made between validation by the authors of the paper themselves and validation by means of a reference to the literature, to the information of the supplier, or to validation information in a database. Validation by the authors implies an experimental check in the laboratory and involves material verification, while the other forms rely on documentation. Reference to the supplier implies a reliance on supplier information about validity, reference to the literature implies a documentation of similar use of these antibodies by other researchers, which is also possible via reference to a database. Example of validation by the authors of an evaluated paper: • Independent antibody strategies of five pillars. ‘‘Our immunostaining in the mouse embryo cerebellum produced a similar labeling pattern as the other FoxP2 antibody’’ (Vibulyaseck et al., 2017). • Independent antibody strategies of five pillars. ‘‘Our immunostaining in the mouse embryo cerebellum produced a similar labeling pattern as the other FoxP2 antibody’’ (Vibulyaseck et al., 2017). Examples of validation by reference to a third party from the evaluated articles: Examples of validation by reference to a third party from the evaluated articles: Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 15/26 • Reference to supplier. ‘‘The rabbit polyclonal anti - FoxP2 antibody (AP5753b, Abgent, San Diego, California, CA, USA) produced a single major band of 80 kDa in the mouse heart tissue lysates in the manufacturer’s western blot analysis.’’ (Vibulyaseck et al., 2017). • Reference to the literature. ‘‘The goat polyclonal anti - EphA4 antibody (AF641, R&D Systems, Minneapolis, MN, USA) recognized a single band of 110 kDa in HEK293 cell lysate transfected with the EphA4 gene by western blot (Hashimoto et al., 2012). Its im- munohistochemical reactivity has been eliminated by preabsorption with the recombinant EphA4 protein (Rosas et al., 2011)’’ (Vibulyaseck et al., 2017). Figure 7 and Tables S8 and S9 show the changes in the prevalence of these types of validation before and after the introduction of guidelines in articles that contain at least one validated antibody. The percentage of articles that use validation by the authors seems extremely high. Change in type of validation It went from 87% to 85% in journals without guidelines and from 94% to 94% in journals with guidelines between 2003 and 2017. However, these percentages might be misleading. These high percentages were often caused by a small number of validated antibodies per article, which is explained by our methodology of evaluating validation per article instead of per antibody. Moreover, this method of validation is often implicitly carried out as a consequence of the experimental method being used. Since the reliability of the rating of this category is questionable (κ = −0.20), we did not perform further statistical analysis on this category. Both the use of the validation methods ‘reference to the literature’ and ‘reference to the antibody supplier’ increased in journals with guidelines (JCN + Nature) after the introduction of these guidelines. These methods increased by 24 percentage points (OR = 2.62, 95% CI = 0.86-INF; adjusted p = 1, one-tailed) and 27 percentage points (OR = INF, 95% CI = 1.96-INF; adjusted p = 0.4, one-tailed) respectively. Before guidelines were installed, there was a small difference in the prevalence of these validation types between journals with and without guidelines. This difference increased after the introduction of guidelines. Before the introduction of guidelines, there was a difference of 13 percentage points (OR = 1.99, 95% CI = 0.36–14.4; adjusted p = 1, two-tailed) in validation by reference to the literature between journals with and without guidelines. After the introduction of guidelines, this difference increased to 43 percentage points (OR = 8.3, 95% CI = 1.8-INF; adjusted p = 0.3, one-tailed). Similarly, there was no difference in the use of validation by reference to the antibody supplier before the introduction of guidelines. However, after guidelines had been installed, there was a difference of 27 percentage points (OR = INF, 95% CI = 1.34-INF; adjusted p = 1, one-tailed) between journals with and without guidelines. It is notable that validation by reference to information provided in an online antibody database is not used at all. Change in type of validation carried out by authors of paper If an antibody was experimentally validated by the article’s authors, we further specified what type of validation was carried out. Figure 8 and Tables S8 and S9 show an overview of the change in reported validation types. Figure 8 contains methods that are mostly used to validate antibodies explicitly. Other types of (often more implicit) validation, Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 16/26 Figure 7 Change in percentage of validated articles reporting different types of validation before (2003) and after (2017) the journals with guidelines implemented antibody reporting guidelines. Sample size (identical for all types of validation unless stated otherwise): with guidelines 2003 n = 23, 2017 n = 27; without guidelines 2003 n = 16, 2017 n = 16; exceptions: reference validation literature with guidelines 2003 n = 22; reference validation supplier with guidelines 2017 n = 26. P-values (one-tailed) were determined with Fisher’s exact test and adjusted using the Holm-Bonferroni correction. Full-size DOI: 10.7717/peerj.9300/fig-7 Figure 7 Change in percentage of validated articles reporting different types of validation before (2003) and after (2017) the journals with guidelines implemented antibody reporting guidelines. Sample size (identical for all types of validation unless stated otherwise): with guidelines 2003 n = 23, 2017 n = 27; without guidelines 2003 n = 16, 2017 n = 16; exceptions: reference validation literature with guidelines 2003 n = 22; reference validation supplier with guidelines 2017 n = 26. P-values (one-tailed) were determined with Fisher’s exact test and adjusted using the Holm-Bonferroni correction. Full-size DOI: 10.7717/peerj.9300/fig-7 such as positive and negative control, were also registered but not taken into account for comparisons because of low interrater reliability. Examples of validation from the evaluated articles: • Pre-adsorption. ‘‘In control experiments, primary antibody was pre-adsorbed with rat ANP (Bachem-Peninsula Laboratories; 1 µM overnight at 4 ◦C) prior to incubation with tissue sections to assess the degree of non-specific staining. Under these conditions, low levels of background staining were observed in cortex’’ (Wiggins, Shen & Gundlach, 2003). • Spatial localization. ‘‘(...) a polyclonal antisera against rat ANP were used in an attempt to visualize ANP-like-IR in the cerebral cortex and to determine the effect of CSD on its level and cellular distribution. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 Change in type of validation carried out by authors of paper Consistent with previous reports, ANP-like-IR was consistently detected in subcortical regions, with a high density of nerve-fibre staining found throughout areas such as the bed nucleus of the stria terminalis (...), the preoptic hypothalamus, areas of the amygdala and in the paraventricular thalamic nucleus (see Kawata et al., 1985, Skofitsch et al., 1985)’’ (Wiggins, Shen & Gundlach, 2003). Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 17/26 Figure 8 Change in percentage of validated articles reporting different types of validation before (2003) and after (2017) the journals with guidelines implemented antibody reporting guidelines. Sample size (identical for all types of validation): with guidelines 2003 n = 20, 2017 n = 23; without guidelines 2003 n = 15, 2017 n = 15. P-values were determined with Fisher’s exact test and adjusted using the Holm-Bonferroni correction. All adjusted p-values are 1. Full-size DOI: 10.7717/peerj.9300/fig-8 Figure 8 Change in percentage of validated articles reporting different types of validation before (2003) and after (2017) the journals with guidelines implemented antibody reporting guidelines. Sample size (identical for all types of validation): with guidelines 2003 n = 20, 2017 n = 23; without guidelines 2003 n = 15, 2017 n = 15. P-values were determined with Fisher’s exact test and adjusted using the Holm-Bonferroni correction. All adjusted p-values are 1. Full-size DOI: 10.7717/peerj.9300/fig-8 Figure 8 Change in percentage of validated articles reporting different types of validation before (2003) and after (2017) the journals with guidelines implemented antibody reporting guidelines. Sample size (identical for all types of validation): with guidelines 2003 n = 20, 2017 n = 23; without guidelines 2003 n = 15, 2017 n = 15. P-values were determined with Fisher’s exact test and adjusted using the Holm-Bonferroni correction. All adjusted p-values are 1. Full-size DOI: 10.7717/peerj.9300/fig-8 • Secondary antibody without primary. ‘‘Additional control incubations with primary antibody omitted were routinely included and resulted in a lack of specific staining’’ (Wiggins, Shen & Gundlach, 2003). • Secondary antibody without primary. ‘‘Additional control incubations with primary antibody omitted were routinely included and resulted in a lack of specific staining’’ (Wiggins, Shen & Gundlach, 2003). • Molecular weight in western blot (and KO). ‘‘The rat monoclonal anti - Pcdh10 antibody (MABT20 clone 5G10, Millipore, Billerica, MA, USA) produced a single band of 137 kDa in the P0 mouse cerebellum and whole brain in our western blot. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 Change in type of validation carried out by authors of paper The band disappeared completely in samples from Pcdh10 - KO mice (OL - KO homozygotes)’’ (Vibulyaseck et al., 2017). • Molecular weight in western blot (and KO). ‘‘The rat monoclonal anti - Pcdh10 antibody (MABT20 clone 5G10, Millipore, Billerica, MA, USA) produced a single band of 137 kDa in the P0 mouse cerebellum and whole brain in our western blot. The band disappeared completely in samples from Pcdh10 - KO mice (OL - KO homozygotes)’’ (Vibulyaseck et al., 2017). Before the introduction of guidelines, all types of validation were used more often in journals without guidelines. However, after the introduction of guidelines, the use of these validation methods decreased in these journals without guidelines while there was a slight, but nonsignificant, increase of their use in the journals with guidelines. This means that the differences in the types of validation that were mostly used increased between journals with guidelines and similar journals without guidelines. After the introduction of guidelines, there was a difference of 10 percentage points in the use of pre-adsorption (OR = 2.87, 95% CI = 0.33-INF; adjusted p = 1, one-tailed), 23 percentage points in the use of spatial localization (OR = 5.9, 95% CI = 0.80-INF; adjusted p-value=1, one-tailed), 32 percentage points in the use of a secondary antibody without a primary one (OR = 8.6, 95% CI = Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 18/26 1.22-INF; adjusted p = 1, one-tailed), and 10 percentage points in the use of molecular weight in western blot (OR = 1.73, 95% CI = 0.39-INF; adjusted p = 1, one-tailed) between similar journals. Interestingly, there is a difference in how often these validation types are used in the two journals that introduced guidelines. In the journal Nature, the methods pre-adsorption and spatial localization were not used at all before and after the introduction of guidelines. The small increase in reporting of spatial localization is thus solely caused by its use in the journal JCN. DISCUSSION These results suggest that journal guidelines might have some positive influences on antibody validation reporting. For example, this is suggested by the large difference in the percentage of articles reporting validation information of all primary antibodies between journals with and without guidelines after the introduction of these guidelines. However, it is notable that in most of our analyses a difference in reporting was already present between the four journals before the introduction of guidelines. This could suggest that the journal itself (e.g., its editorial attention to validation, or earlier publications about validation), regardless of the presence of reporting guidelines, already has an influence on antibody validation reporting. Another explanation might be that there is a difference in validation practices and journal selection between research areas or groups. Moreover, our results provide some insight in how journal guidelines might work. This can be illustrated by comparing the increase in reporting of validation information with identification information of all primary antibodies. On average, the reporting of identification information improved by 58 percentage points, while the reporting of validation information only improved by 23 percentage points in journals that introduced guidelines. This suggest that the introduction of antibody reporting guidelines particularly affected aspects of antibody reporting that are relatively easy to change. Adding identification information for each antibody used in your publication, such as catalogue numbers, is easier than providing evidence of actual validation. From an editorial perspective, the provision of identity information such as catalogue numbers is also easier to check. At least for some of the articles, researchers seem to have opted for the easier solutions to comply with antibody guidelines by providing readily available information. A similar indication for solutions that choose the ‘path of least resistance’ to comply with guidelines, can be found in the use of third-party validation information. Once again, this information is relatively easy to include in a paper with a simple reference and requires no extra experimental work. Our results show that validation by reference to the literature increased by 24 percentage points in journals that introduced guidelines, while this percentage decreased in journals without guidelines. Likewise, validation by reference to the antibody supplier’s information increased by 27 percentage points in journals with guidelines, while this method was never used in journals without guidelines. These results are not significant, but the odds ratios seem to suggest (especially in the case of reference Hoek et al. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 DISCUSSION (2020), PeerJ, DOI 10.7717/peerj.9300 to supplier’s information) that these validation practices merit further investigation. More research assessing how researchers regard journal guidelines, how these guidelines affect their practices, and a replication of this study with a larger sample size is recommended. Because the guidelines mostly seem to influence aspects of antibody reporting that are relatively easy to provide, we concluded that the effects of journal guidelines on antibody reporting are limited. This limited effect of journal guidelines is consistent with findings in similar research on journals’ measures to improve reporting about research resources. A recent evaluation of the 2013 Nature life science checklist shows that it improved the reporting of some items, but also that approximately half of the compliant articles did not actually use the tools recommended by the checklist (Han et al., 2017). Another evaluation of the Nature checklist showed improvements in transparency of reported information, but much more modest improvements in experimental design. Furthermore, the study found that the checklist did not improve antibody reporting (NPQIP Collaborative group, 2019). However, with regard to antibody identifiability, our results are more optimistic than previous studies. Vasilevsky et al. found no relationship between reporting guidelines and identifiability of resource materials in biomedical research. Identifiability of resources was actually higher in journals with no or loose guidelines than in journals with strict guidelines. Antibody identifiability in the Journal of Comparative Neurology was only slightly higher than average across all journals (Vasilevsky et al., 2013). While our results point towards some improvements, it is clear that journal guidelines are not a definitive solution to fix validation and misidentification problems. Much seems to depend on how journal guidelines are implemented. Mere symbolic support for guidelines in editorial instructions may not be enough to improve reporting practices. Baker et al. found that the endorsement of the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines by journals had little to no impact the reporting of animal studies in these journals (Baker et al., 2014). Hair et al. (2019) found that even requiring authors to complete an ARRIVE checklist after submission of their manuscript did not improve compliance. In another study of journal guidelines regarding RRIDs, compliance was the lowest when the journals only provided author instructions. Compliance became higher when authors received mailed instructions, and it became very high when editorial staff assisted authors with their RRIDs (Bandrowski et al., 2016). Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 Limitations Our study has several noteworthy limitations. As an exploratory study, our study was not pre-registered, which we would recommend for possible future confirmatory replications of our work. Furthermore, the labour-intensive nature of scrutinising publications for validation information means our sample and interrater testing are limited. Together with a conservative statistics test (Fisher’s exact), this might have increased our Type II error. Having a low power can have several consequences for the reliability of results, such as an overestimation of the effect size. Readers of our study should be cautious of this limitation while interpreting our results. In addition, coding of articles inevitably implies some level of arbitrary convention and interpretation, for example as we decided to code on the level of articles and not individual antibodies. No distinction was made between implicit and explicit validation, giving the researchers the benefit of the doubt with respect to their validation efforts, which may overestimate the beneficial effect of guidelines and how often certain validation methods are used. The room for interpretation about whether specific forms of experimental validation were actually used, is also illustrated by the lower agreement between raters in this respect. This again means that our results should be regarded with caution and a replication of this study with a larger sample size and multiple assessors is recommended. Because of these limitations, we tried to be as open as possible about the decisions we made during the coding process, by illustrating these choices with examples from the evaluated articles in this manuscript, and by providing an elaborate explanation of our choices in the coding table. Last, our pseudo-experimental research design has some limitations. Because of the substantial time period from which we took our samples (2003 and 2017), the time between the intervention of implementing the guidelines and the measurement of changed behaviour is stretched. We chose this approach because of the implementation of the guidelines by the two journals at different times. However, another way to approach this could be by sampling over time, for example by using interrupted time series analysis. Moreover, the quasi-experimental design makes the effect of journal guideline introduction hard to isolate from wider influences, such as a growing awareness of the urgency of antibody validation, or the growing knowledge stored in databases such as the Antibodyregistry. DISCUSSION These results suggest that journal guidelines may have a beneficial effect, but that they require additional measures to make them effective. Another interesting observation from our study is that, in contrast to the reporting of antibody validation, the reporting of information on the identity of all primary antibodies increased in all four journals between 2003 and 2017. Moreover, this percentage increased significantly by 37 percentage points in the total sample of all four journals combined. Although the improvement was larger in journals that introduced guidelines, it is likely that this improvement is at least partly caused by a growing awareness of antibody problems. That this growing awareness only seems to have an effect on improving antibody identification reporting and not on antibody validation reporting might be due to the current attention for reproducibility in science. Although antibody validity is important for the overall validity of research, detailed reporting of which antibodies were used is Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 20/26 important to ensure reproducibility. However, as we have argued, reproducible research is not necessarily valid research. We would therefore urge not to forget the validity question in the replication crisis debate. Furthermore, it would be interesting to study researchers’ motivation for this change in reporting behaviour to see whether and how it relates to the reproducibility crisis. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 CONCLUSIONS This study suggests that the implementation of antibody reporting guidelines by a journal might have some positive influences on the reporting of antibody validation information, as we hypothesized. The percentage of validated antibodies per article, the percentage of articles which reported validation and identification of all primary antibodies, and different types of validation all increased in journals with guidelines after the introduction of these guidelines. However, improvements were particularly visible for forms of antibody information that are relatively easy to provide, such as providing complete identification information. Strictly speaking, such information supports the reliability of antibodies, and it is conceivable that securely identified and previously validated antibodies can be used reliably in identical applications. Nevertheless, improvements of more robust experimental validation were modest, at best. Combined with the results of previous studies on journal guidelines, this suggests that the effect of journal antibody guidelines by themselves may be limited and may require additional measures to ensure effective implementation. In light of the quasi-experimental and exploratory nature of our study, we need to be careful with drawing these conclusions. Other factors, such as a general shift in research practices, a change in publication behavior, a preference for publishing in a journal that adheres to the values of a researcher, or changed editorial practices might also play a role. More research would therefore be needed to study how researchers make decisions with regard to antibody validation and how they adjust their research practices in response to changing journal guidelines. Limitations Fortunately, our results do show signs of an overall growing awareness of antibody problems, since the reporting of antibody identification information significantly improved in the entire sample. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 ACKNOWLEDGEMENTS We would like to thank Jeffrey Stuart, Nicole Vasilevsky, Malcolm MacLeod and one anonymous reviewer for their constructive feedback on our manuscript; Ger Pruijn, Serge Horbach, Freek Oude Maatman, Merle-Marie Pittelkow, Jasmine Muradchanian and Ymkje Anna de Vries for comments; and the Research Quality Team for inspiring discussions. Competing Interests The authors declare there are no competing interests. Funding The authors received no funding for this work. The authors received no funding for this work. Competing Interests ADDITIONAL INFORMATION AND DECLARATIONS Funding Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 Data Availability The following information was supplied regarding data availability: The following information was supplied regarding data availability: We provide our data and R code in the supplementary files. Our data can also be found at 10.17026/dans-xhk-74m4. Author Contributions • Joyce M. Hoek conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. • Joyce M. Hoek conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. • Wytske M. Hepkema performed the experiments, authored or reviewed drafts of the paper, and approved the final draft. • Wytske M. Hepkema performed the experiments, authored or reviewed drafts of the paper, and approved the final draft. Hoek et al. (2020), PeerJ, DOI 10.7717/peerj.9300 22/26 • Willem Halffman conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft. • Willem Halffman conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft. Data Availability Data Availability Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.9300#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.9300#supplemental-information. REFERENCES Changing order: replication and induction in scientific practice. London: SAGE Publications. Crane D. 1967. The gatekeepers of science: some factors affecting the selection of articles for scientific journals. The American Sociologist 2:195–201. Freedman LP, Cockburn IM, Simcoe TS. 2015. The economics of reproducibility in preclinical research. 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https://openalex.org/W4391448086	https://www.nature.com/articles/s41598-024-51886-y.pdf	English	null	Near-infrared spectroscopy for kidney oxygen monitoring in a porcine model of hemorrhagic shock, hemodilution, and REBOA	Scientific reports	2,024	cc-by	10,192	Near‑infrared spectroscopy for kidney oxygen monitoring in a porcine model of hemorrhagic shock, hemodilution, and REBOA Natalie A. Silverton 1,2, Lars R. Lofgren 1, Kai Kuck 1, Gregory J. Stoddard 3, Russel Johnson 4, Ali Ramezani 1 & Guillaume L. Hoareau 5 OPEN Acute kidney injury is a common complication of trauma and hemorrhagic shock. In a porcine model of hemorrhagic shock, resuscitative endovascular balloon aortic occlusion (REBOA) and hemodilution, we hypothesized that invasive kidney oxygen concentration measurements would correlate more strongly with noninvasive near infra-red spectroscopy (NIRS) oxygen saturation measurements when cutaneous sensors were placed over the kidney under ultrasound guidance compared to placement over the thigh muscle and subcutaneous tissue. Eight anesthetized swine underwent hemorrhagic shock 4 of which were resuscitated with intravenous fluids prior to the return of shed blood (Hemodilution protocol) and 4 of which underwent REBOA prior to resuscitation and return of shed blood (REBOA protocol). There was a moderate correlation between the NIRS and kidney tissue oxygen measurements (r = 0.61 p < 0.001; r = 0.67 p < 0.001; r = 0.66 p < 0.001for left kidney, right kidney, and thigh NIRS respectively). When the animals were separated by protocol, the Hemodilution group showed a weak or nonsignificant correlation between NIRS and kidney tissue oxygen measurements (r = 0.10 p < 0.001; r = 0.01 p = 0.1007; r = 0.28 p < 0.001 for left kidney, right kidney, and thigh NIRS respectively). This contrasts with the REBOA group, where left and right kidney as well as thigh NIRS were moderately correlated with kidney tissue oxygen (r = 0.71 p < 0.001; r = 0.74 p < 0.001; r = 0.70 p < 0.001; for left kidney, right kidney, and thigh NIRS respectively). There was a strong correlation between both kidney NIRS signals and thigh NIRS measurements (r = 0.85 p < 0.001; r = 0.88 p < 0.001;for left kidney vs thigh and right kidney vs thigh respectively). There was also a strong correlation between left and right kidney NIRS (r = 0.90 p < 0.001). These relationships were maintained regardless of the resuscitation protocol. These results suggest that kidney NIRS measurements were more closely related to thigh NIRS measurements than invasive kidney tissue oxygen concentration. Trauma is the leading cause of death for Americans under the age of 46 and is the third leading cause across all ages, accounting for approximately 100,000 deaths per year1,2. Hemorrhage is responsible for 30–40% of trauma mortality in the United States3. Hemorrhage that does not immediately lead to the death of a trauma victim, how- ever, often results in acute kidney injury (AKI). www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ other organs below the balloon, such as the kidneys. While a life-saving measure, the impact of prolonged bal- loon inflation on kidney hypoxia may be profound. REBOA also provides a unique opportunity to provide a translational model of survivable and profound renal ischemia-injury with direct clinical correlates. A real-time bedside monitor of kidney oxygenation during hemorrhage and REBOA might improve the care of critically ill trauma patients by allowing for early diagnosis of kidney hypoxia. p y g y g y yp Near-infrared spectroscopy (NIRS) is a noninvasive technology used to determine regional tissue oxygen satu- ration. NIRS relies on a sensor attached to the skin that emits light in the near-infrared spectrum (700–900 nm). This wavelength of light can penetrate underlying tissues and is within the range of absorption of oxygenated and deoxygenated hemoglobin. While pulse oximetry measures arterial hemoglobin saturation, NIRS measures a mixture of arterial and venous capillary oxygenation, making it well-suited to determine tissue oxygen delivery and perfusion5. NIRS is routinely used to measure the capillary oxygen saturation of the brain’s frontal lobe when patients are undergoing cardiac or vascular surgery. The NIRS sensor consists of a single light emitter and two light sensors at various distances from the emitter. The depth of penetration of NIRS is related to the distance from the light emitter to the detector. Subtracting the closer detector signal from that of the far detector, in theory, allows for measurements that are specific to the underlying brain tissue rather than the superficial skin or the skull6,7. Cerebral oximetry measured with NIRS correlates with jugular venous oxygen saturation suggesting that this noninvasive technology reflects tissue oxygenation8. A large body of literature describes the use of NIRS in neonates and infants both as a systemic perfusion monitor and a regional monitor of kidney perfusion5,9–13. The maximum depth of penetration of NIRS, however, is thought to be only 2.5 cm, thereby limiting the use of NIRS as a kidney perfusion monitor in adults14–16. Recently, however, two studies in adult cardiac surgery patients using ultrasound guided placement of NIRS sensors over the kidney found that intraoperative regional tissue oxygen desaturations were associated with the subsequent development of AKI17,18. A third study found a correlation between NIRS oxygen saturation measurements over the kidney and renal venous oxygen saturation in adult cardiac surgery patients19. www.nature.com/scientificreports/ In all three studies, patients were excluded for body mass index > 30 kg/m2 or if the surface of their kidney was > 4 cm from the skin. y We have developed a porcine model of hemorrhage and REBOA in which we measure kidney oxygen con- centration directly by placing an oxygen sensor in the renal medulla20. Using this oxygen sensor, we can detect renal hypoxia during the hemodynamic changes that occur with hemorrhage and REBOA. We designed the current study to determine whether invasive kidney tissue oxygen concentration correlates with noninvasive NIRS oxygen saturation when these sensors are placed on the skin over the kidney using ultrasound guidance. REBOA creates a profound ischemic condition, leading to severe vasodilation and hemodynamic instability even after resuscitation with whole blood21–23. We, therefore, created a second model of hemorrhage and hemodilution to assess the relationship between invasive and noninvasive kidney oxygen monitoring in the setting of more subtle hemodynamic changes that may apply to a broader patient population. We hypothesized that measure- ments from NIRS sensors placed on the skin over the kidney would correlate more strongly with direct medullar kidney oxygen measurements than those obtained from NIRS sensors placed on the skin over the thigh. We also hypothesized that this relationship would hold true under both the extreme hemodynamic instability that occurs with REBOA and the less severe changes that occur with hemorrhage and hemodilution. Methods Overviewh The animal research: reporting of in vivo experiments guidelines (ARRIVE) were used to prepare this manu- script. The Institutional Animal Care and Use Committee at the University of Utah approved this study (Protocol number 00001819; Approved 10/28/2021) and all experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act Regulations and Standards. An overview of the experimental protocols can be seen in Fig. 1. Animal preparation p p Ten castrate male Yorkshire swine or non-pregnant females weighing 53–60 kg and 6–8 months old were accli- matized in temperature- and light-controlled enclosures with given access to environmental enrichment for at least 7 days. Animals were fasted overnight but allowed free access to water prior to induction of anesthesia. Anesthesia was induced with a combined intramuscular injection of ketamine (2.2 mg/kg, Vedco, Saint Joseph, MO) and xylazine (2.2 mg/kg, Vedco, Saint Joseph, MO) and after endotracheal intubation, anesthesia was maintained at 1–2.5% isoflurane (Dechra, Northwich, United Kingdom) in 2 L/min of oxygen. Animals were mechanically ventilated with a positive end-expiratory pressure of 4 cm H2O and a fraction of inspired oxygen between 40 and 100% to maintain pulse oximetry > 95%. Tidal volumes of 6–8 ml/kg were used with the respira- tory rate adjusted to maintain end-tidal CO2 of 35–45 mmHg. Balanced isotonic fluids (Plasmalyte 148, Baxter, Deerfield, IL) were administered at 5 ml/kg/hr intravenously as maintenance fluid. Ionized calcium, potassium, and glucose were monitored throughout the experiment and treated as needed. Warming blankets were used to maintain normothermia. Near‑infrared spectroscopy for kidney oxygen monitoring in a porcine model of hemorrhagic shock, hemodilution, and REBOA Natalie A. Silverton 1,2, Lars R. Lofgren 1, Kai Kuck 1, Gregory J. Stoddard 3, Russel Johnson 4, Ali Ramezani 1 & Guillaume L. Hoareau 5 OPEN This complication occurs in 43% of critically injured individuals and is associated with significant increases in mortality4. Reduced oxygen delivery to the kidneys secondary to bleeding and hypovolemia is the cornerstone of AKI after critical injury. g ypt j y Resuscitative Endovascular Balloon Aortic Occlusion (REBOA) is a potential therapy to treat patients with severe injuries and profound blood loss due to non-compressible torso hemorrhage. A balloon-tipped catheter is inserted through the femoral artery and positioned in the aorta. The balloon can then be inflated to maintain blood flow to the major organs of the upper body, such as the heart and brain, at the expense of blood flow to 1Department of Anesthesiology, University of Utah, Helix Building 5050, 30N Mario Capecchi Dr., Salt Lake City, UT 84132, USA. 2Geriatric Research, Education and Clinical Center, VA Medical Center, 500 Foothill Dr, Salt Lake City, UT 84148, USA. 3Division of Epidemiology, Department of Internal Medicine, University of Utah, 295 Chipeta Way, Rm 1N433, Salt Lake City, UT 84132, USA. 4Department of Emergency Medicine, Helix Building 5050, 30 N Mario Capecchi, Room 2S240, Level 2, South, Salt Lake City, UT 84132, USA. 5Department of Emergency Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, Helix Building 5050, 30 N Mario Capecchi, Room 2S240, Level 2, South, Salt Lake City, UT 84132, USA. *email: natalie.silverton@hsc.utah.edu \| https://doi.org/10.1038/s41598-024-51886-y Scientific Reports \| (2024) 14:2646 www.nature.com/scientificreports/ Surgical preparation Lidocaine was used for local infiltration. Percutaneous vascular access was established with ultrasound guid- ance and the Seldinger technique in the following locations. A 6 Fr carotid artery catheter was placed for upper body blood pressure monitoring. A 7 Fr brachial artery catheter was placed for controlled hemorrhage and blood sampling. A 12 Fr femoral artery catheter was inserted to introduce a custom-made 7 Fr REBOA catheter (Custom-made, Certus Critical Care). This femoral artery catheter was also used for lower body blood pres- sure monitoring. In 4 animals, a REBOA balloon was positioned immediately superior to the diaphragm. This https://doi.org/10.1038/s41598-024-51886-y Scientific Reports \| (2024) 14:2646 \| www.nature.com/scientificreports/ ntificreports/ Figure 1. (a) Description of the Hemodilution protocol. Each animal underwent surgical instrumentation followed by 3 separate periods of hemorrhage where 250 ml of blood was removed from the brachial artery. Each hemorrhage period was followed a 500 ml bolus of balanced IV fluids. At the end of the 3 hemorrhage periods, all 750 ml of shed blood was returned to the animal. This was followed by a resuscitation period in which mean arterial pressure was maintained > 65 mmHg with fluid boluses and norepinephrine as needed. (b). Description of the REBOA protocol. Each animal underwent surgical instrumentation followed by a single hemorrhage period when 750 ml of blood was removed from the brachial artery. This was followed by a 30-min period of anemia. Resuscitative endovascular balloon occlusion of the aorta (REBOA) was then maintained for 45 min followed by gradual balloon deflation and simultaneous return of shed blood. This was followed by a resuscitation period in which mean arterial pressure was maintained > 65 mmHg with fluid boluses and norepinephrine as needed. Figure 1. (a) Description of the Hemodilution protocol. Each animal underwent surgical instrumentation followed by 3 separate periods of hemorrhage where 250 ml of blood was removed from the brachial artery. Each hemorrhage period was followed a 500 ml bolus of balanced IV fluids. At the end of the 3 hemorrhage periods, all 750 ml of shed blood was returned to the animal. This was followed by a resuscitation period in which mean arterial pressure was maintained > 65 mmHg with fluid boluses and norepinephrine as needed. (b). Description of the REBOA protocol. Each animal underwent surgical instrumentation followed by a single hemorrhage period when 750 ml of blood was removed from the brachial artery. Surgical preparation This was followed by a 30-min period of anemia. Resuscitative endovascular balloon occlusion of the aorta (REBOA) was then maintained for 45 min followed by gradual balloon deflation and simultaneous return of shed blood. This was followed by a resuscitation period in which mean arterial pressure was maintained > 65 mmHg with fluid boluses and norepinephrine as needed. placement was confirmed with fluoroscopy. A 9 Fr introducer catheter was placed in the internal jugular vein to deliver fluids and autologous blood and then a triple-lumen catheter was inserted through the sheath for central venous pressure monitoring and medication infusions. A 9 Fr introducer sheath was placed in a femoral vein for fluid bolus administration. A midline laparotomy was performed, and 20 Fr Foley catheter was placed in the bladder via a cystotomy. A splenectomy was performed to prevent auto-transfusion.ht y y p y p p The left kidney was dissected, a 20 g IV catheter was then placed in the renal medulla under ultrasound guidance, and a fiberoptic probe was threaded through the catheter until it was visualized in the renal medulla (Fig. 2a). This probe is a luminescent oxygen sensor and a Doppler-based flow sensor designed for oxygen and perfusion monitoring (Oxford Optronix, Abington, UK). The probe and IV catheter were then glued in place (Vetbond, 3 M, St Paul, MN). The left kidney was then returned to the retroperitoneal space. The abdomen was then closed until the end of the experiment. The surgical set-up described for this model has been previously published by our group20. p y g p Surface ultrasound was used to locate both kidneys, skin-to-kidney surface measurements were recorded, and NIRS sensors (Foresight, Edwards Lifesciences, Irvine, CA) were placed on the skin directly over the kidneys bilaterally (Fig. 2b). A NIRS sensor was also placed over the left thigh. These were Large ForeSight NIRS sensors (model number FSESL) and were connected to a Foresight Elite monitor (CAS Medical Systems, Branford, CT). The source-detector distances for these sensors are 15 mm and 50 mm for shallower and deeper tissue measure- ments respectively. Flank mode was used on the monitor for NIRS sensors placed on the skin over the left and right kidney and thigh mode was used for the NIRS sensors placed on the skin over the thigh. Hemorrhage and resuscitation protocols g p Before starting the experimental procedure, there was a stabilization period of at least 10 min. During this time, mean arterial pressure (MAP) was maintained at > 65 mmHg. If below that threshold, 5/ml/kg boluses of bal- anced isotonic crystalloid were given. After 2 boluses, an infusion of norepinephrine (starting dose 0.02 mcg/ kg/min) was titrated until target MAP was achieved. Animals were then randomly allocated into one of two hemorrhage and resuscitation protocols. Scientific Reports \| (2024) 14:2646 \| https://doi.org/10.1038/s41598-024-51886-y www.nature.com/scientificreports/ Figure 2. (a). Ultrasound image acquired by placing the linear transducer directly over surface of the left kidney. The 20 g IV can be seen in long axis with the tip of the oxygen sensor extending out into renal medullary tissue. (b). Ultrasound image acquired by placing the linear transducer over the skin of the left flank just below the last rib. Subcutaneous muscle and fat are seen overlying the surface of the kidney. This area was marked and a near infra-red spectroscopy sensor was placed on the skin in this location. Figure 2. (a). Ultrasound image acquired by placing the linear transducer directly over surface of the left kidney. The 20 g IV can be seen in long axis with the tip of the oxygen sensor extending out into renal medullary tissue. (b). Ultrasound image acquired by placing the linear transducer over the skin of the left flank just below the last rib. Subcutaneous muscle and fat are seen overlying the surface of the kidney. This area was marked and a near infra-red spectroscopy sensor was placed on the skin in this location. In the hemodilution protocol (Fig. 1a.), 250 ml of whole blood (approximately 7% of blood volume estimated as 60 mL/kg x body weight in kg) was removed from the brachial artery catheter over 15 min. Drawn blood was collected in citrated blood collection bags under constant agitation. Blood was stored in a warm water bath at 38 °C. At the end of each hemorrhage period, a 500 ml bolus of balanced isotonic crystalloid was given over 5 min. This procedure was repeated 3 times. After the third bolus of balanced isotonic crystalloid, all 750 ml (approximately 21% of blood volume) of shed blood were returned, and the animal was observed for 20–30 min. In the REBOA protocol (Fig. 1b), all 750 ml of blood were removed over 30 min. Hemorrhage and resuscitation protocols Animals then remained untreated for an additional 30 min, after which they were then subjected to 45 min of REBOA. At the end of the REBOA period, the balloon was slowly deflated over 10 min as the animals were transfused their shed blood. A critical care period ensued during which animals were resuscitated with intravenous balanced crystalloids and ( i h i ) di ifi d l i h (S l l Fi 1) f 255 i il In the hemodilution protocol (Fig. 1a.), 250 ml of whole blood (approximately 7% of blood volume estimated as 60 mL/kg x body weight in kg) was removed from the brachial artery catheter over 15 min. Drawn blood was collected in citrated blood collection bags under constant agitation. Blood was stored in a warm water bath at 38 °C. At the end of each hemorrhage period, a 500 ml bolus of balanced isotonic crystalloid was given over 5 min. This procedure was repeated 3 times. After the third bolus of balanced isotonic crystalloid, all 750 ml (approximately 21% of blood volume) of shed blood were returned, and the animal was observed for 20–30 min. pp y In the REBOA protocol (Fig. 1b), all 750 ml of blood were removed over 30 min. Animals then remained untreated for an additional 30 min, after which they were then subjected to 45 min of REBOA. At the end of the REBOA period, the balloon was slowly deflated over 10 min as the animals were transfused their shed blood. A critical care period ensued during which animals were resuscitated with intravenous balanced crystalloids and vasopressors (norepinephrine) according to a pre-specified algorithm (Supplemental Fig. 1) for 255 min until the end of the experiment. At the end of each experiment, the animal was euthanized. Data collection Upper body (carotid) and lower body (femoral) MAP were calculated from the continuous arterial blood pressure traces which were resampled to 1 Hz throughout the experiment. Renal medullary tissue oxygen concentra- tion was recorded using the Oxford Optronix luminescent oxygen probe and Doppler blood flow sensor. Data from these sensors were sampled every second (PowerLab data acquisition platform, ADInstruments, Colorado Springs, CO). Regional tissue oxygen saturation using NIRS was recorded over the kidneys bilaterally and the left thigh (CAS Medical Systems, Branford, CT). Data from the NIRS sensors was sampled every 2 s. Serum analysis y Arterial whole blood was sampled at baseline, after each hemorrhage period in the Hemodilution protocol, and every 30 min in the REBOA protocol. Plasma lactate concentration, hemoglobin concentration, blood gases and electrolytes concentrations were measured at these intervals (iStat, Abbott, Chicago, IL). Results ll In all animals, the surface of the kidney was between 1.5 and 2.0 cm from the skin. One animal in the REBOA protocol suffered a cardiac arrest immediately after surgical instrumentation and data could not be obtained. A second animal in the Hemodilution group was excluded from the analysis because the kidney oxygen sen- sor malfunctioned. The individual NIRS saturation and kidney oxygen concentration data for all 8 animals are presented in Fig. 3. One animal in the Hemodilution group only underwent two hemorrhage periods because the hemoglobin concentration decreased to 5.0 mg/dL after the second hemorrhage, and it was thought that a third hemorrhage might be fatal. Animals 1 and 4 in the hemodilution protocol appeared to have a temporal In all animals, the surface of the kidney was between 1.5 and 2.0 cm from the skin. One animal in the REBOA protocol suffered a cardiac arrest immediately after surgical instrumentation and data could not be obtained. A second animal in the Hemodilution group was excluded from the analysis because the kidney oxygen sen- sor malfunctioned. The individual NIRS saturation and kidney oxygen concentration data for all 8 animals are presented in Fig. 3. One animal in the Hemodilution group only underwent two hemorrhage periods because the hemoglobin concentration decreased to 5.0 mg/dL after the second hemorrhage, and it was thought that a third hemorrhage might be fatal. Animals 1 and 4 in the hemodilution protocol appeared to have a temporal g gt g , g third hemorrhage might be fatal. Results ll H FB H FB BR 0 20 40 60 80 Oxygen (% or mmHg) 0 20 40 60 80 Time (minutes) Animal 1 H FB H FB H FB BR 0 20 40 60 80 Oxygen (% or mmHg) 0 20 40 60 80 Time (minutes) Animal 2 H FB H FB H FB BR 0 20 40 60 80 Oxygen (% or mmHg) 0 20 40 60 80 Time (minutes) Animal 3 H FBH FBH FB BR 0 20 40 60 80 Oxygen (% or mmHg) 0 20 40 60 80 100 Time (minutes) Animal 4 Hemodilution Protocol H FB H FB BR 0 20 40 60 80 Oxygen (% or mmHg) 0 20 40 60 80 Time (minutes) Animal 1 H FB H FB H FB BR 0 20 40 60 80 Oxygen (% or mmHg) 0 20 40 60 80 Time (minutes) Animal 2 Hemodilution Protocol Time (minutes) Time (minutes) H FB H FB H FB BR 0 20 40 60 80 Time (minutes) Animal 3 H FBH FBH FB BR 0 20 40 60 80 Oxygen (% or mmHg) 0 20 40 60 80 100 Time (minutes) Animal 4 H AO AR 0 20 40 60 80 100 Oxygen (% or mmHg) 0 50 100 150 200 Time (minutes) Animal 5 250 H AO AR 0 20 40 60 80 100 Oxygen (% or mmHg) 0 50 100 150 200 250 Time (minutes) Animal 6 Animal 7 Animal 8 REBOA Protocol AO AR 50 100 150 200 Time (minutes) Animal 7 250 H AO AR 0 20 40 60 80 100 Oxygen (% or mmHg) 0 50 100 150 200 250 Time (minutes) Animal 8 dney NIRS Right Kidney NIRS Thigh NIRS Kidney Oxygen Figure 3. Left kidney, right kidney, and thigh near infra-red spectroscopy (NIRS) are compared to direct kidney tissue oxygen measurements over time for 4 animals undergoing the Hemodilution protocol. The red line marked “H” represents the start of each hemorrhage. The gray line marked “F” represents the end of each hemorrhage and the start of a bolus of balanced crystalloid. The orange line marked “BR” represents the time when shed blood was returned to the animal. For the resuscitative endovascular balloon aortic occlusion (REBOA) protocol, the red line marked “H” represents the start of the hemorrhage period. The gray line marked “AO” represents the start of aortic occlusion. Statistics Descriptive statistics were reported as mean (standard deviation). Mean arterial pressure data were collapsed to a single mean value for each animal for the three time periods reported (baseline, hemorrhage, and resuscitation). For the baseline and resuscitation characteristics, a Student’s t-test was used for continuous data and a Fisher exact test for categorical data. Our primary comparison was between direct kidney tissue oxygen concentration and the three NIRS measurements (left kidney, right kidney, and thigh). These data were compared using a “within subjects” correlation coefficient, which accounts for the lack of independence among repeated measures by removing the variation between subjects24. A mixed-effects linear regression was also used to model the rela- tionship between NIRS oxygen saturation measurements and direct kidney medullary oxygen concentration. This also accounts for lack of independence of repeated measurements within the same animal. A Bland–Alt- man analysis for clustered data was performed for comparison of measurements with the same units in order to determine the mean difference and 95% limits of agreement. All significance tests were two-tailed, with p < 0.05 considered statistically significant. The analysis used STATA version 17.0 (Statcorp, College Station, TX). https://doi.org/10.1038/s41598-024-51886-y Scientific Reports \| (2024) 14:2646 \| www.nature.com/scientificreports/ Results ll Animals 1 and 4 in the hemodilution protocol appeared to have a temp H FB H FB BR 0 20 40 60 80 Oxygen (% or mmHg) 0 20 40 60 80 Time (minutes) Animal 1 H FB H FB H FB BR 0 20 40 60 80 Oxygen (% or mmHg) 0 20 40 60 80 Time (minutes) Animal 2 H FB H FB H FB BR 0 20 40 60 80 Oxygen (% or mmHg) 0 20 40 60 80 Time (minutes) Animal 3 H FBH FBH FB BR 0 20 40 60 80 Oxygen (% or mmHg) 0 20 40 60 80 100 Time (minutes) Animal 4 Hemodilution Protocol H AO AR 0 20 40 60 80 100 Oxygen (% or mmHg) 0 50 100 150 200 Time (minutes) Animal 5 250 H AO AR 0 20 40 60 80 100 Oxygen (% or mmHg) 0 50 100 150 200 250 Time (minutes) Animal 6 H AO AR 0 20 40 60 80 100 Oxygen (% or mmHg) 0 50 100 150 200 Time (minutes) Animal 7 250 H AO AR 0 20 40 60 80 100 Oxygen (% or mmHg) 0 50 100 150 200 250 Time (minutes) Animal 8 REBOA Protocol Left Kidney NIRS Right Kidney NIRS Thigh NIRS Kidney Oxygen Figure 3. Left kidney, right kidney, and thigh near infra-red spectroscopy (NIRS) are compared to direct kidney tissue oxygen measurements over time for 4 animals undergoing the Hemodilution protocol. The red line marked “H” represents the start of each hemorrhage. The gray line marked “F” represents the end of each hemorrhage and the start of a bolus of balanced crystalloid. The orange line marked “BR” represents the time when shed blood was returned to the animal. For the resuscitative endovascular balloon aortic occlusion (REBOA) protocol, the red line marked “H” represents the start of the hemorrhage period. The gray line mar “AO” represents the start of aortic occlusion. The blue line marked “AR” represents the end of aortic occlusion when the aortic balloon was gradually released. For all graphs, the red solid and dashed lines indicate left and right kidney NIRS respectively and the blue solid line indicates thigh NIRS. The black solid line represents kidney tissue oxygen concentration. NIRS data were sampled every 2 s and kidney tissue oxygen was sampled every second. www.nature.com/scientificreports/ relationship between invasive kidney oxygen concentration and NIRS measurements. In animals 2 and 3 how- ever, there appeared to be very little variation in NIRS measurements despite large changes in kidney oxygen concentration. In the REBOA protocol, animals 5, 6, and 7 had abrupt decreases in kidney oxygen concentration and NIRS measurements with balloon inflation and these were followed by increases in oxygenation after release of the balloon. In animal 8, kidney oxygen concentration initially remained elevated after balloon inflation. This was thought to be due to an incomplete occlusion of blood flow to the renal arteries rather than a measurement artifact. The aortic balloon was then hyperinflated and subsequently, kidney oxygen concentration decreased. relationship between invasive kidney oxygen concentration and NIRS measurements. In animals 2 and 3 how- ever, there appeared to be very little variation in NIRS measurements despite large changes in kidney oxygen concentration. In the REBOA protocol, animals 5, 6, and 7 had abrupt decreases in kidney oxygen concentration and NIRS measurements with balloon inflation and these were followed by increases in oxygenation after release of the balloon. In animal 8, kidney oxygen concentration initially remained elevated after balloon inflation. This was thought to be due to an incomplete occlusion of blood flow to the renal arteries rather than a measurement artifact. The aortic balloon was then hyperinflated and subsequently, kidney oxygen concentration decreased. hl Table 1 summarizes the baseline and resuscitation data for the animals in each experimental protocol. Baseline characteristics were similar between the two groups. Peak plasma lactate concentration in the REBOA group was significantly higher than in the hemodilution group, otherwise resuscitation characteristics were also similar between the two groups.h g p The relationship between NIRS oxygen saturations and direct kidney tissue oxygen measurements is shown in Fig. 3. Pooled data from all 8 animals in both groups showed a moderate correlation between the kidney NIRS and kidney tissue oxygen measurements (r = 0.61 p < 0.001; r = 0.67 p < 0.001; for left and right kidney NIRS respectively). The correlation between thigh NIRS and kidney tissue oxygen, however, was similar (r = 0.66 p < 0.001). www.nature.com/scientificreports/ When the animals were separated by protocol, the Hemodilution group showed a weak or nonsignifi- cant correlation between left or right kidney NIRS and kidney tissue oxygen measurements (r = 0.10 p < 0.001; r = 0.01 p = 0.1007; left and right kidney NIRS respectively). The correlation between thigh NIRS and kidney tissue oxygen was also poor (r = 0.28 p < 0.001). This contrasts with the REBOA group, where left and right kidney as well as thigh NIRS were moderately correlated with kidney tissue oxygen (r = 0.71 p < 0.001; r = 0.74 p < 0.001; r = 0.70 p < 0.001; for left kidney, right kidney, and thigh NIRS respectively). pt y g y g p y) Figure 4 shows the comparison of kidney NIRS to thigh NIRS measurements when data are pooled from all 8 animals. There was a strong correlation between both kidney NIRS signals and thigh NIRS measurements (r = 0.85 p < 0.001;r = 0.88 p < 0.001;for left kidney vs thigh and right kidney vs thigh respectively). There was also a strong correlation between left and right kidney NIRS (r = 0.90 p < 0.001). These relationships were maintained regardless of the resuscitation protocol (Hemodillution group r = 0.89 p < 0.001; r = 0.83 p < 0.001; for left kidney vs thigh and right kidney vs thigh respectively and REBOA group r = 0.86 p < 0.001; r = 0.88 p < 0.001; for left vs thigh and right vs thigh respectively) Left and right kidney NIRS were also highly correlated regardless of protocol (Hemodillution group r = 0.82 p < 0.001 and REBOA group r = 0.93 p < 0.001) (Fig. 5). g p p g p p g Supplemental Fig. 2 shows the Bland–Altman analysis for the comparison of NIRS measurements with each other. The mean difference and limits of agreement of left kidney NIRS vs thigh NIRS and right kidney NIRS vs thigh NIRS were 0.02% (− 20.42, 20.45) and − 1.24% (− 15.26, 12.79) respectively. The mean difference and limits of agreement when comparing left and right kidney NIRS were 1.25% (− 17.64, 20.14). Results ll The blue line marked “AR” represents the end of aortic occlusion when the aortic balloon was gradually released. For all graphs, the red solid and dashed lines indicate left and right kidney NIRS respectively and the blue solid line indicates thigh NIRS. The black solid line represents kidney tissue oxygen concentration. NIRS data were sampled every 2 s and kidney tissue oxygen was sampled every second. https://doi.org/10.1038/s41598-024-51886-y Scientific Reports \| (2024) 14:2646 \| www.nature.com/scientificreports/ Table 1. Baseline and resuscitation data. Hgb = hemoglobin; IQr = interquartile range; kg = kilograms; MAp = mean arterial pressure; n = number of observations in the comparison; REBOA = resuscitative endovascular balloon aortic occlusion; SD = standard deviation. Significant values are in [bold]. Discussion In our animal model of hemorrhage, hemodilution, and REBOA, we showed a moderate correlation between kidney tissue oxygen and regional NIRS oxygen saturations but this relationship was only apparent during the extreme hemodynamic changes that occurred with balloon occlusion of the aorta. The correlation was poor or insignificant during more subtle changes, such as hemorrhage and hemodilution. Moreover, in all animals, the strongest correlations found were between the NIRS measurements themselves (kidney vs thigh and left vs right kidney) regardless of the experimental protocol. Taken together, these data suggest that regional NIRS measurements made with sensors carefully placed over the kidney with ultrasound were more closely related to Table 1. Baseline and resuscitation data. Hgb = hemoglobin; IQr = interquartile range; kg = kilograms; MAp = mean arterial pressure; n = number of observations in the comparison; REBOA = resuscitative endovascular balloon aortic occlusion; SD = standard deviation. Significant values are in [bold]. All Animals (8) Hemodilution Protocol (4) REBOA Protocol (4) P Weight (kg) Mean (SD) 56.4 (3.0) 57.2 (2.9) 55.7 (3.2) 0.530 Male n (%) 3 /8 (37.5) 2 /4 (50) 1/4 (25) > 0.999 Baseline MAP (mmHg) Mean (SD) 85 (10) 82 (9) 89 9) 0.296 Baseline Hgb (mg/dL) Mean (SD) 10.1 (1.5) 10.4 (1.8) 9.6 (1.1) 0.508 MAP during hemorrhage (mmHg) Mean (SD) 68 (12) 70 8) 66 (15) 0.633 Nadir Hgb (mg/dL) Mean (SD) 6.6 (1.1) 5.9 (0.6) 7.3 (1.1) 0.058 MAP after return of blood (mmHg) Mean (SD) 82 (14) 90 (17) 74 (5) 0.121 Peak Lactate (mmol/L) Mean (SD) 7.7 (4.7) 3.5 (1.8) 11.8 (0.6) < 0.001 Table 1. Baseline and resuscitation data. Hgb = hemoglobin; IQr = interquartile range; kg = kilograms; MAp = mean arterial pressure; n = number of observations in the comparison; REBOA = resuscitative endovascular balloon aortic occlusion; SD = standard deviation. Significant values are in [bold]. https://doi.org/10.1038/s41598-024-51886-y Scientific Reports \| (2024) 14:2646 \| www.nature.com/scientificreports/ Figure 4. Left kidney, right kidney, and thigh near infra-red spectroscopy (NIRS) are compared to direct kidney tissue oxygen measurements for all 8 animals (grey symbols), the 4 animals undergoing the Hemodilution protocol (maroon, orange, bright red, dark red) and the 4 animals undergoing the REBOA protocol (green, teal, light blue, dark blue). In graphs d– i, each individual animal is represented by a different color. Discussion The mixed effect linear regression model is plotted as a solid black line and the equation of that line is reported. The within group correlation coefficient is reported as “r”. NIRS data were sampled every 2 s and kidney tissue oxygen was sampled every second. Figure 4. Left kidney, right kidney, and thigh near infra-red spectroscopy (NIRS) are compared to direct kidney tissue oxygen measurements for all 8 animals (grey symbols), the 4 animals undergoing the Hemodilution protocol (maroon, orange, bright red, dark red) and the 4 animals undergoing the REBOA protocol (green, teal, light blue, dark blue). In graphs d– i, each individual animal is represented by a different color. The mixed effect linear regression model is plotted as a solid black line and the equation of that line is reported. The within group correlation coefficient is reported as “r”. NIRS data were sampled every 2 s and kidney tissue oxygen was sampled every second. Figure 4. Left kidney, right kidney, and thigh near infra-red spectroscopy (NIRS) are compared to direct kidney tissue oxygen measurements for all 8 animals (grey symbols), the 4 animals undergoing the Hemodilution protocol (maroon, orange, bright red, dark red) and the 4 animals undergoing the REBOA protocol (green, teal, light blue, dark blue). In graphs d– i, each individual animal is represented by a different color. The mixed effect linear regression model is plotted as a solid black line and the equation of that line is reported. The within group correlation coefficient is reported as “r”. NIRS data were sampled every 2 s and kidney tissue oxygen was sampled every second. thigh NIRS than invasive kidney oxygen concentration, even when the surface of the kidney was between 1.5 and 2 cm from the skin. Components of biological tissues, including hemoglobin and water, can absorb near-infrared light. Hemo- globin, for instance, exhibits strong absorption peaks within the near-infrared range25. This allows NIRS sensors to assess tissue capillary hemoglobin concentration and oxygenation saturation. Light scattering occurs when near-infrared photons interact with cellular and subcellular structures, causing deviations in the photons’ tra- jectories that are challenging to predict and lead to variations in the light’s penetration depth. As a result, the measured signal is a weighted average of oxygen levels in the various tissues beneath the skin’s surface. Scientific Reports \| (2024) 14:2646 \| Discussion The mixed effect linear regression model is plotted as a solid black line and the equation of that line is reported. The within group correlation coefficient is reported as “r”. NIRS data were sampled every 2 s and kidney tissue oxygen was sampled every second. Figure 5. Left kidney, right kidney, and thigh near infra-red spectroscopy (NIRS) are compared to each other for all 8 animals (grey symbols), the 4 animals undergoing the Hemodilution protocol (maroon, orange, bright red, dark red) and the 4 animals undergoing the REBOA protocol (green, teal, light blue, dark blue). In graphs d–i, each individual animal is represented by a different color. The mixed effect linear regression model is plotted as a solid black line and the equation of that line is reported. The within group correlation coefficient is reported as “r”. NIRS data were sampled every 2 s and kidney tissue oxygen was sampled every second. significant component of the weighted average of tissue oxygen measured by NIRS was due to medullary tis- sue oxygenation and thus may explain why the NIRS measurements we obtained over the kidney were more closely related to thigh NIRS than direct medullary tissue oxygen concentration. Any correlation we found likely represents a relationship of kidney perfusion and global perfusion determinants such as mean arterial pressure and cardiac output. p Clinical studies placing NIRS sensors on the skin over the kidney in adult cardiac surgery patients have shown an association of NIRS oxygenation measurements and the subsequent development of AKI17,18. In these stud- ies patients were excluded if the surface of the kidney was > 4 cm from the skin by ultrasound. The distance of the kidney from the skin in these adult human studies was therefore greater than in our animal model and the contribution of medullary tissue oxygen to the weighted average of their NIRS measurements was likely even less than in our model. The relationship these studies found between regional tissue oxygen measured using NIRS and outcome therefore may be related to the ability of NIRS to monitor global perfusion and capillary tissue oxygenation of the subcutaneous tissue in these patients. Indeed, a study in adult cardiac surgery patients showed that NIRS sensors placed over the thigh predicted subsequent AKI with receiver operator characteristic analysis suggesting an optimal cut-off of 67% and an area under the curve of 0.8428. Discussion Absorption and scattering of light by tissues also limits the maximum penetration depth of the near-infrared light employed by NIRS sensors. Moreover, the NIRS measurement is made through diffuse reflectance rather than direct transmission, further limiting the depth of tissues that can be measured. The manufacturer of the NIRS sensor used in this study states that the maximum depth of penetration is 2.5 centimeters26. The depth of penetration of NIRS measurements, however, likely depends on the optical properties of the tissue being meas- ured, the size and type of sensor used, the source-detector separation, and the specific algorithm used7,27. Patil et al. modeled NIRS experimentally and found a nonlinear relationship between source-detector separation and depth of penetration such that a source-detector separation of 20 mm resulted in a depth of penetration of 10 mm but a source-detector separation of 40 mm resulted in a depth of penetration of 15mm7. The sensors we used had one emitter and two detectors with source-detectors distances of 15 mm and 50 mm. If the results of Patil’s experimental model can be applied to our sensors and the optical properties of subcutaneous and kidney tissue, then extrapolating from their data, the range of penetration for our study should have been between 8 and 17 mm. I i l d l th f f th kid b t 1 5 d 2 (15 20 ) b l th ki Th In our animal model, the surface of the kidney was between 1.5 and 2 cm (15–20 mm) below the skin. The weighted average of tissue oxygen measured by NIRS, therefore, was likely to be substantially more affected by subcutaneous tissue than kidney tissue. In addition, the anatomy of the porcine and adult human kidney is such that the medullary tissue is 1–2 cm from the surface of the kidney. This makes it even less likely that any Scientific Reports \| (2024) 14:2646 \| https://doi.org/10.1038/s41598-024-51886-y www.nature.com/scientificreports/ Figure 5. Left kidney, right kidney, and thigh near infra-red spectroscopy (NIRS) are compared to each other for all 8 animals (grey symbols), the 4 animals undergoing the Hemodilution protocol (maroon, orange, bright red, dark red) and the 4 animals undergoing the REBOA protocol (green, teal, light blue, dark blue). In graphs d–i, each individual animal is represented by a different color. www.nature.com/scientificreports/ A porcine hemorrhage and REBOA study demonstrated a reduction in serum lactate concentration and less duodenal necrosis with 50% occlusion compared to complete REBOA34. Our data suggest that thigh NIRS might be a noninvasive and clinically available surrogate for lower body perfusion dur- ing REBOA. Future studies will evaluate the efficacy of titrating partial REBOA to maximize upper and lower body oxygen delivery using NIRS guidance. y yg y g g One limitation of our study is that we only compared NIRS oxygen saturations to medullary kidney tissue oxygen concentration. While the surface of the kidney was only 1.5–2 cm from the skin in all of our animals, the medulla was often 1–2 cm deeper than this. Therefore, if we had measured kidney tissue oxygen in the more superficial cortex tissue, we might have found a closer relationship with NIRS oxygen saturation measurements from sensors placed immediately above the kidney. We measured medullary oxygen because this kidney region has the lowest oxygen reserve and is thought to be responsible for most AKI caused by hypoperfusion35,36. A second limitation relates to the methodology of comparing two different technologies for measuring tissue oxygenation. Measurements using the same technology are subject to similar errors and therefore are likely to show a higher correlation compared to measurements from differing technologies. This may have contributed to the closer correlation when the various NIRS measurements were compared to each other rather than when each NIRS measurement was compared to invasive kidney oxygen concentration. A third limitation of the study is that we had such an abundance of data (collected every 1–2 s for hours in each animal) that statistical significance was easily obtained even when the correlation between variables was poor. This large dataset also precluded a statistical comparison between correlation coefficients because all the relationships were statistically significantly different even when the correlation coefficients differed by 0.01 (a clinically insignificant difference). In addition, the variability in NIRS measurements between animals was sometimes as large or larger than the variation over time within an individual animal. This large variability in NIRS measurements between animals might be partially explained by the two very different resuscitation protocols. Subtle differences in NIRS sensor placement might have also been a factor. www.nature.com/scientificreports/ saturation in 13 adult cardiac surgery patients with a continuous cardiac output pulmonary catheter placed in the renal vein19. The correlation coefficients found in this adult study, however, ranged from 0.51 to 0.75 and so were similar to the results we found between NIRS and direct kidney tissue oxygen measurements regardless of whether NIRS sensors were placed over the kidney or over the thigh. If it is true that thigh NIRS monitoring may be as effective at predicting poor perfusion and AKI as placing sensors over the kidney, then NIRS monitoring could be used in adult patients regardless of their BMI. Future studies are needed to ascertain whether or not changes in clinical management based on these subcutaneous tissue oxygen measurements can improve kidney oxygenation or reduce the incidence of AKI in critically ill patients. saturation in 13 adult cardiac surgery patients with a continuous cardiac output pulmonary catheter placed in the renal vein19. The correlation coefficients found in this adult study, however, ranged from 0.51 to 0.75 and so were similar to the results we found between NIRS and direct kidney tissue oxygen measurements regardless of whether NIRS sensors were placed over the kidney or over the thigh. If it is true that thigh NIRS monitoring may be as effective at predicting poor perfusion and AKI as placing sensors over the kidney, then NIRS monitoring could be used in adult patients regardless of their BMI. Future studies are needed to ascertain whether or not changes in clinical management based on these subcutaneous tissue oxygen measurements can improve kidney oxygenation or reduce the incidence of AKI in critically ill patients. yg y We found the most robust relationship between NIRS oxygen monitoring (whether over the kidney or the thigh) and kidney tissue oxygen was during aortic occlusion and release with REBOA. This relationship was likely because of the extreme decrease in blood flow to all regions distal to the aortic balloon. REBOA creates precise and reproducible aortic occlusion. While it is a useful tool to evaluate renal tissue perfusion across a wide range of renal blood flows, it is also a technique with direct clinical applicability. Many studies have established that REBOA is best performed by partially deflating the balloon after a brief period of full occlusion31–33. Partial REBOA prevents both total lower-body ischemia and the deleteriously high blood pressure in the upper extremity that occur with complete REBOA. www.nature.com/scientificreports/ This inter-subject variability was accounted for statistically by using a “within-subject” correlation coefficient that removes the variation between subjects, and by using a mixed effect linear regression which accounts for the lack of independence among repeated measures when mak- ing between-subjects comparisons. Nonetheless, the correlations we found between NIRS measurements and invasive kidney oxygen concentration, at best suggest that NIRS could be used as a trend monitor suggesting changes in kidney oxygen rather than a precise indicator of kidney oxygen concentration. Finally, our study only assessed the correlation between invasive kidney oxygen measurements and noninvasive NIRS monitoring. We did not evaluate the ability of noninvasive NIRS monitoring to predict actual kidney injury, nor did we ascertain whether hemodynamic management based on NIRS monitoring could prevent AKI. Even if NIRS placed over the kidney is only a subcutaneous tissue monitor, skin and muscle are highly vascular organs, and maintaining their perfusion may ensure the perfusion of other vital organs, such as the kidney. Future work should focus on whether an intervention based on regional oxygen monitoring with NIRS can reduce the incidence or severity of AKI in critically ill and injured patients. Conclusion In our porcine model of hemorrhage, hemodilution, and resuscitation, NIRS measurements from sensors placed on the skin over both the kidney and the thigh showed a moderate correlation with invasive kidney oxygen concentrations during extreme hemodynamic changes such as aortic occlusion. During the more mild hemody- namic changes of hemodilution the correlation was poor or insignificant. Furthermore, kidney NIRS and thigh NIRS measurements were closely related suggesting that both might reflect subcutaneous tissue oxygenation even when the surface of the kidney is 1.5–2 cm from the skin. These data shed light on other human studies of kidney NIRS which have shown association with renal vein oxygen saturation and outcome. Further studies are needed to determine whether this subcutaneous tissue oxygen monitoring with NIRS can be used to guide adult hemodynamic management or the implementation of partial REBOA. Discussion It is therefore possible that regional oxygen saturation measurements of subcutaneous tissue are good predictors of global malperfusion, whether they are placed over the subcutaneous tissue of the kidney or the thigh. y p y g Several studies have compared NIRS measurements to either direct tissue oxygen measurements or venous oxygen saturation. Skowno et al. compared transcutaneous hepatic tissue oxygen saturation using NIRS to direct hepatic tissue oxygen measurements in juvenile pigs and found that in animals weighing 15-20 kg, hepatic artery clamping was associated with an 84% decrease in direct hepatic oxygen concentration compared to a 6% decrease in NIRS measurements29. In 37 children undergoing cardiac catheterization, Ortmann et al. found that measurements from NIRS sensors placed over the flank in children < 10 kg were strongly correlated with renal vein oxygen saturation (r = 0.821; p = 0.002) but in children > 10 kg there was no significant correlation30. 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Prevalence and risk factors for acute kidney injury among trauma patients: a multicenter cohort study. Crit Care 22(1), 344. https://doi.org/10.1186/s13054-018-2265-9 (2018). 22(1), 344. https://doi.org/10.1186/s13054-018-2265-9 (2018). p g 5. Booth, E. A., Dukatz, C., Ausman, J. & Wider, M. Cerebral and somatic venous oximetry in adults and infants. Surg. Neurol. Int 1, 75. https://doi.org/10.4103/2152-7806.73316 (2010). 6. Benni, P. B., MacLeod, D., Ikeda, K. & Lin, H. M. A validation method for near-infrared spectroscopy based tissue oximeters for cerebral and somatic tissue oxygen saturation measurements. J. Clin. Monit. Comput. 32(2), 269–284. https://doi.org/10.1007/ s10877-017-0015-1 (2018). ( ) 7. Patil, A. V., Safaie, J., Moghaddam, H. A., Wallois, F. & Grebe, R. Experimental investigation of NIRS spatial sensitivity. Bio Opt Express. 2(6), 1478–1493. https://doi.org/10.1364/BOE.2.001478 (2011).f p p p g 8. Nagdyman, N. et al. Comparison of different near-infrared spectroscopic cerebral oxygenation indices with central venous and jugular venous oxygenation saturation in children. Paediatr. Anaesth. 18(2), 160–166. https://doi.org/10.1111/j.1460-9592.2007. 02365.x (2008). 9. Bruckner, M. et al. Normal regional tissue oxygen saturation in neonates: a systematic qualitative review. . Pediatr. Res. https://doi. org/10.1038/s41390-021-01786-y (2021).t g y 10. Gil-Anton, J. et al. References Combined cerebral and renal near-infrared spectroscopy after congenital heart surgery. Pediatr. Cardiol. 36(6), 1173–1178. https://doi.org/10.1007/s00246-015-1139-z (2015). p g 11. Gist, K. M. et al. A decline in intraoperative renal near-infrared spectroscopy is associated with adverse outcomes in chil following cardiac surgery. Pediatr. Crit. Care Med. 17(4), 342–349. https://doi.org/10.1097/PCC.0000000000000674 (2016).ff 12. Hanson, S. J., Berens, R. J., Havens, P. L., Kim, M. K. & Hoffman, G. M. 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Anesth. 34(12), 3300–3305. https://doi.org/10.1053/j.jvca. 2020.04.044 (2020). 20. Lofgren, L. R., Hoareau, G. L., Kuck, K. & Silverton, N. A. Noninvasive and invasive renal hypoxia monitoring in a porcine model f h h i h k J Vi E p http //d i /10 3791/64461 (2022) 2020.04.044 (2020). 20. Lofgren, L. R., Hoareau, G. L., Kuck, K. & Silverton, N. A. Noninvasive and invasive renal hypoxia monitoring in a porcine model of hemorrhagic shock. J. Vis. Exp. https://doi.org/10.3791/64461 (2022). 0. Lofgren, L. R., Hoareau, G. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Received: 1 November 2023; Accepted: 10 January 2024 Data availability Th d d y The datasets during and/or analyzed during the current study are available from the corresponding author upon reasonable request. https://doi.org/10.1038/s41598-024-51886-y Scientific Reports \| (2024) 14:2646 \| Fundingh g The research reported in this publication was supported in part by the National Kidney Foundation of Utah and Idaho, by the University of Utah Department of Anesthesiology Research Council, by a grant from the International Anesthesia Research Society, by the Study Design and Biostatistics Center (SDBC) with fund- ing in part from the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UM1TR004409, and the U.S. Department of Defense–Peer Reviewed Medical Research Program (Award W81XWH-20–1-0180). The research reported in this publication was also supported in part by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR002538. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Defense. These organizations were not involved in study design, analysis, interpretation, or manuscript creation. Acknowledgements g We would like to acknowledge the University of Utah Anesthesiology Department Research Council for the support of this project and the University of Utah Department of Emergency Medicine for use of the lab facili- ties. We would like to particularly acknowledge the help of Amy Taylor and Calli Carroll for their research and clinical support of the work. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 5. Russo, R. M. et al. Partial resuscitative endovascular balloon occlusion of the aorta in swine model of hemorrhagic shock. J. Am Coll. Surg. 223(2), 359–368. https://doi.org/10.1016/j.jamcollsurg.2016.04.037 (2016).l g p g j j g 36. Evans, R. G. et al. Haemodynamic influences on kidney oxygenation: clinical implications of integrative physiology. Clin. Pharmacol. Physiol. 40(2), 106–122. https://doi.org/10.1111/1440-1681.12031 (2013). Author contributions N.S., K.K, and G.H. conceived the study, participated in data collection and data analysis. G.H. provided surgi- cal expertise, advice on experimental design, and coordinated the execution of the project and the laboratory facilities. N.S. and G.S. did the statistical analysis. N.S. wrote the manuscript. A.R., R.J., and L.L. participated in data collection and contributed to manuscript preparation. All authors reviewed and contributed to editing the manuscript. Competing interests p g Natalie Silverton, Kai Kuck, and Lars Lofgren are inventors on a patent application for a urine oxygen and flow sensing technology. This prototype is under development for commercial consideration by Natalie Silverton and Kai Kuck but as of yet no commercial activity has occurred. This technology is unrelated to near-infrared spectroscopy monitoring. Nonetheless, this work was performed under a conflict of interest management plan approved by the University of Utah Conflict of Interest Office. This included disclosure of conflict of interest to patients and collaborators and an independent peer review of the manuscript. Guillaume L. Hoareau is a consult- ant and shareholder of Certus Critical Care. The interpretation and reporting of these data are the responsibility of the authors alone and should not be seen as an official policy of or interpretation by the US Government, nor does this report necessarily represent the official views of the National Institutes of Health. No other authors have any disclosures to declare. References Automated partial versus complete resuscitative endovascular balloon occlusion of the aorta for the manage- ment of hemorrhagic shock in a pig model of polytrauma: A randomized controlled pilot study. Mil. Med. 185(11–12), e1923– e1930. https://doi.org/10.1093/milmed/usaa217 (2020). p g ( ) 33. Williams, T. K. et al. Endovascular variable aortic control (EVAC) versus resuscitative endovascular balloon occlusion of the aorta (REBOA) in a swine model of hemorrhage and ischemia reperfusion injury. J. Trauma. Acute Care Surg. 85(3), 519–526. https:// doi.org/10.1097/TA.0000000000002008 (2018). g ( ) 34. Johnson, M. A. et al. Endovascular perfusion augmentation for critical care: Partial aortic occlusion for treatment of severe ischemia-reperfusion shock. Shock. 51(5), 659–666. https://doi.org/10.1097/SHK.0000000000001199 (2019). g 34. Johnson, M. A. et al. Endovascular perfusion augmentation for critical care: Partial aortic occlusion for treatment of severe ischemia-reperfusion shock. Shock. 51(5), 659–666. https://doi.org/10.1097/SHK.0000000000001199 (2019). https://doi.org/10.1038/s41598-024-51886-y Scientific Reports \| (2024) 14:2646 \| Additional informationh Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-024-51886-y. Correspondence and requests for materials should be addressed to N.A.S. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. https://doi.org/10.1038/s41598-024-51886-y Scientific Reports \| (2024) 14:2646 \|
https://openalex.org/W3128312104	https://www.mdpi.com/2077-0383/10/4/719/pdf?version=1614063699	English	null	Local Anesthetics and Recurrence after Cancer Surgery-What’s New? A Narrative Review	Journal of clinical medicine	2,021	cc-by	11,311	Keywords: local anesthetics; cancer; recurrence; inﬂammation; metastasis Review Local Anesthetics and Recurrence after Cancer Surgery-What’s New? A Narrative Review Sarah D. Müller †, Jonathan S. H. Ziegler † and Tobias Piegeler * Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Sarah D. Müller †, Jonathan S. H. Ziegler † and Tobias Piegeler * Department of Anesthesiology and Intensive Care, University Hospital Leipzig, Liebigstrasse 20, 04103 Leipzig, Germany; sarah.mueller2@medizin.uni-leipzig.de (S.D.M.); jshz@posteo.de (J.S.H.Z.) * Correspondence: tobias.piegeler@medizin.uni-leipzig.de † Denotes equal contribution. Abstract: The perioperative use of regional anesthesia and local anesthetics is part of almost every anesthesiologist’s daily clinical practice. Retrospective analyses and results from experimental studies pointed towards a potential beneﬁcial effect of the local anesthetics regarding outcome—i.e., overall and/or recurrence-free survival—in patients undergoing cancer surgery. The perioperative period, where the anesthesiologist is responsible for the patients, might be crucial for the further course of the disease, as circulating tumor cells (shed from the primary tumor into the patient’s bloodstream) might form new micro-metastases independent of complete tumor removal. Due to their strong anti-inﬂammatory properties, local anesthetics might have a certain impact on these circulating tumor cells, either via direct or indirect measures, for example via blunting the inﬂammatory stress response as induced by the surgical stimulus. This narrative review highlights the foundation of these principles, features recent experimental and clinical data and provides an outlook regarding current and potential future research activities. Citation: Müller, S.D.; Ziegler, J.S.H.; Piegeler, T. Local Anesthetics and Recurrence after Cancer Surgery-What’s New? A Narrative Review. J. Clin. Med. 2021, 10, 719. https://doi.org/10.3390/jcm10040719 Academic Editor: Marco Cascella Received: 31 December 2020 Accepted: 8 February 2021 Published: 11 February 2021 2. Background Concept: Circulating Tumor Cells 2. Background Concept: Circulating Tumor Cells Since 1869, when Thomas Ashworth described malignant cells in the peripheral blood of a patient with metastatic subcutaneous thoraco-abdominal tumors [9], this particular cell population, shed from the primary tumor into the bloodstream, was termed as circulating tumor cells (CTCs) and has gained a lot of scientiﬁc attention over the years and decades. During surgical procedures, CTCs are released into bloodstream [10] and—depending on the tumor entity—prognosis might be negatively correlated with their quantity [11]. Techniques for precise detection and characterization have evolved more in recent years [12]. Some of these techniques are based on the detection of epithelial surface markers such as EpCAM, which is highly expressed, for example, by breast cancer cells [13]. Other methods, such as immunocytochemical characterization, which separate CTCs by their distinct morphological features, are also available [14]. As already mentioned, the detection of CTCs in an individual patient’s blood as well as the number of CTCs has been correlated with metastasis, disease status [15], and clinical outcome, for example, in HER-2-positive breast cancer [16] or thyroid cancer [17]. However, before becoming CTCs, the epithelial tumor cells have to undergo epithelial-to-mesenchymal transition (EMT) to be able to migrate, invade their surroundings, and ﬁnally enter the circulation [18]. After exiting the blood stream at a remote location, the cells might then become epithelial again (mesenchymal-to- epithelial transition, MET) and form new metastatic sites [19] (see also Figure 1). Figure 1. Schematic illustration of metastasis formation by circulating tumor cell. CTC = circulating tumor cells; EMT = epithelial-to-mesenchymal transition; MET = mesenchymal-to-epithelial transition [20]. Figure 1. Schematic illustration of metastasis formation by circulating tumor cell. CTC = circulating tumor cells; EMT = epithelial-to-mesenchymal transition; MET = mesenchymal-to-epithelial transition [20]. It could even be assumed that CTCs might be able to form new metastatic sites even after complete tumor excision by so-called tumor self-seeding. Kim and colleagues investigated the ability of malignant human breast, colon, and melanoma cells to seed a tumor from circulation in mouse model, showing that self-seeding might only require minimal adaptation of CTCs to the recipient microenvironment [21]. The authors could further demonstrate that CTCs sense attraction signals from the tumor and are, furthermore, able to extravasate and invade the surrounding tissue in response to these signals [21]. 1. Introduction Local anesthetics (LA) are well-known substances and a mainstay of anesthesia since the introduction of cocaine in 1884. There is a huge amount of evidence supporting the perioperative use of local anesthetics, either administered systemically or used as part of regional anesthesia techniques for a variety of reasons: the drugs and their associated analgesic procedures are effective regarding pain relief due to their ability to block the voltage-gated sodium channel, thus inhibiting nerve cell depolarization [1,2], they might reduce postoperative nausea and vomiting (PONV) [3,4], and they might pave the way to an early and enhanced recovery after surgery [5]. Additionally, by using local anesthetics and regional anesthesia, opioid consumption might be reduced, thus leading to improved postoperative bowel function, less constipation, and early restoration of oral nutrition [6]. Moreover, a reduction of postoperative morbidity by dampening the surgical stress re- sponse, which could be correlated with perioperative myocardial infarction, pulmonary infection, and thromboembolism, might be another strong advantage of the use of regional anesthesia and LA [7,8]. Received: 31 December 2020 Accepted: 8 February 2021 Published: 11 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. However, there is more to it. The hypothesis that regional anesthesia and LA might be able to inﬂuence cancer recurrence was generated from retrospective studies, and over the last couple of years, several experimental studies—both in vivo and in vitro—have pointed out the importance of the anti-inﬂammatory and even anti-cancer/anti-metastatic effects of LA in this context and have provided insight into potential mechanisms by which the LA might be able to exert their impact on malignant cells. This narrative review will highlight and summarize the current knowledge regarding these potentially beneﬁcial anti-metastatic effects of the LA in order to increase the acceptance of this concept among our fellow anesthesiologists. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 719. https://doi.org/10.3390/jcm10040719 J. Clin. Med. 2021, 10, 719 2 of 14 2. Background Concept: Circulating Tumor Cells Although controversially discussed, several clinical trials were able to show that CTCs might serve as a potential prognostic marker, for example, in colorectal cancer [22,23]. Even the use of CTCs in monitoring tumor response to systemic therapy is widely examined. Hou and colleagues analyzed 97 blood samples from patients suffering from small-cell lung J. Clin. Med. 2021, 10, 719 3 of 14 cancer using the earlier mentioned EpCAM-based immunomagnetic detection method [24]. This particular study demonstrated the importance of both baseline CTC numbers as well as of the changes in CTC numbers after chemotherapy as prognostic factors in patients with small-cell lung cancer [24]. g It has been demonstrated that CTCs are increasingly released during the crucial peri- operative period [25], the short stretch of time in which the anesthesiologist is responsible for the patient. Increasing evidence suggests that this period might be crucial regarding the long-term outcome after cancer surgery, possibly—among other factors—due to a signiﬁcant inﬂammatory response, which then impairs the ability of the innate immune system to detect and destroy CTCs [26,27]. The innate immune system, especially the natural killer (NK) cell activity (NKA), is signiﬁcantly unstable under stress [28]. Under normal circumstances, NK cells are mainly responsible for a phenomenon called immune surveillance, which also includes the detection of CTCs [26]. A signiﬁcant loss of NKA after abdominal surgery, for example, leads to a compromised resistance to tumor devel- opment in rats; Ben-Eliyahu and colleagues were able to show an increased lung tumor retention in rats, which received surgery prior to inoculation with radiolabeled tumor cells in comparison to the unstressed control group [29]. An attenuation of this stress response, e.g., by local anesthetics, might therefore, in turn, be able to decrease the ability of CTCs to metastasize [26,30]. 3. Background Concept: Inﬂammatory Stress Response 3.1. Overview Appropriate activation of the innate and adaptive immune response requires a bal- anced and sufﬁcient cytokine production. Under normal circumstances, i.e., in the absence of stress, the immune system is able to avoid a hyper-inﬂammatory response [31,32]. The current SARS-CoV-2 pandemic has again impressively demonstrated how a dysregulation of cytokine production with excessive high circulating levels of these biological messen- gers can cause systemic collateral damage leading to multi-organ-failure [31]. However, not only pathogens or autoimmune disorders are able to trigger the so-called cytokine storm [31]. Cancer and its therapies, such as surgery or chemotherapy, may as well cause an immune cell hyperactivity [33–35]. As already described, the perioperative period as a vulnerable time has become a focus of attention in clinical and experimental studies over the past decades—especially in cancer surgery [36]. Surgical trauma inevitably leads to an inﬂammatory response [37], unbalancing pro- and anti-inﬂammatory factors [38], ultimately leading to additional immunosuppression [39], which in turn might then favor the CTCs to escape their immune surveillance [26] (Figure 2). Tissue trauma causes the release of vasoactive meditators (e.g., leukotrienes and histamine) and plasma components evoking an inﬂammatory microenvironment and systemic acute phase reaction with the release of pro-inﬂammatory cytokines like interleukin (IL)-1, IL-6 [40,41], IL-8, and tumor necrosis factor α (TNF-α) [42]. These cytokines, however, have distinct effects; high levels of IL-6, for example, lead to the initiation of signal transduction processes, ultimately causing endothelial hyperpermeability and hypotension, an effect that might be important during the pathogenesis of acute lung injury and pulmonary edema [43]. TNF-α not only induces fever and augments systemic inﬂammation, but also regulates parts of the immune system, e.g., by inducing inﬂammatory signaling events involving nuclear factor kappa B (NF-kB) [44]. The activation of this transcription factor leads to an increased expression of pro-inﬂammatory genes, thus further enhancing other inﬂammatory processes [45]. 4 of 14 J. Clin. Med. 2021, 10, 719 Figure 2. Schematic illustration of processes leading to perioperative inﬂammation and immune suppression (surgical stress response) and possible inhibition by local anesthetics (shown as ⊥). LA = local anesthetics; IL = interleukin; TNFα = tumor necrosis factor α; ICAM-1 = intercellular adhesion molecule 1; MHC = major histocompatability complex; NK cell = natural killer cell. Modiﬁed after [46]. Figure 2. Schematic illustration of processes leading to perioperative inﬂammation and immune suppression (surgical stress response) and possible inhibition by local anesthetics (shown as ⊥). 3.2. Inﬂammation, the Inﬂammatory Response, and Cancer Inﬂammation affects disease progression. Virchow hypothesized in 1863 that injury and chronic inﬂammation might serve as the origin of tissue proliferation and cancer. Although the exact mechanisms are still not fully understood, there is increasing evidence that an inﬂammatory environment enhances proliferation, survival, migration, and an- giogenesis of tumor cells and that misguided immune cell recruitment might be related with cancer recurrence [47]. Kim and colleagues were able to provide evidence for the assumption that IL-6 and IL-8 might be tumor-derived attraction signals [21]. High IL-6 serum-levels are associated with poor prognosis for lung, breast, and colon cancer [48–50]. TNF-α-induced NF-kB activation seems to play a major role during these pathophysi- ologic circumstances as well [51]; upregulated NF-kB transcription might be a critical link between inﬂammation and cancer, as inactivation of the NF-kB pathway attenuates the formation of inﬂammation-associated tumors in a colitis-associated cancer model in mice [52]. Another important molecule in terms of cancer and inﬂammation is intercellular adhesion molecule 1 (ICAM-1). It usually serves as a counter receptor for the neutrophil cluster of differentiation (CD) 11b/CD18 on the surface of endothelial cells, but is also expressed by many cancer cell types [53,54]. A tight adherence of neutrophils and tumor cells via the tumor-expressed ICAM-1 activates the neutrophils, weakens the endothelial barrier, and enhances the extravasation of CTCs [55]. Vascular hyper-permeability and endothelial barrier function is mainly regulated by Src tyrosine protein kinase (Src) [56], which also plays an important role for the metastatic potential of tumor cells, due to its ability to regulate, e.g., tumor cell migration and invasion by various signal transduction pathways, including TNF-α [57]. 3. Background Concept: Inﬂammatory Stress Response 3.1. Overview LA = local anesthetics; IL = interleukin; TNFα = tumor necrosis factor α; ICAM-1 = intercellular adhesion molecule 1; MHC = major histocompatability complex; NK cell = natural killer cell. Modiﬁed after [46]. 3.3. Anti-Inﬂammatory Effects of Local Anesthetics Local anesthetics, especially the amide local anesthetics, have strong anti-inﬂammatory properties, which have also been studied extensively [42]. Lidocaine and ropivacaine, for example, were demonstrated to be able to preserve endothelial barrier function by an atten- uation of TNF-α-induced Src activation in vitro, e.g., in pulmonary endothelial cells [58], J. Clin. Med. 2021, 10, 719 5 of 14 which also leads to reduced phosphorylation of ICAM-1 and diminished neutrophil adhe- sion [58]. Two further studies reported a beneﬁcial effect of ropivacaine on experimental lung injury in rats and mice, which was also due to a reduction in Src activation as well as in ICAM-1 expression [59,60]. A decreased phosphorylation of IkB by lidocaine and, therefore, an inhibition of NF-kB activation as observed by Lang and colleagues in epithelial cells in vitro underlines the anti-inﬂammatory effects of the LA once more [61]. It could also be shown that lidocaine and bupivacaine, another amide LA, are able to inhibit the release of leukotriene B4, IL-1 [62], and IL-8 [63] in vitro. Lan and colleagues could even demonstrate an attenuation of IL-1β, IL-6, and IL-8 by lidocaine in activated human umbilical vein en- dothelial cells after TNF-α stimulation under ischemia/reperfusion-injury conditions [64]. Similar results could be observed in a recent clinical study evaluating patients undergoing laparoscopic cholecystectomy; patients who had received an intravenous lidocaine infusion had decreased postoperative serum levels of IL-1, IL-6, interferon γ. and TNF-α when compared to an infusion with normal saline [65]. Most of these effects were observed at clinically relevant, non-toxic concentrations of the drugs. However, although these concentrations might also be reached in plasma via absorption of LA after a regional anesthesia procedure, the experimental data suggest that the systemic use of the drugs might be favorable in terms of the anti-inﬂammatory properties of the drugs [2,58]. 4. Local Anesthetics and Cancer–What Do We Know So Far? 4.1. Historic Clinical Data 4.2. First Experimental Data After the ﬁrst encouraging results from the retrospective analyses had been published, researchers tried to provide evidence for potential beneﬁcial (direct) effects on malignant cells and CTCs. Most of these observed effects were—at least in part—due to the already mentioned anti-inﬂammatory effects of LA and suggested that a systemic administration would be much more important than a local effect at the site of injection. Lidocaine and ropivacaine, for example, inhibited TNF-α-induced Src activation independent of sodium channel blockade in non-small cell lung cancer cells in vitro, thus also reducing tumor cell migration [80]. Further downstream of these signaling events, it appears that both drugs are also be able to inhibit TNF-α-induced signaling events involving focal adhesion kinase and caveolin-1, which explained an also observed reduction in the release of matrix- metalloproteinase (MMP)-9 [81]. MMPs are enzymes utilized by the tumor cells to break up the extracellular matrix in order to invade the surrounding tissue [82]. The inhibition of these signal transduction events ultimately leads to a reduction in the TNF-α-induced invasiveness of the tumor cells in this study [81]. y Tumor growth and apoptosis might also be affected by LA. Treatment of human breast cancer cell lines MCF-7 and MCF-10A with lidocaine and bupivacaine in clinically relevant concentrations revealed an inhibition of cell viability and an induction of apoptosis-related proteins in vitro [83]. Potentially beneﬁcial actions were examined in various different tumors. Xuan and colleagues investigated that bupivacaine possesses an anti-metastatic and anti-proliferative effect on human ovarian and prostate cancer cell lines [84]. There is also experimental data underlining that LA can inhibit the growth of human hepatocellular carcinoma cells [85]. Both lidocaine and ropivacaine can affect the expression of cell-cycle- related genes and induce apoptosis in these cells [85]. It has also been demonstrated that LA might slow down cancer cell growth in vitro and induce cell death at the same time in pancreatic [86] and colon cancer cells [87]. The drugs might be able to directly induce apoptosis via the mitochondrial and p38 mitogen-activate protein kinase MAP-kinase- dependent pathways as Lu and colleagues found out after incubating a neuroblastoma cell line with bupivacaine [88]. 4. Local Anesthetics and Cancer–What Do We Know So Far? 4.1. Historic Clinical Data 2021, 10, 719 6 of 14 the progression of the disease [74]. Negative effects of opioids and volatile anesthetics on the NKA have also been observed and might, therefore, also be important in this regard [30,75]. The reduction in opioids and volatile anesthetics was one of the ﬁrst possible explanations of the observed beneﬁcial clinical effects of the perioperative use of LA in patients undergoing surgery. However, given the more recent evidence— including studies evaluating the effects of sevoﬂurane, e.g., in breast cancer [76]—this hypothesis is more likely to be incorrect. the progression of the disease [74]. Negative effects of opioids and volatile anesthetics on the NKA have also been observed and might, therefore, also be important in this regard [30,75]. The reduction in opioids and volatile anesthetics was one of the ﬁrst possible explanations of the observed beneﬁcial clinical effects of the perioperative use of LA in patients undergoing surgery. However, given the more recent evidence— including studies evaluating the effects of sevoﬂurane, e.g., in breast cancer [76]—this hypothesis is more likely to be incorrect. (2) yp y (2) As outlined above, there is strong evidence that LA and regional anesthesia might be able to reduce perioperative inﬂammation and the stress response as induced by surgery [65,77], and also preserve NKA as one of the most important factors for the detection and destruction of CTCs [78,79]. This systemic effect of the LA might, therefore, have a possible positive impact on perioperative processes lead- ing to new micro-metastases, e.g., by CTCs, thus allowing a prolonged (at least recurrence-free) survival. (2) As outlined above, there is strong evidence that LA and regional anesthesia might be able to reduce perioperative inﬂammation and the stress response as induced by surgery [65,77], and also preserve NKA as one of the most important factors for the detection and destruction of CTCs [78,79]. This systemic effect of the LA might, therefore, have a possible positive impact on perioperative processes lead- ing to new micro-metastases, e.g., by CTCs, thus allowing a prolonged (at least recurrence-free) survival. (3) As the theories regarding the indirect effects, induced by a reduction of potentially harmful circumstances as presented above are not able to completely explain the observed effects in cancer patients, several—mostly experimental—studies examined potential direct effects of LA on malignant cells and CTCs as outlined in the next chapter of this article. 4. Local Anesthetics and Cancer–What Do We Know So Far? 4.1. Historic Clinical Data Several retrospective studies reported a possible beneﬁcial effect of LA on the outcome, i.e., the overall or recurrence-free survival of patients after tumor surgery. One of the ﬁrst reports from Exadaktylos and colleagues retrospectively analyzed metastasis-free-survival of 129 women with breast cancer undergoing mastectomy and axillary clearance [66]. Patients treated with paravertebral anesthesia plus general anesthesia had a signiﬁcant advantage regarding their recurrence-free survival at 12 months compared to women who received general anesthesia only (94% vs. 82%) [66]. This particular study gave rise to several further retrospective analyses. Biki and colleagues focused their analysis on prostate cancer; they compared patients with invasive prostatic carcinoma undergoing surgery between 1994 and 2003 receiving either general anesthesia plus epidural analgesia or general anesthesia with opioid analgesia only and were able to show that the epidural group had an 57% lower risk of recurrence compared with general anesthesia and opioids, even after adjusting the groups towards tumor size, Gleason Score. and prostate-speciﬁc antigene [67]. However, after these ﬁrst encouraging results, there were also several studies reporting no effect; Cummings and colleagues, for example, could not show a difference between patients receiving an epidural analgesia in addition to general anesthesia or not regarding recurrence or survival after resection of gastric cancer [68]. A large randomized trial by Myles and colleagues also concluded that there was no association by the use of epidural anesthesia and cancer-free survival in 503 patients undergoing abdominal surgery [69]. It has to be noted, however, that this particular study was not powered to detect a difference regarding cancer recurrence. Additionally, most studies evaluated cancer recurrence after regional anesthesia. In accordance with the pre-clinical data outlined above, it might be reasonable to hypothesize that the systemic use and application of the LA might be able to exert more pronounced effects. Several theories regarding possible mechanisms of the observed potential beneﬁcial effects of the LA results have been stated: (1) It is well-known that the use of regional anesthesia and LA might lower the use of opioids or volatile anesthetics during general anesthesia [70]. Several studies also suggested that these drugs and anesthetics might promote cancer progression and reduce long-term survival [71], maybe by promoting tumor angiogenesis [72,73]. However, a more recent experimental study evaluating the effect of opioids in a mouse model of breast cancer surgery reported no negative impact of morphine on J. Clin. Med. 5. Recent and Current Data 5.1. Experimental Studies Several research groups have put a lot of effort into a further exploration of these initially presented mechanisms by which the LA might exert their beneﬁcial effects. J. Clin. Med. 2021, 10, 719 7 of 14 Most interestingly, newer evidence points towards a possible synergistic effect of the LA together with chemotherapy; in vitro, lidocaine appears to have an enhancing effect on the chemotoxicity of cisplatin via the demethylation of retinoic acid receptor beta 2 (RARβ2) located in the cell nucleus and tumor suppressor Ras association domain-containing protein 1 (RASSF1) in breast cancer cells [89]. Following these in vitro results, it has subsequently also been shown that lidocaine alone can reduce the tumor size of hepatocellular carcinoma and, moreover, it enhances the sensitivity of the tumor cells against cisplatin in an in vivo murine model [90]. A study from Freeman and colleagues also found a potential metastasis- inhibiting effect of perioperative systemic lidocaine combined with cisplatin in another murine model of triple negative breast cancer with a Stage IV metastatic burden [91]. In this study, 50 animals were treated with cisplatin only or with cisplatin plus lidocaine at clinically relevant concentrations (bolus of 1.5 mg/kg plus 2 mg/kg x h−1 during surgery) and lidocaine treatment lead to fewer metastatic lesions in the animals’ lungs [91]. The same scientiﬁc research group published a study in 2019 focusing on a four-branched murine 4T1 model on the inﬂuence of lidocaine, methylprednisolone, and propofol in combination with general anesthesia with sevoﬂurane on pulmonary metastasis after 14 days after surgery of the primary breast cancer tumor [92]. Here, the hepatic metastasis load was equal in all groups. However, lidocaine and propofol each reduced the post- mortem in vitro cultured pulmonary metastasis colonies [92]. These ﬁndings are consistent with another study comparing the inﬂuence of lidocaine in dependency of sevoﬂurane compared to ketamine/xylazine anesthesia on pulmonary metastasis in a 4T1 mouse model of breast cancer [93]. Of note, the administration of high dose steroids in the 2019 study by Freeman and colleagues [92] even enhanced the pulmonary metastasis burden. The authors hypothesized that these results might be due to a facilitation of the dispersion and metastasis of CTCs by the drug [94]. y g In another recent study by Chamaraux-Tran and colleagues, the effect of a lidocaine treatment was tested in breast cancer cells in vitro and in an in vivo mouse model [95]. 5. Recent and Current Data 5.1. Experimental Studies In accordance with earlier results, there was a direct cytotoxic effect on the tumor cells. The triple-negative cell lines especially were more sensitive to the treatment with lidocaine. Lidocaine had an inhibitory effect on breast cancer cell migration with a predominant effect (again) on the triple-negative cell line. In the mouse experiments, the lidocaine group showed a delay in the development of peritoneal carcinomatosis after injection of MDA-MB-231 cells and repeated injections of the drug into the peritoneal cavity. Of note, no intravenous administration has been examined, but instead, the authors claim a potential beneﬁcial peritoneal administration (in a dose already used for shoulder pain after laparoscopy) to also use the direct cytotoxic effects of lidocaine in case of peritoneal dissemination of tumor cells [95]. Following the data showing that LA are able to induce apoptosis in (for example breast and thyroid) cancer cells by activating caspases and regulating the mitogen-activated pro- tein kinase signaling pathway [96], the apoptosis-inducing effect has also been shown in hepatocellular cancer cells [90]. In a recent study also in lung cancer cells, a poten- tially beneﬁcial effect of lidocaine treatment on cell viability and proliferation has been shown [97]. Here, the authors hypothesized that the mechanism of the lidocaine-anti- tumor-effect might be based on an up-regulation of miR-539 microRNA, which inhibits signaling of the epidermal growth factor receptor (EGFR) through direct binding, thus regulating downstream signaling via extracellular-signal regulated kinase (ERK) and the phosphatidyl-inositol 3-kinases (PI3K)/AKT pathway [97]. The activation of the latter has been found in melanoma cells [98,99] and is known to be blocked by amide LA [58,80]. Another recent study evaluating cell viability of breast cancer cells also found an inhibitory effect of different LA [100]. However, most of the effects in this particular study were only observed at concentrations ten times higher than the clinically relevant plasma concentra- tion. The observed cell alterations might, therefore, be considered to be due to toxic rather than pro-apoptotic effects of the LA in this setting. J. Clin. Med. 2021, 10, 719 8 of 14 The inhibiting effect of lidocaine on Src tyrosine protein kinase (Src) [101–103] indicates that systemically administered local anesthetics might potentially be able to inhibit cancer cell metastasis [26,96,104,105]. 5. Recent and Current Data 5.1. Experimental Studies A recent study by Wall and colleagues in 2019 supported this hypothesis by measuring the metastatic burden in lung and liver as well as MMP-2 levels in dependency of the treatment with lidocaine and the Src-inhibitor bosutinib in a 4T1 mouse tumor model [106]. Bosutinib neutralized the protecting effect of lidocaine regarding lung metastasis and levels of MMP-2. However, the authors claimed that it remains unclear if the ﬁndings are due to a direct Src effect or another pathway [106]. g p y It has also been shown that lidocaine at clinically relevant doses might have demethy- lating effects on breast cancer cell lines [107,108], while at the same time enhancing the effect of the deoxycytidine analog chemotherapeutic decitabine [108]. Besides the thera- peutic effects in acute myeloid leukemia, decitabine is a possible agent in the therapy of breast cancer as well and could be used as a second line therapy in chemotherapy-resistant patients [109,110]. Thus—at least based on the results of these experimental studies—the additional administration of intravenous lidocaine might be able to enhance its therapeutic effect and might be of interest for future studies. Most of the outlined anti-inﬂammatory and potentially anti-metastatic effects of the LA are mediated independent of sodium channel blockade [80]. However, it has also been observed that lidocaine (in clinically relevant doses) is able to block cancer-associated and prognostic relevant variants of voltage-activated sodium channels like Nav1.5 [111]. Interestingly, a recent animal study detected chronic electric activity in solid breast tumor masses in mice [112]. This activity is supposedly of neuronal origin, as it has a connection to the parasympathetic nervous system, which disrupts on injection of lidocaine and chemical sympathectomy [112]. With these neuronal networks, systemically administered lidocaine could, therefore, have a new target in its ﬁrst described mechanism of action, thus disrupting the neuronal membrane potential and neural activity during tumor growth and metastasis. 5.2. Clinical Studies–Systemic Use of Local Anesthetics Clinical studies examining the short- and long-term effects of (amide) LA on perioper- ative pain und its chroniﬁcation continued to increase in number over the past few years. The authors of a recently updated Cochrane analysis, however, were uncertain whether intravenous lidocaine might have beneﬁcial effects on postoperative pain, nausea, or opioid consumption [113]. As the use of intravenous lidocaine is still considered an off-label use in most countries, concerns regarding the risk of intoxications after systemic administration of the drug have been raised repeatedly and were also addressed in a very recent consensus paper [114]; here, the authors provided evidence for the fact that intravenous lidocaine might be considered safe, if clinicians followed several precautions, including correct dosage and a 24 h limit for the duration of the drug infusion, as well as close post-operative monitoring [114], the latter possibly bearing the potential to collide with enhanced recovery after surgery (ERAS) programs, in which intravenous lidocaine has already been established as part of the multimodal analgesic regimen [115,116]. Due to these concerns raised by clinicians, the perioperative systemic use of LA should always be a “risk-beneﬁt” decision depending on the individual patient, her/his co-morbidities, the surgical procedure, and, of course, the available evidence [114]. 5.3. Clinical Studies–Local Anesthetics and Cancer Recurrence A very recent RCT compared the rate of breast cancer recurrence after curative surgery in more than 2000 patients receiving either a propofol-based anesthesia in combination with a paravertebral nerve block or a general anesthesia with sevoﬂurane and an opioid-based analgesic regimen. Unfortunately, there was no difference regarding the primary outcome between these two groups [117]. However, this particular and well-executed study once more underlines the importance for more clinical studies evaluating the potential impact J. Clin. Med. 2021, 10, 719 9 of 14 9 of 14 of the systemic use of the drugs in terms of their anti-inﬂammatory or even anti-metastatic effects. These prospective clinical trials focusing on the outcome of patients undergoing can- cer surgery with or without systemic administration of LA are still lacking. Not only, since the 2014 Cochrane review [118], are we aware of the conﬂicting—and not very convincing— data regarding the impact of the perioperative use of regional anesthesia in cancer patients, but some studies have found an effect in some types of cancer [66,67,119–122], and some studies have not [69,123–126] or only in certain cancers in subpopulations [127]. In various articles, the urgent need for clinical trials evaluating the effects of perioperative, systemic administration of LA, e.g., of lidocaine during the perioperative period of cancer patients, is stressed [26,128,129]. Following this call, there are several clinical trials currently investigating a potentially beneﬁcial effect of lidocaine in cancer patients. However, unfortunately, some of these studies do not focus on the anti-metastatic effect of systemically administered lidocaine and follow a more clinical approach and outcome protocol (NCT00938171, NCT03824808, NCT03530033) or do not compare LA vs. placebo (NCT03134430). Some of the studies are still promising though; a currently recruiting double blinded randomized placebo- controlled clinical trial (NCT04048278) is designed to compare the effects of lidocaine infusions on Src activity in CTCs during the perioperative period in patients undergoing robotic surgery for pancreatic cancer. Another clinical trial (NCT04162535) is recruiting 40 patients and focuses on the secondary end points on the survival comparison of intra- venous lidocaine in combination with a propofol-based total intravenous anesthesia (TIVA) compared to standard treatment (TIVA without lidocaine or sevoﬂurane-based general anesthesia) in colorectal cancer surgery. Planned in much larger (n = 450) dimensions is a quadruple-blinded and randomized clinical trial (NCT02786329) in colorectal cancer, again comparing intravenous lidocaine in combination with TIVA, sevoﬂurane versus TIVA, or sevoﬂurane alone. 5.3. Clinical Studies–Local Anesthetics and Cancer Recurrence This particular trial mainly aims to investigate survival after surgery and the incidence of recurrence within the ﬁrst 5 years following surgery. A clinical trial (NCT02839668) in breast cancer patients has already completed the recruitment phase and investigates the use of intravenous lidocaine (1.5 mg/kg) in addition to either TIVA or sevoﬂurane. 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Author Contributions: Conceptualization: T.P., writing—original draft preparation: all authors, writing—review and editing: T.P. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. 10 of 14 J. Clin. Med. 2021, 10, 719 Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Informed Consent Statement: Not applicable. References Anesthetics, immune cells, and immune responses. J. Anesth. 2008, 22, 263–277. [CrossRef] [PubMed] 34. Ogawa, K.; Hirai, M.; Katsube, T.; Murayama, M.; Hamaguchi, K.; Shimakawa, T.; Naritake, Y.; Hosokawa, T.; Kajiwara, T. p 34. 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https://openalex.org/W4239269391	https://research.rug.nl/files/64758338/6759be65_0923_41ea_8cdb_22f1a02df53a_12583_victoria_jackson_v3.pdf	English	null	Meta-analysis of exome array data identifies six novel genetic loci for lung function	Wellcome open research	2,018	cc-by	23,997	Meta-analysis of exome array data identifies six novel genetic loci for lung function Understanding Society Scientific Group Published in: Wellcome open research DOI: 10.12688/wellcomeopenres.12583.3 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publication date: 2018 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Understanding Society Scientific Group (2018). Meta-analysis of exome array data identifies six novel genetic loci for lung function. Wellcome open research, 3(4). https://doi.org/10.12688/wellcomeopenres.12583.3 Citation for published version (APA): Understanding Society Scientific Group (2018). Meta-analysis of exome array data identifies six novel genetic loci for lung function. Wellcome open research, 3(4). https://doi.org/10.12688/wellcomeopenres.12583.3 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). University of Groningen Meta-analysis of exome array data identifies six novel genetic loci for lung function Understanding Society Scientific Group Published in: Wellcome open research DOI: 10.12688/wellcomeopenres.12583.3 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2018 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Understanding Society Scientific Group (2018). Meta-analysis of exome array data identifies six novel genetic loci for lung function. Wellcome open research, 3(4). https://doi.org/10.12688/wellcomeopenres.12583.3 University of Groningen Meta-analysis of exome array data identifies six novel genetic loci for lung function Understanding Society Scientific Group Published in: Wellcome open research DOI: 10.12688/wellcomeopenres.12583.3 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2018 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Understanding Society Scientific Group (2018). Meta-analysis of exome array data identifies six novel genetic loci for lung function. Wellcome open research, 3(4). https://doi.org/10.12688/wellcomeopenres.12583.3 University of Groningen Meta-analysis of exome array data identifies six novel genetic loci for lung function Understanding Society Scientific Group Copyright Smith , Gail Davies , Kristin M. Burkart , Josyf C. Mychaleckyj , Tobias N. Bonten , Stefan Enroth , Lars Lind , Guy G. Brusselle , Ashish Kumar , Beate Stubbe , Understanding Society Scientific Group, Mika Kähönen , Annah B. Wyss , Bruce M. Psaty , Susan R. Heckbert , Ke Hao , Taina Rantanen , Stephen B. Kritchevsky , Kurt Lohman , Tea Skaaby , Charlotta Pisinger , Torben Hansen , Holger Schulz , Ozren Polasek , Archie Campbell , John M. Starr , Stephen S. Rich , Dennis O. Mook-Kanamori , Åsa Johansson , Erik Ingelsson , André G. Uitterlinden , Stefan Weiss , Olli T. Raitakari , Vilmundur Gudnason , Kari E. North , Sina A. Gharib , Don D. Sin , Kent D. Taylor , George T. O'Connor , Jaakko Kaprio , Tamara B. Harris , Oluf Pederson , Henrik Vestergaard , James G. Wilson , Konstantin Strauch , Caroline Hayward , Shona Kerr , Ian J. Deary , 1 2 1,3 4,5 6 7 8 9 10 11 12 13,14 15,16 15 17 18 19 20,21 22 23,24 25 26 27 28,29 30,31 32 33 34,35 5 36 37 37 38 39,40 9 11 13,41 42 15 16 43-45 46 18 47,48 19 49 20,50 23 22 51,52 26 53 28,54,55 27,30,31,56 57 58,59 60 61,62 63 64,65 66 67 42 43 43 15 47,68 69 12 20,70 22 25,52 26 71,72 54,73 74,75 76,77 4,5 78 79 8,80 81 82,83 11,84,85 86 15 15,16 87 88,89 19 19 20,50 RESEARCH ARTICLE Meta-analysis of exome array data identifies six novel genetic loci for lung function [version 3; referees: 2 approved] Victoria E. Jackson , Jeanne C. Latourelle , Louise V. Wain , Albert V. Smith , Megan L. Grove , Traci M. Bartz , Ma'en Obeidat , Michael A. Province , Wei Gao , Beenish Qaiser , David J. Porteous , Patricia A. Cassano , Tarunveer S. Ahluwalia , Niels Grarup , Jin Li , Elisabeth Altmaier , Jonathan Marten , Sarah E. Harris , Ani Manichaikul , Tess D. Pottinger , Ruifang Li-Gao , Allan Lind-Thomsen , Anubha Mahajan , Lies Lahousse , Medea Imboden , Alexander Teumer , Bram Prins , Leo-Pekka Lyytikäinen , Gudny Eiriksdottir , Nora Franceschini , Colleen M. Sitlani , Jennifer A. Copyright strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of th or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. down policy believe that this document breaches copyright please contact us providing details, and we will remove access to the wo vestigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 RESEARCH ARTICLE Meta-analysis of exome array data identifies six novel genetic loci for lung function [version 3; referees: 2 approved] Victoria E. Jackson , Jeanne C. Latourelle , Louise V. Wain , Albert V. Smith , Megan L. Grove , Traci M. Bartz , Ma'en Obeidat , Michael A. Province , Wei Gao , Beenish Qaiser , David J. Porteous , Patricia A. Cassano , Tarunveer S. Ahluwalia , Niels Grarup , Jin Li , Elisabeth Altmaier , Jonathan Marten , Sarah E. Harris , Ani Manichaikul , Tess D. Pottinger , Ruifang Li-Gao , Allan Lind-Thomsen , Anubha Mahajan , Lies Lahousse , Medea Imboden , Alexander Teumer , Bram Prins , Leo-Pekka Lyytikäinen , Gudny Eiriksdottir , Nora Franceschini , Colleen M. Sitlani , Jennifer A. Brody , Yohan Bossé , Wim Timens , Aldi Kraja , Anu Loukola , Wenbo Tang , Yongmei Liu , Jette Bork-Jensen , Johanne M. Justesen , Allan Linneberg , Leslie A. Lange , Rajesh Rawal , Stefan Karrasch , Jennifer E. Huffman , Blair H. Copyright London 60 23,90 25 26 27,91 54,92,93 30,31 57,94 33 34,35 95 10 6 96 1,3 60 Department of Health Sciences, University of Leicester, Leicester, UK 1 National Institute for Health Research, Leicester Respiratory Biomedical Research Unit, Glenfield Hospital, Leicester, UK Icelandic Heart Association 201 Kopavogur Iceland 3 4 man Genetics Center, Department of Epidemiology, Human Genetics, and Environmental Sciences, School of Pub ersity of Texas Health Science Center at Houston, Houston, TX, 77030, USA y Cardiovascular Health Research Unit, Departments of Medicine and Biostatistics, University of Washington, Seattle, WA, 98101, USA 7 8 diovascular Health Research Unit, Departments of Medicine and Biostatistics, University of Washington, Seattle, W University of British Columbia Centre for Heart Lung Innovation, St Paul’s Hospital, Vancouver, BC, Canada Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA 9 Department of Genetics, Washington University School of Medicine, St. Copyright Brody , Yohan Bossé , Wim Timens , Aldi Kraja , Anu Loukola , Wenbo Tang , Yongmei Liu , Jette Bork-Jensen , Johanne M. Justesen , Allan Linneberg , Leslie A. Lange , Rajesh Rawal , Stefan Karrasch , Jennifer E. Huffman , Blair H. Smith , Gail Davies , Kristin M. Burkart , Josyf C. Mychaleckyj , Tobias N. Bonten , Stefan Enroth , Lars Lind , Guy G. Brusselle , Ashish Kumar , Beate Stubbe , Understanding Society Scientific Group, Mika Kähönen , Annah B. Wyss , Bruce M. Psaty , Susan R. Heckbert , Ke Hao , Taina Rantanen , Stephen B. Kritchevsky , Kurt Lohman , Tea Skaaby , Charlotta Pisinger , Torben Hansen , Holger Schulz , Ozren Polasek , Archie Campbell , John M. Starr , Stephen S. Rich , Dennis O. Mook-Kanamori , Åsa Johansson , Erik Ingelsson , André G. Uitterlinden , Stefan Weiss , Olli T. Raitakari , Vilmundur Gudnason , Kari E. North , Sina A. Gharib , Don D. Sin , Kent D. Taylor , George T. O'Connor , Jaakko Kaprio , Tamara B. Harris , Oluf Pederson , Henrik Vestergaard , James G. Wilson , Konstantin Strauch , Caroline Hayward , Shona Kerr , Ian J. Deary , 1 2 1,3 4,5 6 7 8 9 10 11 12 13,14 15,16 15 17 18 19 20,21 22 23,24 25 26 27 28,29 30,31 32 33 34,35 5 36 37 37 38 39,40 9 11 13,41 42 15 16 43-45 46 18 47,48 19 49 20,50 23 22 51,52 26 53 28,54,55 27,30,31,56 57 58,59 60 61,62 63 64,65 66 67 42 43 43 15 47,68 69 12 20,70 22 25,52 26 71,72 54,73 74,75 76,77 4,5 78 79 8,80 81 82,83 11,84,85 86 15 15,16 87 88,89 19 19 20,50 Page 1 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 R. Graham Barr , Renée de Mutsert , Ulf Gyllensten , Andrew P. Morris , M. Arfan Ikram , Nicole Probst-Hensch , Sven Gläser , Eleftheria Zeggini , Terho Lehtimäki , David P. Strachan , Josée Dupuis , Alanna C. Morrison , Ian P. Hall , Martin D. Tobin , Stephanie J. Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS, 39216, USA 7 Copyright We carried out meta-analyses of exome array data and three lung Methods: function measures: forced expiratory volume in one second (FEV ), forced vital capacity (FVC) and the ratio of FEV to FVC (FEV /FVC). These analyses by the SpiroMeta and CHARGE consortia included 60,749 individuals of European ancestry from 23 studies, and 7,721 individuals of African Ancestry from 5 studies in the discovery stage, with follow-up in up to 111,556 independent individuals. We identified significant (P<2·8x10 ) associations with six SNPs: a Results: nonsynonymous variant in , which is predicted to be damaging, three RPAP1 intronic SNPs ( and ) and two intergenic SNPs near SEC24C, CASC17 UQCC1 to and Expression quantitative trait loci analyses found evidence LY86 FGF10. for regulation of gene expression at three signals and implicated several genes, including and . TYRO3 PLAU Further interrogation of these loci could provide greater Conclusions: understanding of the determinants of lung function and pulmonary disease. Keywords Lung function, respiratory, exome array, GWAS, COPD Martin D. Tobin ( ), Stephanie J. London ( ) Corresponding authors: martin.tobin@le.ac.uk london2@niehs.nih.gov 88 89 90 91 92 93 94 95 96 Referee Status: Invited Referees version 3 published 07 Aug 2018 version 2 published 21 Jun 2018 version 1 published 12 Jan 2018 1 2 report report report , University of Robin Beaumont Exeter, UK , University of Rachel M. Freathy Exeter, UK 1 , Hospital for Sick Children, Lisa Strug Canada , The Hospital for Sick Naim Panjwani Children, Canada 2 12 Jan 2018, :4 (doi: ) First published: 3 10.12688/wellcomeopenres.12583.1 21 Jun 2018, :4 (doi: ) Second version: 3 10.12688/wellcomeopenres.12583.2 07 Aug 2018, :4 (doi: ) Latest published: 3 10.12688/wellcomeopenres.12583.3 v3 1 1 1 -7 Copyright Louis, MO, USA Department of Biostatistics, Boston University School of Public Health, Boston, MA, USA 10 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, FI-00014, Helsinki, Finland 11 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, FI-00014, Helsinki, Finland 11 12 Centre for Genomic & Experimental Medicine, MRC Institute of Genetics & Molecular Medicine, University of Edinburgh, Edinburgh, EH4 2XU, UK 12 Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA 13 Department of Healthcare Policy and Research, Division of Biostatistics and Epidemiology, Weill Cornell Medical College, New York City, NY, USA 14 Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark 15 Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Palo Alto, CA, USA 17 Research Unit of Molecular Epidemiology, Institute of Epidemiology II, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany 18 Environmental Health, 85764 Neuherberg, Germany Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of E 2XU, UK 19 Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, EH8 9JZ, UK 20 Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, EH8 9JZ, UK 20 Centre for Genomic and Experimental Medicine, University of Edinburgh, Edinburgh, EH4 2XU, UK 21 Center for Public Health Genomics, University of Virginia, Charlottesville, VA, USA 22 Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY, USA 23 Department of Preventive Medicine - Division of Health and Biomedical Informatics, Northwestern University - Feinberg School of Medicine, Chicago IL USA 24 Department of Preventive Medicine - Division of Health and Biomedical Informatics, Northwestern University - Feinberg School of Medicine, Chicago, IL, USA 24 g Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, 2333 ZA, Netherlands 25 partment of Clinical Epidemiology, Leiden University Medical Center, Leiden, 2333 ZA, Netherlands Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, 2333 ZA, Netherlands 25 26 Department of Immunology, Genetics, and Pathology, Biomedical Center, SciLifeLab Uppsala, Uppsala Universit Sweden 6 Department of Immunology, Genetics, and Pathology, Biomedical Center, SciLifeLab Uppsala, Uppsala Univ Sweden 26 Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK 7 Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK 27 Respiratory Medicine, Ghent University Hospital, Ghent, BE9000, Belgium 28 Respiratory Medicine, Ghent University Hospital, Ghent, BE9000, Belgium 28 Bioanalysis, Ghent University, Ghent, BE9000, Belgium 29 Swiss Tropical and Public Health Institute, Basel, Switzerland 30 University of Basel, Basel, Switzerland 31 Institute for Community Medicine, University Medicine Greifswald, 17475 Greifswald, Germany 32 Institute for Community Medicine, University Medicine Greifswald, 17475 Greifswald, Germany 32 Human Genetics, Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK 33 Human Genetics, Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK 33 Department of Clinical Chemistry, Fimlab Laboratories, Tampere 33520, Finland 34 Department of Clinical Chemistry, Faculty of Medicine and Life Sciences, Univers 35 Page 2 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, NL9713 GZ, Netherlands Groningen Research Institute for Asthma and COPD, University Medical Center Groningen, University of Groningen, Groningen, Netherlands 39 40 Boehringer Ingelheim , Danbury, CT, USA 41 Boehringer Ingelheim , Danbury, CT, USA 41 Wake Forest School of Medicine, Winston-Salem, North Carolina, USA 42 Wake Forest School of Medicine, Winston-Salem, North Carolina, USA 42 Centre for Clinical Research and Prevention, Bispebjerg and Frederiksberg Hospital, The Capital Region, Co 43 Research and Prevention, Bispebjerg and Frederiksberg Hospital, The Capital Region, Copenhagen, Denmark nical Experimental Research Rigshospitalet 2600 Glostrup Denmark Centre for Clinical Research and Prevention, Bispebjerg and Frederiksberg Hospital, The Capital Region, Copenhagen, Denmark Department of Clinical Experimental Research, Rigshospitalet, 2600 Glostrup, Denmark 43 44 Centre for Clinical Research and Prevention, Bispebjerg and Frederiksberg Hospital, The Capital Region, Copenhagen, Denmark Department of Clinical Experimental Research, Rigshospitalet, 2600 Glostrup, Denmark 44 Department of Clinical Experimental Research, Rigshospitalet, 2600 Glostrup, Denmark 4 Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark Department of Medicine, Division of Bioinformatics and Personalized Medicine, University of Colorado Denver, Aurora, CO, USA Institute of Epidemiology I, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany Institute and Outpatient Clinic for Occupational, Social and Environmental Medicine, Ludwig-Maximilians-Universität, Munich, Germany Division of Population Health Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK 45 46 47 48 49 Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark Department of Medicine, Division of Bioinformatics and Personalized Medicine, University of Colorado Denver, Aurora, CO, USA Institute of Epidemiology I Helmholtz Zentrum München German Research Center for Environmental Health 85764 Neuherberg Germany 45 46 47 Department of Medicine, Division of Bioinformatics and Personalized Medicine, University of Colorado Denver, Aurora, CO, USA 46 p gy , , , g, y Institute and Outpatient Clinic for Occupational, Social and Environmental Medicine, Ludwig-Maximilians-Universität, Munich, Germany Di i i f P l ti H lth S i Ni ll H it l d M di l S h l U i it f D d D d DD1 9SY UK 48 49 Institute and Outpatient Clinic for Occupational, Social and Environmental Medicine, Ludwig-Maximilians-Universität, Munich, Germany 48 Division of Population Health Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee, D 9 Department of Psychology, University of Edinburgh, Edinburgh, EH8 9JZ, UK 50 Department of Psychology, University of Edinburgh, Edinburgh, EH8 9JZ, UK 0 Department of Pulmonology, Leiden University Medical Center, Leiden, 2333 ZA, Netherlands 1 Department of Pulmonology, Leiden University Medical Center, Leiden, 2333 ZA, Netherlands 51 Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, 2333 ZA, Netherlands 2 Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, 233 52 Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden 53 Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden 3 Epidemiology, Erasmus Medical Center, Rotterdam, 3000CA, Netherlands 54 Epidemiology, Erasmus Medical Center, Rotterdam, 3000CA, Netherlands 54 Respiratory Medicine, Erasmus Medical Center, Rotterdam, 3000CA, Netherlands 5 Respiratory Medicine, Erasmus Medical Center, Rotterdam, 3000CA, Netherlands 55 Internal Medicine B, University Medicine Greifswald, Greifswald, 17475, Germany 57 Internal Medicine B, University Medicine Greifswald, Greifswald, 17475, Germany 57 Department of Clinical Physiology, Tampere University Hospital, Tampere, 33521, Finland 58 Department of Clinical Physiology, Tampere University Hospital, Tampere, 33521, Finland 8 Department of Clinical Physiology, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, 33014, Finland 59 Department of Clinical Physiology, Faculty of Medicine and Life Sciences, University of Tamp 59 Epidemiology Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Dept of H Services, Research Triangle Park, NC, 27709, USA 0 Services, Research Triangle Park, NC, 27709, USA , g , , , Cardiovascular Health Research Unit, Departments of Epidemiology, Medicine and Health Services, University of Washington, Seattle, WA, 98101, USA 61 ar Health Research Unit, Departments of Epidemiology, Medicine and Health Services, University of Washington, S Kaiser Permanente Washington Health Research Institute, Seattle, WA, USA 2 Kaiser Permanente Washington Health Research Institute, Seattle, WA, USA 62 Cardiovascular Health Research Unit, Department of Epidemiology, University of Washington, Seattle, WA, 9810 3 Cardiovascular Health Research Unit, Department of Epi Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029-6574, USA 64 Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029-6574, USA 64 epartment of Genetics and Genomic Sciences, Icahn Sch Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 1002 5 Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, 65 Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029-6574, USA Department of Health Sciences, University of Jyväskylä, Jyväskylä, Fl-40014, Finland 66 Department of Health Sciences, University of Jyväskylä, Jyväskylä, Fl-40014, Finland 6 Department of Health Sciences, University of Jyväskylä, Jyväskylä, Fl-40014, Finland 66 Sticht Center on Aging, Wake Forest School of Medicine, Winston-Salem, NC, USA 7 Sticht Center on Aging, Wake Forest School of Medicine, Winston-Salem, NC, USA 67 mprehensive Pneumology Center Munich (CPC-M), Member of the German Center for Lung Research, Munich, G Comprehensive Pneumology Center Munich (CPC-M), Member of the German Center for Lung Research, Munich, Germany Faculty of Medicine University of Split Split Croatia 68 69 Comprehensive Pneumology Center Munich (CPC-M), Member of the German Center 68 Comprehensive Pneumology Center Munich (CPC-M), M Faculty of Medicine, University of Split, Split, Croatia 69 zheimer Scotland Research Centre, University of Edinburgh, Edinburgh, EH8 9JZ, UK Alzheimer Scotland Research Centre, University of Edinburgh, Edinburgh, EH8 9JZ, UK 70 Alzheimer Scotland Research Centre, University of Ed 70 Department of Medical Sciences, Molecular Epidemiology and Science for Life Laboratory, Uppsala Universit 71 Department of Medical Sciences, Molecular Epidemiology and Science for Life Laboratory, Uppsala University, Uppsala, Sweden 71 Department of Medicine, Division of Cardiovascular Medicine, Stanford Universit 72 Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanfo 72 Internal Medicine, Erasmus Medical Center, Rotterdam, 3000CA, Netherlands 73 Internal Medicine, Erasmus Medical Center, Rotterdam, 3000CA, Netherlands 73 Internal Medicine, Erasmus Medical Center, Rotterdam, 3000CA, Netherlands 73 aculty Institute for Genetics and Functional Genomics, Un or Genetics and Functional Genomics, University Medicin DZHK (German Centre for Cardiovascular Research), partner site: Greifswald, Greifswald, Germany 75 DZHK (German Centre for Cardiovascular Research), partner site: Greifswald, Greifswald, Germany 75 Department of Clinical Physiology and Nuclear Medicine, Turku University Hospital, Turku, 20521, Finland 76 Research Centre of Applied and Preventative Cardiovascular Medicine, University of Turku, Turku, 20014, Finland 77 Research Centre of Applied and Preventative Cardiovascular Medicine, University of Turku, Turku, 20014, Fi 77 Department of Epidemiology and Carolina Center for Genome Science, University of North Carolina, Chapel H 78 Computational Medicine Core, Center for Lung Biology, UW Medicine Sleep Center, Department of Medicine, University of Washington, Seattle, WA, 98109, USA 79 Computational Medicine Core, Center for Lung Biology, UW Medicine Sleep Center, Department of Medicine, Un Seattle, WA, 98109, USA 9 Respiratory Division, Department of Medicine, University of British Columbia, Vancouver, BC, Canada 80 Institute for Translational Genomics and Population Sciences and Department of Pediatrics, Los Angeles Biomedical Research Institute at 81 Institute for Translational Genomics and Population Sciences and Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, 90502, USA 81 Institute for Translational Genomics and Population Sciences and Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, 90502, USA 81 Harbor-UCLA Medical Center, Torrance, CA, 90502, USA Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, MA, 02118, USA 82 Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, MA, 02118, USA 82 Pulmonary Center, Department of Medicine, Boston Univ National Heart, Lung and Blood Institute's and Boston University's Framingham Heart Study, Framingham, MA, 0 3 National Heart, Lung and Blood Institute's and Boston University's Framin 83 Department of Health, University of Helsinki, Helsinki, FI-00014, Finland 84 Department of Public Health, National Institute for Health and Welfare, Helsinki, FI-00271, Finland 85 Department of Public Health, National Institute for Health and Welfare, Hel 85 National Institute on Aging, National Institutes of Health, Bethesda, MD, 20892, USA 86 g g, , , , , Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS, 39216, USA 87 Page 3 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 Open Peer Review Discuss this article (0) Comments Institute of Genetic Epidemiology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, 85764, Germany Chair of Genetic Epidemiology, IBE, Faculty of Medicine, LMU Munich, Munich, 81377, Germany Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, NY, 10032, USA Department of Biostatistics, University of Liverpool, Liverpool, L69 3GL, UK Radiology, Erasmus Medical Center, Rotterdam, 3000CA, Netherlands Neurology, Erasmus Medical Center, Rotterdam, 3000CA, Netherlands Department of Internal Medicine - Pulmonary Diseases, Vivantes Klinikum Spandau Berlin, Berlin, 13585, Germany Population Health Research Institute, St George's, University of London, London, SW17 0RE, UK NIHR Nottingham Biomedical Research Centre and Division of Respiratory Medicine, University of Nottingham, Nottingham, NG7 2UH, UK Abstract Over 90 regions of the genome have been associated with lung Background: function to date, many of which have also been implicated in chronic obstructive pulmonary disease. Keywords Lung function, respiratory, exome array, GWAS, COPD Abstract Over 90 regions of the genome have been associated with lung Background: function to date, many of which have also been implicated in chronic obstructive pulmonary disease. We identified significant (P<2·8x10 ) associations with six SNPs: a Results: nonsynonymous variant in , which is predicted to be damaging, three RPAP1 intronic SNPs ( and ) and two intergenic SNPs near SEC24C, CASC17 UQCC1 to and Expression quantitative trait loci analyses found evidence LY86 FGF10. for regulation of gene expression at three signals and implicated several genes, including and . TYRO3 PLAU -7 Further interrogation of these loci could provide greater Conclusions: understanding of the determinants of lung function and pulmonary disease. Martin D. Tobin ( ), Stephanie J. Abstract London ( ) Corresponding authors: martin.tobin@le.ac.uk london2@niehs.nih.gov Page 4 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 : Formal Analysis, Writing – Original Draft Preparation; : Formal Analysis, Writing – Review & Editing; Author roles: Jackson VE Latourelle JC : Formal Analysis, Supervision, Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Review & Editing; Wain LV Smith AV Grove : Data Curation, Writing – Review & Editing; : Formal Analysis, Writing – Review & Editing; : Formal Analysis, Writing – ML Bartz TM Obeidat M Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Formal Analysis, Writing – Review & Province MA Gao W Editing; : Formal Analysis, Writing – Review & Editing; : Data Curation; : Data Curation, Formal Analysis, Qaiser B Porteous DJ Cassano PA Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Conceptualization, Data Ahluwalia TS Grarup N Curation, Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Review & Editing; : Formal Analysis, Writing – Li J Altmaier E Review & Editing; : Formal Analysis, Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Review & Editing; Marten J Harris SE : Data Curation, Formal Analysis, Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Review & Manichaikul A Pottinger TD Editing; : Data Curation, Formal Analysis, Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Li-Gao R Lind-Thomsen A Review & Editing; : Formal Analysis, Writing – Review & Editing; : Conceptualization, Data Curation, Formal Analysis, Mahajan A Lahousse L Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Review & Editing; : Data Curation, Formal Analysis, Imboden M Teumer A Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Review & Editing; : Data Curation, Formal Analysis, Prins B Lyytikäinen LP Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Formal Analysis, Eiriksdottir G Franceschini N Writing – Review & Editing; : Formal Analysis, Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Review Sitlani CM Brody JA & Editing; : Data Curation, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Data Curation, Bossé Y Timens W Kraja A Formal Analysis, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Data Curation, Formal Analysis, Loukola A Tang W Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Review & Editing; : Conceptualization, Data Curation, Liu Y Bork-Jensen J Writing – Review & Editing; : Formal Analysis, Writing – Review & Editing; : Conceptualization, Writing – Review & Justesen JM Linneberg A Editing; : Data Curation, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Data Curation, Lange LA Rawal R Karrasch S Writing – Review & Editing; : Formal Analysis, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; Huffman JE Smith BH : Data Curation, Writing – Review & Editing; : Conceptualization, Writing – Review & Editing; : Data Davies G Burkart KM Mychaleckyj JC Curation, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Bonten TN Enroth S Review & Editing; : Data Curation, Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; Lind L Brusselle GG : Data Curation, Formal Analysis, Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; Kumar A Stubbe B : Conceptualization, Data Curation, Writing – Review & Editing; : Conceptualization, Formal Analysis, Writing – Review & Kähönen M Wyss AB Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Psaty BM Heckbert SR Hao K Data Curation, Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Rantanen T Kritchevsky SB Conceptualization, Data Curation, Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Review & Editing; : Lohman K Skaaby T Conceptualization, Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Pisinger C Hansen T Conceptualization, Data Curation, Formal Analysis, Writing – Review & Editing; : Conceptualization, Writing – Review & Editing; Schulz H Polasek : Conceptualization, Data Curation, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Data Curation, O Campbell A Starr JM Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Conceptualization, Data Rich SS Mook-Kanamori DO Curation, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Data Curation, Writing – Review & Johansson Å Ingelsson E Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Uitterlinden AG Weiss S Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Conceptualization, Formal Analysis, Raitakari OT Gudnason V Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Writing – Review & Editing; : Data Curation, North KE Gharib SA Sin DD Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; Taylor KD O'Connor GT : Conceptualization, Data Curation, Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; Kaprio J Harris TB : Data Curation, Formal Analysis, Writing – Review & Editing; : Data Curation, Formal Analysis, Writing – Review & Pederson O Vestergaard H Editing; : Data Curation, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Wilson JG Strauch K Hayward C Conceptualization, Data Curation, Formal Analysis, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Data Kerr S Deary IJ Curation, Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Conceptualization, Barr RG de Mutsert R Data Curation, Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Data Curation, Gyllensten U Morris AP Formal Analysis, Writing – Review & Editing; : Conceptualization, Writing – Review & Editing; : Conceptualization, Ikram MA Probst-Hensch N Data Curation, Formal Analysis, Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Gläser S Zeggini E Conceptualization, Writing – Review & Editing; : Conceptualization, Data Curation, Writing – Review & Editing; : Lehtimäki T Strachan DP Conceptualization, Data Curation, Writing – Review & Editing; : Formal Analysis, Supervision, Writing – Review & Editing; : Dupuis J Morrison AC Formal Analysis, Writing – Review & Editing; : Conceptualization, Formal Analysis, Supervision, Writing – Review & Editing; : Hall IP Tobin MD Conceptualization, Formal Analysis, Supervision, Writing – Review & Editing; : Conceptualization, Formal Analysis, Supervision, London SJ Writing – Review & Editing Writing – Review & Editing; : Data Curation, Formal Analysis, Writin Prins B No competing interests were disclosed. Abstract Competing interests: MDT has been supported by MRC fellowships G0501942 and G0902313. MDT and LVW are supported by the MRC Grant information: (MR/N011317/1). IPH is supported by the MRC (G1000861). ALW and SJL are supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (ZIA ES 043012). We acknowledge use of phenotype and genotype data from the British 1958 Birth Cohort DNA collection, funded by the Medical Researanch Council grant G0000934 and the Wellcome Trust grant 068545/Z/02. APM was a Wellcome Trust Senior Fellow in Basic Biomedical Science (grant number WT098017) and was also supported by Wellcome Trust grant WT064890. EI is supported by the Swedish Research Council (2012-1397), Knut och Alice Wallenberg Foundation (2013.0126) and the Swedish Heart-Lung Foundation (20140422). JK is supported by Academy of Finland Center of Excellence in Complex Disease Genetics grants 213506, 129680 and Academy of Finland grants 265240, 263278. The Finnish Twin Cohort is supported by the Welcome Trust Sanger Institute, UK. The Lothian Birth Cohort is supported by Age UK (The Disconnected Mind Project), the UK Medical Research Council (MR/K026992/1) and The Royal Society of Edinburgh. ÅJ is supported by the Swedish Society for Medical Research (SSMF), The Kjell och Märta Beijers Foundation, The Marcus Borgström Foundation, The Åke Wiberg foundation and The Vleugels Foundation. UG is supported by Swedish Medical Research Council grants K2007-66X-20270-01-3 and 2011-2354 and European Commission FP6 (LSHG-CT-2006-01947). SHIP is part of the Community Medicine Page 5 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 Research net of the University of Greifswald, Germany, which is funded by the Federal Ministry of Education and Research, the Ministry of Cultural Affairs as well as the Social Ministry of the Federal State of Mecklenburg-West Pomerania, and the network ‘Greifswald Approach to Individualized Medicine (GANI_MED)’ funded by the Federal Ministry of Education and Research, and the German Asthma and COPD Network (COSYCONET) (grant no.01ZZ9603, 01ZZ0103, 01ZZ0403, 03IS2061A, BMBF 01GI0883). ExomeChip data have been supported by the Federal Ministry of Education and Research (grant no. 03Z1CN22) and the Federal State of Mecklenburg-West Pomerania. The University of Greifswald is a member of the Caché Campus program of the InterSystems GmbH. UKHLS is supported by grants WT098051 (Wellcome Trust) and ES/H029745/1 (Economic and Social Research Council). Y.B. holds a Canada Research Chair in Genomics of Heart and Lung Diseases. Abstract Lies Lahousse is a Postdoctoral Fellow of the Research Foundation - Flanders (FWO grant G035014N). The Rotterdam Study is funded by Erasmus Medical Center and Erasmus University, Rotterdam, the Netherlands Organization for Scientific Research (NOW), the Netherlands Organization for the Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. Genotyping in the Rotterdam study was supported by Netherlands Organization for Scientific Research (NOW grants 175.010.2005.011 ; 911-03-305 012), the Research Institute for Diseases in the Elderly (RIDE2 grants 014-93-015) and Netherlands Genomics Initiative (NGI)/Netherlands Consortium for Healthy Aging (NCHA grant050-060-810). MESA/MESA SHARe is supported by HHS (HHSN268201500003I), NIH/NHLBI (contracts N01-HC-95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169) and HIH/NCATS (contracts UL1-TR-000040, UL1-TR-001079, UL1-TR-001881, DK063491). MESA SHARe is funded by NIH/NHLBI contract N02-HL-64278, MESA Air is funded by US EPA (RD831697) and MESA Spirometry funded by NIH/NHLBI (R01-HL077612). SSR and BMP are supported by NIH/NHLBI grant rare variants and NHLBI traits in deeply phenotyped cohorts (R01-HL120393). Cardiovascular Health Study: This CHS research was supported by NHLBI contracts HHSN268201200036C, HHSN268200800007C, HHSN268201800001C, HHSN268200960009C, N01HC55222, N01HC85079, N01HC85080, N01HC85081, N01HC85082, N01HC85083, N01HC85086; and NHLBI grants U01HL080295, R01HL068986, R01HL087652, R01HL105756, R01HL103612, R01HL120393, and R01HL130114 with additional contribution from the National Institute of Neurological Disorders and Stroke (NINDS). Additional support was provided through R01AG023629 and R01HL085251 from the National Institute on Aging (NIA). The provision of genotyping data was suprovidedpported in part by the National Center for Advancing Translational Sciences, CTSI grant UL1TR001881, and the National Institute of Diabetes and Digestive and Kidney Disease Diabetes Research Center (DRC) grant DK063491 to the Southern California Diabetes Endocrinology Research Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The Atherosclerosis Risk in Communities (ARIC) study is carried out as a collaborative study supported by the National Heart, Lung, and Blood Institute (NHLBI) contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C). Funding support for “Building on GWAS for NHLBI-diseases: the U.S. CHARGE consortium” was provided by the NIH through the American Recovery and Reinvestment Act of 2009 (ARRA) (5RC2HL102419). DOMK received funding from the Dutch Science Organisation (ZonMW-VENI Grant 916.14.023). Abstract The genotyping in the NEO study was supported by the Centre National de Génotypage (Paris, France), headed by Jean-François Deleuze. The NEO study is supported by the participating Departments, the Division and the Board of Directors of the Leiden University Medical Center, and by the Leiden University, Research Profile Area Vascular and Regenerative Medicine. SAPALDIA was supported by the Swiss National Science Foundation (grants no 33CS30-148470/1, 33CSCO-134276/1, 33CSCO-108796, , 324730_135673, 3247BO-104283, 3247BO-104288, 3247BO-104284, 3247-065896, 3100-059302, 3200-052720, 3200-042532, 4026-028099, PMPulDP3_129021/1, PMPDP3_141671/1), the Federal Office for the Environment, the Federal Office of Public Health, the Federal Office of Roads and Transport, the canton's government of Aargau, Basel-Stadt, Basel-Land, Geneva, Luzern, Ticino, Valais, and Zürich, the Swiss Lung League, the canton's Lung League of Basel Stadt/ Basel Landschaft, Geneva, Ticino, Valais, Graubünden and Zurich, Stiftung ehemals Bündner Heilstätten, SUVA, Freiwillige Akademische Gesellschaft, UBS Wealth Foundation, Talecris Biotherapeutics GmbH, Abbott Diagnostics, European Commission 018996 (GABRIEL), Wellcome Trust WT 084703MA. The Novo Nordisk Foundation Center for Basic Metabolic Research is an independent Research Center at the University of Copenhagen partially funded by an unrestricted donation from the Novo Nordisk Foundation (www.metabol.ku.dk). Generation Scotland received core support from the Chief Scientist Office of the Scottish Government Health Directorates [CZD/16/6] and the Scottish Funding Council [HR03006]. Genotyping of the GS:SFHS samples was carried out by the Genetics Core Laboratory at the Edinburgh Clinical Research Facility, University of Edinburgh, Scotland and was funded by the Medical Research Council UK.. The Croatia KORCULA study was supported by the Ministry of Science, Education and Sport in the Republic of Croatia (108-1080315-0302). JD, JCL, WG and GTOC are supported by NIH/NHLBI Contract HHSN268201500001I. Genotyping, quality control and calling of the Illumina HumanExome BeadChip in the Framingham Heart Study was supported by funding from the National Heart, Lung and Blood Institute Division of Intramural Research (Daniel Levy and Christopher J. O’Donnell, Principle Investigators). The AGES study is supported by the NIH (N01-AG012100), the Iceland Parliament (Alþingi) and the Icelandic Heart Association. HABC was supported by NIA contracts N01AG62101, N01AG62103, and N01AG62106; NIA grant R01-AG028050, and NINR grant R01- NR012459 and was supported in part by the Intramural Research Program of the NIH, National Institute on Aging. The HABC genome-wide association study was funded by NIA grant 1R01AG032098- 01A1 and genotyping services were provided by the Center for Inherited Disease Research (CIDR). Jackson VE, Latourelle JC, Wain LV How to cite this article: et al. Meta-analysis of exome array data identifies six novel genetic loci for Wellcome Open Research 2018, :4 (doi: ) lung function [version 3; referees: 2 approved] 3 10.12688/wellcomeopenres.12583.3 12 Jan 2018, :4 (doi: ) First published: 3 10.12688/wellcomeopenres.12583.1 Amendments from Version 2 REVISED Amendments from Version 2 REVISED We have added a further limitation to the discussion of the paper outlining a recently highlighted issue regarding the trait transformation undertaken in our replication analyses. We show through sensitivity analyses that our results are not affected by this issue (Supplementary Figure 4), but note that future studies should avoid such a transformation. Meta-analyses of single variant associations We first evaluated single variant associations between FEV1, FVC and FEV1/FVC and the 179,215 SNPs that passed study level quality control and were polymorphic in both consortia. These analyses identified 34 SNPs in regions not previously associated with lung function, showing association with at least one trait at overall P<10-5, and showing association with consistent direction and P<0·05 in both consortia (full results in Supplementary Table 2, quantile-quantile and Manhattan plots shown in Supplementary Figure 1). We followed up these SNP associations in a replication analysis comprising 3 studies with 111,556 indi- viduals. Combining the results from the discovery and replication stages in a meta-analysis identified six SNPs in total that were independent to known signals and met the pre-defined signifi- cance threshold (P<2·8×10-7) overall in, or near to FGF10, LY86, SEC24C, RPAP1, CASC17 and UQCC1 (Table 2, Supplementary Figure 2). A SNP near to the CASC17 signal (rs11654749, r2=0·3 with rs1859962) has previously been associated with FEV1 in a genome-wide analysis of gene-smoking interactions, although this association was not replicated at the time21; the present analysis provides the first evidence for independent replication of this signal. A seventh signal was also identified in LCT (Table 2, Supplementary Figure 2); whilst this locus has not previously been implicated in lung function, this SNP is known to vary in frequency across European populations22, and we cannot rule out that this association is not an artefact of population structure. Our discovery analysis furthermore identified associations (P<10-5) in 25 regions previously associated with one or more of FEV1, FVC and FEV1/FVC (Supplementary Table 3). See referee reports See referee reports Abstract CIDR is fully funded through a federal contract from the National Institutes of Health to The Johns Hopkins University, contract number HHSN268200782096C. We thank the Jackson Heart Study (JHS) participants and staff for their contributions to this work. The JHS is supported by contracts HHSN268201300046C, HHSN268201300047C, HHSN268201300048C, HHSN268201300049C, HHSN268201300050C from the National Heart, Lung, and Blood Institute and the National Institute on Minority Health and Health Disparities. JGW is supported by U54GM115428 from the National Institute of General Medical Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. e funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. © 2018 Jackson VE . This is an open access article distributed under the terms of the , Copyright: et al Creative Commons Attribution Licence which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The author(s) is/are employees of the US Government and therefore domestic copyright protection in USA does not apply to this work. The work may be protected under the copyright laws of other jurisdictions when used in those jurisdictions. Jackson VE, Latourelle JC, Wain LV How to cite this article: et al. Meta-analysis of exome array data identifies six novel genetic loci for Wellcome Open Research 2018, :4 (doi: ) lung function [version 3; referees: 2 approved] 3 10.12688/wellcomeopenres.12583.3 12 Jan 2018, :4 (doi: ) First published: 3 10.12688/wellcomeopenres.12583.1 Page 6 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 Page 7 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 associations, which test for the joint effect of several rare variants in a gene (see Methods for details). Introduction Measures of lung function act as predictors of mortality and morbidity and form the basis for the diagnosis of several diseases, most notably chronic obstructive pulmonary disease (COPD), one of the leading causes of death globally1. Environmental factors, including smoking and exposure to air pollution play a significant role in lung function; however there has also been shown to be a genetic component, with estimates of the narrow sense heritability ranging between 39–66%2–5. Genome- wide association studies (GWAS) of lung function have iden- tified associations between single nucleotide polymorphisms (SNPs) and lung function at over 150 independent loci to date6–14. Associations have also been identified in GWAS of COPD15–19; however, the identification of disease associated SNPs has been restricted by limited sample sizes. Many signals first identi- fied in powerful studies of quantitative lung function traits, have been found to be associated with risk of COPD, highlighting the potential clinical usefulness of comprehensive identification of lung function associated SNPs13. Low frequency (minor allele frequency (MAF) 1–5%) and rare (MAF<1%) variants have been largely underexplored by GWAS to date. Exome arrays have been designed to facilitate the investigation of these low frequency and rare variants, predomi- nately within coding regions, in large sample sizes. Alongside a core content of rare coding SNPs, the exome array additionally includes common variation, including tags for previously identi- fied GWAS hits, ancestry informative SNPs, a grid of markers for estimating identity by descent and a random selection of synonymous SNPs20. Generally, the observed effect of the SNPs at the novel signals were similar in ever and never smokers; the exception was rs1448044 near FGF10, which showed a significant association with FVC only in ever smokers in our discovery analysis (ever smokers P=1·49×10-6; never smokers P=0·695, Supplementary Table 4 and Supplementary Figure 3). In the replication analysis, however, this association was observed in both ever and never smokers (ever smokers P=3·14×10-5; never smokers P=1·40×10-4, Supplementary Table 5). For rs1200345 (RPAP1) and rs1859962 (CASC17), associations were most statistically significant in the analyses restricted to individuals of European Ancestry (Supplementary Table 4 and Supplementary Figure 3), as was the association with rs2322659 (LCT), giving further support that this association may be due to population stratification. An earlier version of this article can be found on bioRxiv (https://doi.org/10.1101/164426) Table 1. Sample characteristics of 11 SpiroMeta and 12 CHARGE studies contributing to the discovery analyses and three studies contributing to the replication analyses. Table 1. Sample characteristics of 11 SpiroMeta and 12 CHARGE studies contributing to the discovery analyses and three studies contributing to the replication analyses. Table 1. Sample characteristics of 11 SpiroMeta and 12 CHARGE studies contributing to the discovery analyses and three studies contributing to the replication analyses. Discovery studies SpiroMeta studies Total sample n (%) Male Ever smokers, n (%) Age, mean (SD) FEV1, litres. mean (SD) FVC, litres. mean (SD) FEV1/FVC, mean (SD) 1958 British Birth Cohort (B58C) 5270 2961 (56·2%) 2866 (53·3%) 44·00 (0·00) 3·35 (0·79) 4·29 (1·03) 0·788 (0·09) Generation Scotland (GS:SFHS) 8164 3413 (41·8%) 3806 (46·6%) 51·59 (13·33) 2·78 (0·87) 3·91 (1·01) 0·710 (0·12) Cooperative Health Research in the Region of Augsburg (KORA F4) 1447 701 (48·5%) 900 (62·2%) 54·82 (9·66) 3·24 (0·85) 4·20 (1·04) 0·771 (0·07) CROATIA-Korcula cohort (KORCULA) 791 296 (36·8%) 418 (52·0%) 55·56 (13·69) 2·72 (0·83) 3·29 (0·95) 0·829 (0·10) Lothian Birth Cohort 1936 (LBC1936) 974 501 (50·6%) 554 (55·9%) 69·55 (0·84) 2·38 (0·67) 3·04 (0·87) 0·787 (0·10) Study of Health in Pomerania (SHIP) 1681 831 (49·4%) 955 (56·8%) 52·25 (13·43) 3·29 (0·88) 3·88 (1·03) 0·848 (0·07) Northern Swedish Population Health Study (NSPHS) 880 407 (46·3%) 122 (13·9%) 49·13 (19·96) 2·93 (0·90) 3·53 (1·06) 0·831 (0·09) Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) 836 413 (49·4%) 426 (51·0%) 70·20 (0·17) 2·44 (0·68) 3·20 (0·87) 0·76 (0·10) Swiss study on Air Pollution and Lung Disease in adults (SAPALDIA) 2707 1379 (50·9%) 1399 (51·7%) 40·86 (10·92) 3·65 (0·83) 4·62 (1·04) 0·794 (0·07) The Cardiovascular Risk in Young Finns Study (YFS) 434 198 (47·3%) 186 (44·4%) 38·88 (5·07) 3·73 (0·75) 4·68 (0·99) 0·800 (0·06) Finnish Twin Cohort (FTC) 214 0 (0%) 0 (0%) 68·73 (3·31) 2·18 (0·47) 2·79 (0·58) 0·786 (0·08) Total 23,398 CHARGE studies (European Ancestry) Total sample n (%) Male Ever smokers, n (%) Age, mean (SD) FEV1, litres. mean (SD) FVC, litres. Results We carried out a meta-analysis of exome array data and three lung function measures: forced expiratory volume in one second (FEV1), forced vital capacity (FVC) and the ratio of FEV1 to FVC (FEV1/FVC). These analyses included 68,470 individuals from the SpiroMeta and CHARGE consortia in a discovery analysis, with follow-up in an independent sample of up to 111,556 indi- viduals. All studies are listed with their study-specific sample characteristics in Table 1, with full study descriptions, including details of spirometry and other measurements described in the Supplementary Note. The genotype calling procedures imple- mented by each study (Supplementary Table 1) and quality con- trol of genotype data are described in the Supplementary Methods. We have undertaken both single variant analyses, and gene-based Meta-analyses of gene-based associations We undertook Weighted Sum Tests (WST)23 and Sequence Kernel Association tests (SKAT)24 to assess the joint effects of multiple low frequency variants within genes on lung function traits. In our discovery analyses of all 68,470 individuals, we tested up to 14,380 genes that had at least two variants with MAF<5% and met the inclusion criteria (exonic or loss of function [LOF], see Methods for definitions) in both consortia. The SKAT analy- ses identified 16 genes associated (P<0·05 in both consortia and overall P<10-4) with FEV1, FVC or FEV1/FVC (Supplementary Table 6), whilst the WST analyses identified 12 genes Page 8 of 28 Page 8 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 Table 1. Sample characteristics of 11 SpiroMeta and 12 CHARGE studies contributing to the discovery analyses and three studies contributing to the replication analyses. mean (SD) FEV1/FVC, mean (SD) AGES-Reykjavik study (AGES) 1566 649 (41·4%) 900 (57·5%) 76·1 (5·62) 2·13 (0·70) 2·87 (0·86) 0·744 (0·09) Atherosclerosis Risk in Communities Study (ARIC) 10,680 5015 (47·0%) 631 (59·1%) 54·3 (5·70) 2·94 (0·77) 3·98 (0·98) 0·738 (0·07) Cardiovascular Health Study (CHS) 3967 1737 (43·8%) 2089 (52·7%) 72·8 (5·55) 2·11 (0·66) 3·00 (0·86) 0·702 (0·10) NHLBI Family Heart Study (FAMHS) 1651 718 (43·5%) 698 (42·3) 53·5 (12·60) 2·91 (0·853) 3·89 (1·05) 0·746 (0·08) Framingham Heart Study (FHS) 7113 3241 (45·5%) 3780 (53·1) 50·7 (14·12) 3·10 (0·925) 4·09 (1·12) 0·755 (0·08) Health Aging and Body Composition Study (HABC) 1457 786 (53·2%) 831 (56·5%) 73·7 (2·83) 2·31 (0·66) 3·11 (0·81) 0·741 (0·08) Health2006 Study 2714 1217 (44·8%) 1577 (58·1%) 49·4 (13·04) 3·13 (0·82) 3·99 (0·99) 0·784 (0·07) Health2008 Study 687 297 (43·2%) 384 (55·9%) 46·7 (8·22) 3·27 (0·79) 4·13 (0·97) 0·791 (0·06) Inter99 Study (without pack-years) 1115 549 (49·2%) 1115 (100%) 47·2 (7·76) 3·26 (0·71) 4·12 (0·92) 0·796 (0·07) Inter99 Study (with pack-years) 4179 2027 (48·5%) 2307 (55·2%) 45·8 (7·95) 3·21 (0·76) 4·10 (0·97) 0·788 (0·08) Multi-Ethnic Study of Atherosclerosis (MESA) 1323 654 (49·4%) 751 (56·8%) 66·0 (9·8) 2·57 (0·76) 3·51 (0·10) 0·733 (0·08) The Rotterdam Study (RS) 546 299 (54·8%) 382 (70·0%) 79·4 (5·00) 2·27 (0·68) 3·03 (0·86) 0·750 (0·08) Total 36,998 Page 9 of 28 Page 9 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 Discovery studies CHARGE studies (African Ancestry) Total Sample n (%) Male Ever smokers, n (%) Age, mean (SD) FEV1, litres. mean (SD) FVC, litres. mean (SD) FEV1/FVC, mean (SD) Atherosclerosis Risk in Communities Study (ARIC) 3180 1183 (37·2%) 1680 (59·1%) 53·6 (5·83) 2·48 (0·65) 3·25 (0·82) 0·765 (0·08) Cardiovascular Health Study (CHS) 624 232 (37·2%) 340 (54·4%) 73·2 (5·49) 1·76 (0·58) 2·48 (0·80) 0·717 (0·11) Health Aging and Body Composition Study (HABC) 943 433 (45·9%) 543 (57·6%) 73·4 (2·90) 1·96 (0·57) 2·61 (0·71) 0·749 (0·09) Jackson Heart Study (JHS) 2143 793 (36·8%) 688 (31·9%) 52·8 (12·6) 2·43 (0·72) 3·02 (0·86) 0·807 (0·09) Multi-Ethnic Study of Atherosclerosis (MESA) 861 404 (46·9%) 467 (54·2%) 65·6 (9·6) 2·19 (0·66) 2·92 (0·86) 0·756 (0·09) Total 7721 Replication studies Study name Total Sample n (%) Male Ever smokers, n (%) Age, mean (SD) FEV1, litres. mean (SD) FVC, litres. Table 1. Sample characteristics of 11 SpiroMeta and 12 CHARGE studies contributing to the discovery analyses and three studies contributing to the replication analyses. mean (SD) FEV1/FVC, mean (SD) UK Biobank 98,657 45,166 (45·8%) 56,404 (57·2%) 56·7 (7·92) 2·75 (0·80) 3·67 (0·98) 0·75 (0·07) UK Household Longitudinal Study (UKHLS) 7443 3293 (44·2%) 4509 (60·5%) 53·10 (15·94) 2·89 (0·90) 3·83 (1·08) 0·753 (0·09) Netherlands Epidemiology of Obesity study (NEO) 5456 2672 (48·0%) 3674 (66·0%) 55·9 (5·9) 3·26 (0·80) 4·26 (1·02) 0·77 (0·07) Total 111,556 (Supplementary Table 8), including TYRO3, one of the TAM family of receptor tyrosine kinases. TYRO3 regulates several processes including cell survival, migration and differentiation and is highly expressed in lung macrophages (Supplementary Table 10). Evidence of association with gene expression was found at two more of the novel signals (sentinel SNPs rs3849969 and rs6088813), implicating a further 16 genes. Of note, in blood expression quantitative trait loci (eQTL) databases, a proxy of a SNP in complete linkage disequilibrium (r2=1) with rs3849969 (rs3812637) was an eQTL for plasminogen activator, urokinase (PLAU). (Supplementary Table 7). There was one gene (LY6G6D) that was identified in both analyses. These genes were followed up in UK Biobank, with two genes, GPR126 and LTBP4, showing evidence of replication in the exonic SKAT analysis (P<3·5×10-6); however conditional analyses in UK Biobank showed that both these associations were driven by single SNPs, that were identified in the single variant association analyses and have been previously reported in GWAS of these traits (Supplementary Table 6 and Supplementary Table 7). (Supplementary Table 7). There was one gene (LY6G6D) that was identified in both analyses. These genes were followed up in UK Biobank, with two genes, GPR126 and LTBP4, showing evidence of replication in the exonic SKAT analysis (P<3·5×10-6); however conditional analyses in UK Biobank showed that both these associations were driven by single SNPs, that were identified in the single variant association analyses and have been previously reported in GWAS of these traits (Supplementary Table 6 and Supplementary Table 7). Functional characterization of novel loci In order to gain further insight into the six loci identified in our analyses of single variant associations (excluding LCT), we employed functional annotation and assessed whether identi- fied SNPs in these regions were associated with gene expression levels. One of the identified novel SNPs was nonsynonymous, three intronic and two were intergenic. We found evidence that three of the SNPs may be involved in cis-acting regulation of the expression of several genes in multiple tissues (Supplementary Table 8). Discussion We undertook an analysis of 68,470 individuals from 23 studies with data from the exome array and three lung function traits, following up the most significant single SNP and gene-based associations in an independent sample of up to 111,556 indi- viduals. There were six SNPs which reached P<10-5 in the discovery stage meta-analysis of single variant associations, and subsequently met the Bonferroni corrected significance threshold for independent replication (P<1·47×10-3, corrected for 34 SNPs being tested). In the combined analyses of our discovery and replication analyses, these six SNPs met the exome chip-wide significance threshold (P<2·8×10-7). One of the SNPs is in a region that has previously been implicated in lung function (near KCJN2/SOX9)21, whilst the remaining five SNPs, although all common, have not previously been identified in other GWAS of lung function. In a recent 1000 Genomes imputed analysis of lung function (which includes some of the studies contributing to SNP rs1200345 in RPAP1 is a nonysynomous variant, pre- dicted to be deleterious by both SIFT (deleterious) and Polyphen (possibly damaging) (Supplementary Table 9); RPAP1 is ubiq- uitously expressed, with high levels of protein detected in the lung (Supplementary Table 10). SNP rs1200345 or proxies (r2>0·8) were also found to be amongst the most strongly associ- ated SNPs with expression levels of RPAP1 in several tissues, including lung, and with a further six genes in lung tissue Page 10 of 28 Page 10 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 ble 2. Novel loci associated with lung function traits. Results are shown for variant in novel loci associated (P<2·7×10-7) with lung function traits in a two stage meta-analysis consisting of up 8,470 individuals from the SpiroMeta and CHARGE Consortia in the discovery analyses, with follow-up in up to 111,556 individuals from UK Biobank, UKHLS and NEO. For each SNP, the result the trait-smoking-ancestry combination which resulted in the most statistically significant association is given. The results for these SNPs and all three traits are shown in Supplementary Table 12. a values from SpiroMeta (βSp) reflect effect-size estimates on an inverse-normal transformed scale after adjustments for age, age2, sex, height and ancestry principal components, and stratified ever smoking status (Analysis of All individuals only). Beta values from CHARGE (βCH) reflect effect-size estimates on an untransformed scale (litres for FEV1 and FVC; ratio for FEV1/FVC). Discussion PLAU has also been found to be upregulated in lung epithelial cells subjected to cyclic strain34 and in patients with COPD and lung cancer, PLAU was found to be expressed in alveolar macrophages and epithelial cells31. We further identified a seventh association with rs2322659 in LCT (MAF=23·5%; combined discovery + replication P=1·70×10-9). Given SNPs in this region are known to vary in frequency across European populations, we cannot dismiss the possibility that this association may be confounded by population stratification; hence we do not report this signal as a novel lung function locus. For SNPs at 7 loci that have been shown to have differences in allele frequency between individuals from different regions of the UK25, and subsequently European populations (including the LCT locus), we undertook a look-up of associations with lung function in our discovery analyses. and subsequently across European populations26. Aside from the association between the LCT locus and FVC, no significant associations were observed between SNPs at these loci and any lung function trait, in either the analyses restricted to European Ancestry (EA) individuals, or in the analysis of EA and African Ancestry (AA) individuals combined (Supplementary Table 11); this suggests popula- tion structure was generally accounted for adequately in our analyses. The final two signals were with common intergenic SNPs close to LY86 (MAF=36·8%; P=9·74×10-23) and FGF10 (MAF=35·6%; P=2·22×10-11). LY86 (lymphocyte antigen 86) interacts with the Toll-like receptor signalling pathway, to form a heterodimer, when bound with RP10535. The sentinel SNP in the present analysis (rs1294421) has previously shown association with waist-hip ratio36, whilst an intronic SNP within LY86 (rs7440529, r2=0·005 with rs1294421) has been implicated in asthma in two studies of individuals of Han Chinese ancestry37,38. FGF10 is a member of the fibroblast growth factor family of proteins, and is involved in a range of biological processes, including embryonic development and morphogenesis, cell growth and repair, tumor growth and invasion. Specifically, the FGF10 signalling pathway is thought to play an criticial role in the development of the lung and in lung epithelial renewal39. A deficiency in Fgf10 has been demonstrated to lead to a fatal disruption of branching morphogenesis during lung development in mice40. One of the novel signals was with a nonsynonymous SNP, rs1200345 in RPAP1, (MAF=48·8%; P=2·33×10-13), which is predicted to be deleterious. Discussion This SNP and proxies with r2>0·8 were also associated with expression in lung tissue of seven genes, including RPAP1 and the TAM receptor TYRO3. TAM receptors play a role in the inhibition of Toll-like receptors (TLRs)- mediated innate immune response by initiating the transcription of cytokine signalling genes (SOCS-1 and 3), which limit cytokine overproduction and inflammation27,28. It has been shown that influenza viruses H5N1 and H7N9 can cause downregula- tion of Tyro3, resulting in an increased inflammatory cytokine response28. Our discovery analyses included individuals of both EA and AA. Two of the identified six novel signals showed inconsistent effects in the AA and EA individuals. For these SNPs, the associations in AA individuals were not statistically significant, and we report associations from the analysis restricted to EA individuals only. For the remaining four SNPs similar effects were observed in both the EA and AA individuals (Supplementary Figure 3). We also examined the effects of the novel SNPs in ever smokers and never smokers separately and found these to be broadly similar, with the exception of rs1448044 in FGF10, which in the discovery analysis showed significant association with FVC in ever smokers, whilst showing no association in never smokers (P=0·695). However, in our replication stage analyses, similar effects were seen in both ever and never smokers for this SNP, and the combined analysis of discovery and replication stages for this SNP, including both ever and never smokers, met the exome chip-wide significance level overall (P=4·22×10-9). We also considered whether this signal could be driven by smoking behaviour in our discovery stage as our primary analyses in SpiroMeta did not adjust for smoking quantity. We undertook a look-up of this SNP in the publicly available results of a GWAS of several smoking behaviour traits41; there was only weak evidence that this SNP was associated with ever versus never smoking (P=0·039), and no evidence for association with amount smoked (cigarettes per day, P=0·10). Three further signals were with intronic SNPs in SEC24C (MAF=29·4%; P=4·99×10-12), CASC17 (MAF=48·2%; P=4·10×10-11), and UQCC1 (MAF=36·7%; P=4·90×10-19). Two of these intronic SNPs have previously been implicated in GWAS of other traits: rs1859962 in CASC17 with prostate cancer29 and rs6088813 in UQCC1 with height30. The CASC17 locus, near KCNJ2/SOX9 has also previously been implicated in lung func- tion, showing significant association with FEV1 in a genome-wide analysis of gene-smoking interactions; however, this association was not formally replicated21. Discussion Samples es (N), Z-statistics (Z) and two-sided P-values (P) are given for the combined discovery analysis and the replication analysis. Two-sided P-values are also given for the full two-stage combined alyses (discovery + replication). Consortium results Combined discovery meta-analysis Replication Two-stage combined NP Chr:Pos (Nearest) gene(s) Trait Smoking Ancestry Effect/other allele Effect allele frequency (Discovery) βCH βSp Ndisc Zdisc Pdisc Nrep Zrep Prep Pmeta 2322659 2:136555659 LCT (nonsynonymous) FVC All Individuals EA Only T/C 23·5% 27·34 0·032 55,591 5·597 2·18×10-8 12,899 2·286 0·0223 1·70 ×10-9 448044 5:44296986 FGF10(dist=8111), NNT(dist=591,318) FVC Ever Smokers EA+AA A/G 35·6% 18·63 0·057 30,966 4·813 1·49 ×10-6 64,400 4·805 1·55 ×10-6 2·22 ×10-11 294421 6:6743149 LY86(dist=87,933), RREB1(dist=364,681) FEV1/ FVC All Individuals EA+AA T/G 36·8% -0·222 -0·038 68,099 -5·479 4·27 ×10-8 111,556 -8·171 3·06 ×10-16 9·74 ×10-23 3849969 10: 75525999 SEC24C (intronic) FEV1 All Individuals EA+AA T/C 29·4% 13·10 0·036 68,116 4·767 1·87 ×10-6 111,556 5·042 4·60 ×10-7 4·99 ×10-12 200345 15: 41819716 RPAP1 (nonsynonymous) FEV1/ FVC All Individuals EA only C/T 48·8% -0·217 -0·025 60,381 -4·586 4·51 ×10-6 111,556 -5·725 1·03 ×10-8 2·33 ×10-13 859962 17: 69108753 CASC17 (intronic) FEV1 All Individuals EA only G/T 48·2% 15·39 0·026 60,395 4·876 1·08 ×10-6 111,554 4·612 3·99 ×10-6 4·10 ×10-11 6088813 20: 33975181 UQCC1 (intronic) FVC All Individuals EA+AA C/A 36·7% -16·16 -0·023 68,115 -4·634 3·58×10-6 111,556 -7·688 1·50 ×10-14 4·90 ×10-19 Wellcome Open Research Page 11 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 the present discovery analysis), all of these SNPs showed at least suggestive association (2·97×10-3>P>1·28×10-5) with one or more lung function trait, but none reached the required threshold (P<5×10-6) to be taken forward for replication in that analysis12. associated with expression of PLAU in blood. The plasmino- gen activator, urokinase (PLAU) plays a role in fibrinolysis and immunity, and with its receptor (PLAUR) is involved in degrada- tion of the extra cellular matrix, cell migration, cell adhesion and cell proliferation31. A study of preterm infants with respiratory distress syndrome, a condition characterised by intra-alveolar fibrin deposition, found PLAU and its inhibitor SERPINE1 to be expressed in the alveolar epithelium, and an increased ratio of SERPINE1 to PLAU was associated with severity of disease32. Studies in mice have also shown that increased expression of Plau may be protective against lung injury, by reducing fibrosis33. Methods Study design, cohorts and genotyping Study design, cohorts and genotyping The SpiroMeta analysis included 23,751 individuals of EA from 11 studies, and the CHARGE analysis comprised 36,998 EA individuals and a further 7,721 individuals of AA from 12 studies. Follow-up analyses were conducted in an independent sample of up to 111,556 individuals from UK Biobank (2015 interim release), the UK Household Longitudinal Study (UKHLS) and the Netherlands Epidemiology of Obesity (NEO) Study (Figure 1). All studies (excluding UK Biobank) were genotyped using either the Illumina Human Exome BeadChip v1 or the Illumina Infinium HumanCoreExome-12 v1·0 BeadChip. UK Biobank samples were genotyped using the Affymetrix Axiom UK BiLEVE or UK Biobank arrays. Statistical analyses Consortium level analyses: Within the SpiroMeta Consortium, each study contributing to the discovery analyses calculated single-variant score statistics, along with covariance matrices describing correlations between variants, using RAREMETAL- WORKER48 or rvtests49. For each trait, these summary statistics were generated separately in ever and never smokers. Traits were adjusted for sex, age, age2 and height, and inverse normally transformed prior to association testing. For studies with unrelated individuals, SNP-trait associations were tested using linear models, with adjustments made for the first 10 ancestry principal components, whilst studies with related individuals utilised linear mixed models to account for familial relationships and underlying population structure. In our replication analyses using UK Biobank, we applied adjust- ment for covariates including ancestry principal components, before undertaking inverse-normal transformations of the lung function phenotypes. Association analyses were then performed using these transformed phenotypes. It has recently been shown that such transformation has the potential to introduce correlations between principal components and phenotypes47; we undertook sensitivity analyses for the six reported SNPs by repeating the association analyses with phenotypes that had been transformed without prior adjustment, with covariate adjustment made as part of the SNP-trait association test. We found there to be some difference in P-values for some SNP- trait combinations; however, the six novel SNP associations we report all met the replication P-value threshold (P<1·47×10-3) in the sensitivity analyses (Supplementary Figure 4). This issue may also be relevant to the gene-based tests; however no replicated novel gene-based associations were identified in this study. Future studies should avoid undertaking adjustment for principal components of ancestry prior to trait transformation, in order to avoid this potential bias. Within the CHARGE Consortium, each study generated equiva- lent summary statistics using the R package SeqMeta50. Discussion Whilst the individuals utilised in the discovery stage of this analysis overlap with those included in this previous interaction analysis, the replication stage of the present study provides the first evidence of replication for this signal in independent cohorts. In the present analysis, there was no evidence that the results differed by smoking status. SNPs rs6088813 in UQCC1 and rs3849969 in SEC24C were identified as eQTLs for multiple genes. Whilst our eQTL analysis did not include formal tests of colocalisation, a SNP in complete linkage disequilibrium with rs3849969 (rs3812637, r2=1) was Page 12 of 28 Page 12 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 lung function and lung disease, and could provide novel targets for therapeutic interventions. Through the use of the exome array, we aimed to identify asso- ciations with low frequency and rare functional variants, thereby potentially uncovering some of the missing heritability of lung function. However, whilst our discovery analyses identified single SNP associations with 23 low frequency variants (Supplementary Table 2), we did not replicate any of these findings. Eleven of these 23 SNPs we were unable to follow-up in our replication studies, due to them either being not genotyped, or monomorphic. Overall, limited statistical power is likely to explain our lack of convincing single variant associations with rare variants, in particular if those variants exhibit only modest effects42. We addi- tionally investigated the joint effects of low frequency and rare variants within genes, on lung function trait, by employing SKAT and WST gene-based tests. These analyses identified associations with a number of genes that could not attributed to the effect of a single SNP. Replication of these gene-based signals proved difficult however, as again a number of SNPs included in the discovery stage of these analyses were monomorphic, or had not been not genotyped in the replication studies. This lead to a disparity in the gene unit being tested in our discovery and replication samples; hence interpretation of these results was not clear-cut. In the end, we were able to replicate only findings with common SNPs. Discussion This finding is in line with several other stud- ies of complex traits and exome array data that have been unable to report robust associations with low frequency variants43–45 and it is clear that future studies will require increasingly larger sample sizes in order to fully evaluate the effect of variants across the allele frequency spectrum. The identification of common SNPs remains important, however, as such findings have the potential to highlight drug targets46, and these variants collectively could have utility in risk prediction. Methods CHARGE: Meta-analysis of FEV1, FVC and FEV1/FVC in 36,998 EA and 7721 AA individuals from 12 studies. Consortium- level analysis Overall Meta-analysis of FEV1, FVC and FEV1/FVC in 60,749 EA and 7721 AA individuals. Combined meta-analysis 34 SNPs selected with P<10-5 with at least one trait in combined meta-analysis and P<0.05 in both consortium - level analysis. SNPs followed up in an independent sample of up to 111,556 individuals from UK Biobank, UKHLS and NEO. Replication analysis Results from discovery meta-analysis and replication analysis combined to give overall result. Identification of novel associations 6 SNPs meeting exome chip- wide significance (P<2.8×10-7) overall identified. 6 SNPs meeting exome chip- wide significance (P<2.8×10-7) overall identified. 0 Genes meeting exome chip- wide significance (P<3-5×10-6) overall identified. Figure 1. Study design. all SNPs meeting the LOF analysis criteria in addition to all other nonsynonymous variants with consortium wide MAF<5% (exonic analysis). Variants were annotated to genes using dbNSFP v2·651 on the basis of the GRCh37/hg19 database. consortium level P-values. The consortium-level results were then combined using sample size weighted z-score meta-analysis. The λ was again calculated for the meta-analysis results and genomic control applied, as appropriate. λ values at the consortium and meta-analysis level are shown in Supplementary Table 13. Since we were interested in identifying low frequency and rare variants, we applied no MAF or minor allele count (MAC) filter. We identified SNPs of interest as those with an overall P<10-5 and a consistent direction of effect and P<0·05 observed in both consortia. Rather than using a strict Bonferroni correction for defining the significance threshold, we adopted the more lenient P<10-5 threshold in order to increase the power to detect variants with modest effect in our discovery analyses, whilst the requirement for consistency in results from the two consortia aimed to limit false positives. All SNPs meeting these thresholds were followed up in independent replication cohorts. Where we identified a SNP within 1Mb of a previously identified For both single variant and gene-based associations, consortium-level results were generated for ever smokers and never smokers separately, and in all individuals combined. Within the CHARGE Consortium, results were combined separately for the EA and AA studies and also in a trans-ethnic analysis of both ancestries. Methods For each trait, summary statistics were generated in ever and never smokers separately, and in all individuals combined. The untrans- formed traits were used for all analyses, adjusted for smoking sta- tus and pack-years, age, age2, sex, height, height2, centre/cohort. Models for FVC were additionally adjusted for weight. Linear regression models, with adjustment for principal components of ancestry were used for studies with unrelated individuals, and linear mixed models were used for family-based studies. Within each consortium we used the score statistics and variance- covariance matrices generated by each study to construct both single variant and gene-based tests using either RAREMETAL48 (SpiroMeta) or SeqMeta50 (CHARGE). For single variant associations, score statistics were combined in fixed effects meta-analyses. Two gene-based tests were constructed: first, the Weighted Sum Test (WST) using Madsen Browning weightings23, and secondly, the Sequence Kernel Association Test (SKAT)24. We performed the SKAT and WST tests using two subsets of SNPs: 1) including all SNPs with an overall consortium-wide MAF<5% that were annotated as splicing, stopgain, stoploss, or frameshift (loss of function [LOF] analysis), and 2) including This study has identified six common SNPs, independent to signals previously implicated in lung function. Additional interrogation of these loci could lead to greater understanding of Page 13 of 28 Page 13 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 gure 1 Study design SpiroMeta:Meta-analysis of FEV1, FVC and FEV1/FVC in 23,751 EA individuals from 11 Studies. CHARGE: Meta-analysis of FEV1, FVC and FEV1/FVC in 36,998 EA and 7721 AA individuals from 12 studies. Overall Meta-analysis of FEV1, FVC and FEV1/FVC in 60,749 EA and 7721 AA individuals. Consortium- level analysis Combined meta-analysis Replication analysis Identification of novel associations 34 SNPs selected with P<10-5 with at least one trait in combined meta-analysis and P<0.05 in both consortium - level analysis. SNPs followed up in an independent sample of up to 111,556 individuals from UK Biobank, UKHLS and NEO. 6 SNPs meeting exome chip- wide significance (P<2.8×10-7) overall identified. Results from discovery meta-analysis and replication analysis combined to give overall result. 0 Genes meeting exome chip- wide significance (P<3-5×10-6) overall identified. Genes followed up in an independent sample of up to 98,657 individuals from UK Biobank. 27 Genes selected with P<10-4 with at least one trait in combined meta-analysis and P<0.05 in both consortium - level analysis. Characterization of findings Characterization of findings In order to gain further insight into the loci identified in our analyses of single variant associations, we assessed whether these regions were associated with gene expression levels in various tissues (FDR of 5%, or q-value<0·05), by querying a publically available blood eQTL database53 and the GTEx project54 for the sentinel SNPs, or any proxy (r2>0·8). We further assessed SNPs of interest (and proxies) within a lung eQTL resource based on non-tumour lung tissues of 1,111 individuals55–57. Descriptions of these resources and further details of the look-ups are provided in the Supplementary Methods. Moreover, all sentinel SNPs and proxies with r2>0.8 were annotated using ENSEMBL’s Variant Effect Predictor (VEP)58; potentially deleterious coding variants were identified as those annotated as ‘deleterious’ by SIFT59 or ‘probably damaging’ or ‘possibly damaging’ by PolyPhen-260. For all genes implicated through the expression data or functional annotation, we searched for evidence of protein expression in the respiratory system by querying the Human Protein Atlas61. Replication analyses: All SNP and gene-based associa- tions were followed up for the trait with which they showed the most statistically significant association only. For associations identified through the smoking subgroup analyses, we followed up associations in the appropriate smoking strata; however, no ancestry stratified follow-up was undertaken as replication studies included only a sufficient number of individuals of European Ancestry. Single variant associations in UK Biobank were tested in ever smokers and never smokers separately, and stratified by genotyping array (UK BiLEVE array or UK Biobank array) using the score test as implemented in SNPTEST v2·5b452. Traits were adjusted for age, age2, height, sex, ten principal compo- nents and pack-years (ever smokers only), and the adjusted traits were inverse normally transformed. Correlations between prin- cipal components and transformed phenotypes may be intro- duced where adjustment is made prior to transformation. In this analysis, we found any introduced correlations to have no impact on the conclusion of our replication analyses; however future studies should apply transformation of phenotypes prior to covariate adjustment, to avoid this issue. For UKHLS, analyses were undertaken analogously to the SpiroMeta discovery studies using RAREMETALWORKER, while for NEO, analyses were undertaken in the same way as was done in the CHARGE discovery studies using SeqMeta. The single variant results from all replication studies were combined using sample size weighted Z-score meta-analysis. Characterization of findings Subsequently, we combined the results from the discovery and replication stage analyses and we report SNPs with overall exome-wide significance of P<2·8×10-7 (Bonferroni corrected for the original 179,215 SNPs tested). Methods Combined meta-analysis: The single variant association results from the SpiroMeta and CHARGE consortia were combined as follows: The genomic inflation statistic (λ) was calculated for SNPs with consortium-wide MAF>1%; where λ had a value greater than one, genomic control adjustment was applied to the Page 14 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 lung function SNP, we deemed the SNP to represent an independent signal if it had r2<0·2 with the known SNP, and if it retained a P <10-5, when conditional analyses were carried out with the known SNP, or a genotyped proxy, using data from the SpiroMeta Consortium, or UK Biobank. Our primary meta-analysis included all individuals; we additionally carried out analyses in smoking subgroups (ever and never smokers), and in the subgroup of individuals of European ancestry only. an overall combined meta-analysis using either z-score meta- analysis (WST) or Fisher’s Method (SKAT). We declared genes with overall P<3·5×10-6 (Bonferroni corrected for 14,380 genes tested) in our combined meta-analysis to be statistically significant. For these statistically significant genes, we carried out additional analyses using the UK Biobank data in which we conditioned on the most significantly associated individual SNP within that gene, to determine whether this was a true gene-based signal, or whether the association could be ascribed to the single SNP (if the conditional P<0·01, then association was deemed to not be driven by the single SNP). For genes which contained at least 2 polymorphic SNPs in both consortia, we combined the results of the consortium level gene based tests using either z-score meta-analysis (for the WST analysis) or Fisher’s Method for combining P-values (in the case of SKAT). We identified genes of interest as those with P<0·05 observed in both consortia and an overall P<10-4, thresh- olds again chosen to limit both false positive and false negative findings. As in the analyses of single variant associations, our primary meta-analyses included all individuals, with secondary analyses undertaken in smoking and ancestry specific subgroups. Author contributions Ordered alphabetically: ABW, AGE, AL, BMP, BS, CH, CP, DOMK, DPS, EZ, GGB, HS, IPH, JBJ, JK, KMB, LL, MAI, MAP, MDT, MK, NG, NMPH, OP, OTR, RdM, RGB, SBK, SG, SJL, SSR, TA, Data availability Summary level results for all analyses are available on OSF: https://doi.org/10.17605/OSF.IO/NSDPJ62 Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). This research has been conducted using the UK Biobank Resource. The genetic and phenotypic UK Biobank data are available upon application to the UK Biobank (https://www. ukbiobank.ac.uk/) to all registered health researchers. These data are from Understanding Society: The UK Household Longitudi- nal Study (UKHLS), which is led by the Institute for Social and Economic Research at the University of Essex and funded by the Economic and Social Research Council. The data were collected by NatCen and the genome wide scan data were analysed by the Wellcome Trust Sanger Institute. Information on how to access the data can be found on the Understanding Society website https://www.understandingsociety.ac.uk/. We followed up genes of interest (P<10-4) using data from UK Biobank only. Summary statistics for UK Biobank were generated using RAREMETALWORKER, with gene-based tests then constructed using RAREMETAL. Finally, we combined the results from the discovery analysis with the replication results in Author contributions Ordered alphabetically: ABW, AGE, AL, BMP, BS, CH, CP, DOMK, DPS, EZ, GGB, HS, IPH, JBJ, JK, KMB, LL, MAI, MAP, MDT, MK, NG, NMPH, OP, OTR, RdM, RGB, SBK, SG, SJL, SSR, TA, Page 15 of 28 Page 15 of 28 Page 15 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 TBH, TH, TL, TR, TS, UG contributed to study concept and designs. AC, AJ, A.Manichaikul, BHS, BMP, BS, CP, DJP, DPS, EI, GGB, GTOC, IJD, JBJ, JGW, JK, JMS, KS, LAL, LL, LL, MAP, MI, MK, NG, NMPH, OP, OTR, PAC, RdM, RGB, RR, SBK, SE, SEH, SG, SK, SK, TA, TBH, TDP, TL, TNB, TR, UG, WT, WT contributed to phenotype data acquisition and quality control. AGE, AJ, AK, AK, ALT, ALT, A.Manichaikul, APM, AT, BMP, BP, CH, DOMK, EI, GD, HV, IJD, JAB, JCM, JGW, JL, KDT, KEN, KL, L-PL, LAL, LL, MAP, MI, MLG, NMPH, OP, RGB, RLG, RR, SBK, SE, SEH, SRH, SSR, SW, TBH, TDP, TH, TL, YL contributed to genotype data acquisition and quality control. DDS, KH, WT, YB contrib- ute to eQTL data acquisition and quality control. Data availability MESA/MESA SHARe is supported by the US Department of Health and Human Services (HHS) (HHSN268201500003I), NIH/National Heart, Lung and Blood Institute (NHLBI; N01-HC-95159, N01-HC-95160, N01-HC- 95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01- HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169) and NIH/National Center for Advancing Translation Studies (NCATS) (UL1-TR-000040, UL1-TR-001079, UL1-TR-001881, DK063491). MESA SHARe is funded by NIH/NHLBI contract N02-HL-64278, MESA Air is funded by US Environmental Protection Agency (RD831697) and MESA Spirometry funded by NIH/NHLBI (R01-HL077612). SSR and BMP are supported by NIH/NHLBI grant rare variants and NHLBI traits in deeply phenotyped cohorts (R01-HL120393). The CHS research was supported by NHLBI (contracts: HHSN268201200036C, HHSN268200800007C, HHSN268201800001C, HHSN268200960009C, N01HC55222, N01HC85079, N01HC85080, N01HC85081, N01HC85082, N01HC85083, N01HC85086; grants: U01HL080295, R01HL068986, R01HL087652, R01HL105756, R01HL103612, R01HL120393, R01HL130114), with additional contribution from the National Institute of Neurological Disorders and Stroke. Additional support was provided through R01AG023629 and R01HL085251 from the National Institute on Aging (NIA). The provision of genotyping data was supported in part by the NCATS, CTSI (UL1TR001881), and the National Institute of Diabetes and Digestive and Kidney Disease Diabetes Research Center (DK063491) to the Southern California Diabetes Endocrinology Research Center. The content is solely the responsibility of the Competing interests No competing interests were disclosed. No competing interests were disclosed. Data availability MESA SHARe is funded by NIH/NHLBI contract N02-HL-64278, MESA Air is funded by US Environmental Protection Agency (RD831697) and MESA Spirometry funded by NIH/NHLBI (R01-HL077612). SSR and BMP are supported by NIH/NHLBI grant rare variants and NHLBI traits in deeply phenotyped cohorts (R01-HL120393). The CHS research was supported by NHLBI (contracts: HHSN268201200036C, HHSN268200800007C, HHSN268201800001C, HHSN268200960009C, N01HC55222, N01HC85079, N01HC85080, N01HC85081, N01HC85082, N01HC85083, N01HC85086; grants: U01HL080295, R01HL068986, R01HL087652, R01HL105756, R01HL103612, R01HL120393, R01HL130114), with additional contribution from the National Institute of Neurological Disorders and Stroke. Additional support was provided through R01AG023629 and R01HL085251 from the National Institute on Aging (NIA). The provision of genotyping data was supported in part by the NCATS, CTSI (UL1TR001881), and the National Institute of Diabetes and Digestive and Kidney Disease Diabetes Research Center (DK063491) to the Southern California Diabetes Endocrinology Research Center The content is solely the responsibility of the European Commission FP6 (LSHG-CT-2006-01947). SHIP is part of the Community Medicine Research net of the University of Greifswald, Germany, which is funded by the Federal Ministry of Education and Research, the Ministry of Cultural Affairs, as well as the Social Ministry of the Federal State of Mecklenburg- West Pomerania, and the network ‘Greifswald Approach to Individualized Medicine’ funded by the Federal Ministry of Education and Research, and the German Asthma and COPD Network (01ZZ9603, 01ZZ0103, 01ZZ0403, 03IS2061A, BMBF 01GI0883). ExomeChip data have been supported by the Federal Ministry of Education and Research (03Z1CN22) and the Federal State of Mecklenburg-West Pomerania. The University of Greifswald is a member of the Caché Campus program of the InterSystems GmbH. UKHLS is supported by the Wellcome Trust (098051) and Economic and Social Research Council (ES/ H029745/1). Y.B. holds a Canada Research Chair in Genomics of Heart and Lung Diseases. Lies Lahousse is a Postdoctoral Fellow of the Research Foundation - Flanders (G035014N). The Rotterdam Study is funded by Erasmus Medical Center and Erasmus Univer- sity, Rotterdam, the Netherlands Organization for Scientific Research (NOW), the Netherlands Organization for the Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the Euro- pean Commission (DG XII), and the Municipality of Rotterdam. Genotyping in the Rotterdam study was supported by NOW (175.010.2005.011, 911-03-305 012), RIDE2 (014-93-015) and Netherlands Genomics Initiative/Netherlands Consortium for Healthy Aging (050-060-810). Data availability ABW, ACM, AK, AK, ALT, A.Mahajan, A.Manichaikul, APM, AT, BP, BQ, CH, CMS, EA, HV, IPH, JAB, JCL, JD, JEH, JL, JM, JMJ, KL, L-PL, LL, LVW, MDT, MI, MO, NF, NMPH, OP, PAC, RLG, SE, SEH, SJL, SW, TDP, TH, TMB, VEJ, WG, WT, YL contributed to data analysis. All authors contributed to writing and/or critical review of the manuscript. The ‘Understanding Society Scientific Group’ include the following: Understanding Society Scientific Group: Michaela Benzeval, Jonathan Burton, Nicholas Buck, Annette Jäckle, Meena Kumari, Heather Laurie, Peter Lynn, Stephen Pudney, Birgitta Rabe, Shamit Saggar, Noah Uhrig, Dieter Wolke. European Commission FP6 (LSHG-CT-2006-01947). SHIP is part of the Community Medicine Research net of the University of Greifswald, Germany, which is funded by the Federal Ministry of Education and Research, the Ministry of Cultural Affairs, as well as the Social Ministry of the Federal State of Mecklenburg- West Pomerania, and the network ‘Greifswald Approach to Individualized Medicine’ funded by the Federal Ministry of Education and Research, and the German Asthma and COPD Network (01ZZ9603, 01ZZ0103, 01ZZ0403, 03IS2061A, BMBF 01GI0883). ExomeChip data have been supported by the Federal Ministry of Education and Research (03Z1CN22) and the Federal State of Mecklenburg-West Pomerania. The University of Greifswald is a member of the Caché Campus program of the InterSystems GmbH. UKHLS is supported by the Wellcome Trust (098051) and Economic and Social Research Council (ES/ H029745/1). Y.B. holds a Canada Research Chair in Genomics of Heart and Lung Diseases. Lies Lahousse is a Postdoctoral Fellow of the Research Foundation - Flanders (G035014N). The Rotterdam Study is funded by Erasmus Medical Center and Erasmus Univer- sity, Rotterdam, the Netherlands Organization for Scientific Research (NOW), the Netherlands Organization for the Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the Euro- pean Commission (DG XII), and the Municipality of Rotterdam. Genotyping in the Rotterdam study was supported by NOW (175.010.2005.011, 911-03-305 012), RIDE2 (014-93-015) and Netherlands Genomics Initiative/Netherlands Consortium for Healthy Aging (050-060-810). MESA/MESA SHARe is supported by the US Department of Health and Human Services (HHS) (HHSN268201500003I), NIH/National Heart, Lung and Blood Institute (NHLBI; N01-HC-95159, N01-HC-95160, N01-HC- 95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01- HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169) and NIH/National Center for Advancing Translation Studies (NCATS) (UL1-TR-000040, UL1-TR-001079, UL1-TR-001881, DK063491). Grant information The NEO study is supported by the participating Departments, the Division and the Board of Directors of the Leiden University Medical Center, and by the Leiden University, Research Profile Area Vascular and Regenerative Medicine. SAPALDIA was supported by the Swiss National Science Foundation (33CS30-148470/1, 33CSCO-134276/1, 33CSCO-108796, 324730_135673, authors and does not necessarily represent the official views of the National Institutes of Health. ARIC study is carried out as a collaborative study supported by the NHLBI (contracts: HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, HHSN268201100012C). Funding support for “Building on GWAS for NHLBI-diseases: the U.S. CHARGE consortium” was provided by the NIH through the American Recov- ery and Reinvestment Act of 2009 (5RC2HL102419). DOMK received funding from the Dutch Science Organisation (ZonMW-VENI Grant 916.14.023). The genotyping in the NEO study was supported by the Centre National de Génotypage (Paris, France), headed by Jean-François Deleuze. The NEO study is supported by the participating Departments, the Division and the Board of Directors of the Leiden University Medical Center, and by the Leiden University, Research Profile Area Vascular and Regenerative Medicine. SAPALDIA was supported by the Swiss National Science Foundation (33CS30-148470/1, 33CSCO-134276/1, 33CSCO-108796, 324730_135673, by the Center for Inherited Disease Research (CIDR). CIDR is fully funded through a federal contract from the National Institutes of Health to The Johns Hopkins University (HHSN268200782096C). We thank the Jackson Heart Study (JHS) participants and staff for their contributions to this work. The JHS is supported by contracts HHSN268201300046C, HHSN268201300047C, HHSN268201300048C, HHSN268201300049C, by the Center for Inherited Disease Research (CIDR). CIDR is fully funded through a federal contract from the National Institutes of Health to The Johns Hopkins University (HHSN268200782096C). We thank the Jackson Heart Study (JHS) participants and staff for their contributions to this work. The JHS is supported by contracts HHSN268201300046C, HHSN268201300047C, HHSN268201300048C, HHSN268201300049C, HHSN268201300050C from the National Heart, Lung, and Blood Institute and the National Institute on Minority Health and Health Disparities. JGW is supported by U54GM115428 from the National Institute of General Medical Sciences. HHSN268201300050C from the National Heart, Lung, and Blood Institute and the National Institute on Minority Health and Health Disparities. JGW is supported by U54GM115428 from the National Institute of General Medical Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgements The authors would like to thank the staff at the Quebec Respira- tory Health Network Tissue Bank for their valuable assistance with the lung eQTL dataset at Laval University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. A full list of principal CHS investigators and institutions can be found at https://CHS-NHLBI.org. The authors thank the staff and participants of the ARIC study for their important contributions. The authors of the NEO study thank all individuals who participated in the Netherlands Epidemiology in Obesity study, all participating general practitioners for inviting eligible participants and all research nurses for collection of the data. We thank the NEO study group, Pat van Beelen, Petra Noordijk and Ingeborg de Jonge for the coordination, lab and data management of the NEO study. SAPALDIA could not have been done without the help of the study participants, technical and administrative support and the medical teams and field workers at the local study sites. Local fieldworkers: Aarau: M Broglie, M Bünter, D Gashi; Basel: R Armbruster, T Damm, U Egermann, M Gut, L Maier, A Vögelin, L Walter; Davos: D Jud, N Lutz; Geneva: M Ares, M Bennour, B Galobardes, E Namer; Lugano: B Baumberger, S Boccia Soldati, E Gehrig-Van Essen, S Ronchetto; Montana: C Bonvin, C Burrus; Payerne: S Blanc, AV Ebinger, ML Fragnière, J Jordan; Wald: R Gimmi, N Kourk- oulos, U Schafroth. Administrative staff: N Bauer, D Baehler, C Gabriel, R Gutknecht. SAPALDIA Team: Study directorate: NM Probst Hensch, T Rochat, N Künzli, C Schindler, JM Gaspoz; Scientific team: JC Barthélémy, W Berger, R Bettschart, A Bircher, G Bolognini, O Brändli, C Brombach, M Brutsche, L Burdet, M Frey, U Frey, MW Gerbase, D Gold, E de Groot, W Karrer, R Keller, B Knöpfli, B Martin, D Miedinger, U Neu, L Nicod, M Pons, F Roche, T Rothe, E Russi, P Schmid-Grendelmeyer, A Schmidt-Trucksäss, A Turk, J Schwartz, D. Stolz, P Straehl, JM Tschopp, A von Eckardstein, E Zemp Stutz; Scientific team at coordinating centers: M Adam, E Boes, PO Bridevaux, D Carballo, E Corradi, I Curjuric, J Dratva, A Di Pasquale, L Grize, D Keidel, S Kriemler, A Kumar, M Imboden, N Maire, A Mehta, F Meier, H Phuleria, E Schaffner, GA Thun, A Ineichen, M Ragettli, M Ritter, T Schikowski, G Stern, M Tarantino, M Tsai, M Wanner. Grant information SAPALDIA was supported by the Swiss National Science Foundation (33CS30-148470/1, 33CSCO-134276/1, 33CSCO-108796, 324730_135673, 3247BO-104283, 3247BO-104288, 3247BO-104284, 3247- 065896, 3100-059302, 3200-052720, 3200-042532, 4026-028099, PMPDP3_129021/1, PMPDP3_141671/1), the Federal Office for the Environment, the Federal Office of Public Health, the Federal Office of Roads and Transport, the canton’s government of Aargau, Basel-Stadt, Basel-Land, Geneva, Luzern, Ticino, Valais, and Zürich, the Swiss Lung League, the Canton’s Lung League of Basel Stadt/Basel Landschaft, Geneva, Ticino, Valais, Graubünden and Zurich, Stiftung ehemals Bündner Heilstätten, SUVA, Freiwillige Akademische Gesellschaft, UBS Wealth Foundation, Talecris Biotherapeutics GmbH, Abbott Diagnostics, European Commission 018996 (GABRIEL), Wellcome Trust (084703). The Novo Nordisk Foundation Center for Basic Meta- bolic Research is an independent Research Center at the University of Copenhagen partially funded by an unrestricted donation from the Novo Nordisk Foundation (www.metabol.ku.dk).� Generation Scotland received core support from the Chief Scientist Office of the Scottish Government Health Directorates [CZD/16/6] and the Scottish Funding Council [HR03006]. Genotyping of the GS:SFHS samples was carried out by the Genetics Core Laboratory at the Edinburgh Clinical Research Facility, University of Edinburgh, Scotland, and was funded by the MRC. The Croatia KORCULA study was supported by the Ministry of Science, Education and Sport in the Republic of Croatia (108-1080315-0302). JD, JCL, WG and GTOC are supported by NIH/NHLBI (HHSN268201500001I). Genotyping, quality control and calling of the Illumina HumanExome BeadChip in the Framingham Heart Study was supported by funding from the National Heart, Lung and Blood Institute Division of Intramural Research (Daniel Levy and Christopher J. O’Donnell, Principle Investigators). The AGES study is supported by the NIH (N01-AG012100), the Iceland Parliament (Alþingi) and the Icelandic Heart Association. HABC was supported by NIA (contracts: N01AG62101, N01AG62103, N01AG62106; grant: R01-AG028050), and NINR (grant R01- NR012459), and was authors and does not necessarily represent the official views of the National Institutes of Health. ARIC study is carried out as a collaborative study supported by the NHLBI (contracts: HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, HHSN268201100012C). Funding support for “Building on GWAS for NHLBI-diseases: the U.S. CHARGE consortium” was provided by the NIH through the American Recov- ery and Reinvestment Act of 2009 (5RC2HL102419). DOMK received funding from the Dutch Science Organisation (ZonMW-VENI Grant 916.14.023). The genotyping in the NEO study was supported by the Centre National de Génotypage (Paris, France), headed by Jean-François Deleuze. This research used the ALICE and SPECTRE High Performance Computing Facilities at the University of Leicester. Grant information This article presents independent research funded partially by the National Institute for Health Research (NIHR). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. MDT has been supported by Medical Research Council (MRC) fellowships G0501942 and G0902313. MDT and LVW are supported by the MRC (MR/N011317/1). IPH is supported by the MRC (G1000861). ALW and SJL are supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (ZIA ES 043012). We acknowledge use of phenotype and genotype data from the British 1958 Birth Cohort DNA collection, funded by the MRC (G0000934) and the Wellcome Trust (068545). APM was a Wellcome Trust Senior Fellow in Basic Biomedical Science (098017) and was also supported by Wellcome Trust grant 064890. EI is supported by the Swedish Research Council (2012-1397), Knut och Alice Wallenberg Foundation (2013.0126) and the Swedish Heart-Lung Foundation (20140422). JK is supported by Academy of Finland Center of Excellence in Complex Disease Genetics (213506, 129680) and Academy of Finland (265240, 263278). The Finnish Twin Cohort is supported by the Welcome Trust Sanger Institute, UK. The Lothian Birth Cohort is supported by Age UK (The Dis- connected Mind Project), the MRC (MR/K026992/1) and The Royal Society of Edinburgh. ÅJ is supported by the Swedish Soci- ety for Medical Research, The Kjell och Märta Beijers Foundation, The Marcus Borgström Foundation, The Åke Wiberg foundation and The Vleugels Foundation. UG is supported by Swedish Medi- cal Research Council (K2007-66X-20270-01-3, 2011-2354) and Page 16 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 authors and does not necessarily represent the official views of the National Institutes of Health. ARIC study is carried out as a collaborative study supported by the NHLBI (contracts: HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, HHSN268201100012C). Funding support for “Building on GWAS for NHLBI-diseases: the U.S. CHARGE consortium” was provided by the NIH through the American Recov- ery and Reinvestment Act of 2009 (5RC2HL102419). DOMK received funding from the Dutch Science Organisation (ZonMW-VENI Grant 916.14.023). The genotyping in the NEO study was supported by the Centre National de Génotypage (Paris, France), headed by Jean-François Deleuze. The NEO study is supported by the participating Departments, the Division and the Board of Directors of the Leiden University Medical Center, and by the Leiden University, Research Profile Area Vascular and Regenerative Medicine. Acknowledgements 33CSCO 134276/1, 33CSCO 108796, 324730_135673, 3247BO-104283, 3247BO-104288, 3247BO-104284, 3247- 065896, 3100-059302, 3200-052720, 3200-042532, 4026-028099, PMPDP3_129021/1, PMPDP3_141671/1), the Federal Office for the Environment, the Federal Office of Public Health, the Federal Office of Roads and Transport, the canton’s government of Aargau, Basel-Stadt, Basel-Land, Geneva, Luzern, Ticino, Valais, and Zürich, the Swiss Lung League, the Canton’s Lung League of Basel Stadt/Basel Landschaft, Geneva, Ticino, Valais, Graubünden and Zurich, Stiftung ehemals Bündner Heilstätten, SUVA, Freiwillige Akademische Gesellschaft, UBS Wealth Foundation, Talecris Biotherapeutics GmbH, Abbott Diagnostics, European Commission 018996 (GABRIEL), Wellcome Trust (084703). The Novo Nordisk Foundation Center for Basic Meta- bolic Research is an independent Research Center at the University of Copenhagen partially funded by an unrestricted donation from the Novo Nordisk Foundation (www.metabol.ku.dk).� Generation Scotland received core support from the Chief Scientist Office of the Scottish Government Health Directorates [CZD/16/6] and the Scottish Funding Council [HR03006]. Genotyping of the GS:SFHS samples was carried out by the Genetics Core Laboratory at the Edinburgh Clinical Research Facility, University of Edinburgh, Scotland, and was funded by the MRC. The Croatia KORCULA study was supported by the Ministry of Science, Education and Sport in the Republic of Croatia (108-1080315-0302). JD, JCL, WG and GTOC are supported by NIH/NHLBI (HHSN268201500001I). Genotyping, quality control and calling of the Illumina HumanExome BeadChip in the Framingham Heart Study was supported by funding from the National Heart, Lung and Blood Institute Division of Intramural Research (Daniel Levy and Christopher J. O’Donnell, Principle Investigators). The AGES study is supported by the NIH (N01-AG012100), the Iceland Parliament (Alþingi) and the Icelandic Heart Association. HABC was supported by NIA (contracts: N01AG62101, N01AG62103, N01AG62106; grant: R01-AG028050), and NINR (grant R01- NR012459), and was supported in part by the Intramural Research Program of the NIA. The HABC genome-wide association study was funded by NIA (1R01AG032098- 01A1) and genotyping services were provided This research used the ALICE and SPECTRE High Performance Computing Facilities at the University of Leicester. Page 17 of 28 Page 17 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 5. Wilk JB, Djousse L, Arnett DK, et al.: Evidence for major genes influencing pulmonary function in the NHLBI Family Heart Study. Genet Epidemiol. 2000; 19(1): 81–94. PubMed Abstract \| Publisher Full Text 6. Wilk JB, Chen TH, Gottlieb DJ, et al.: A Genome-Wide Association Study of Pulmonary Function Measures in the Framingham Heart Study. PLoS Genet. 2009; 5(3): e1000429. PubMed Abstract \| Publisher Full Text \| Free Full Text 7. Repapi E, Sayers I, Wain LV, et al.: Genome-wide association study identifies five loci associated with lung function. Nat Genet. 2010; 42(1): 36–44. PubMed Abstract \| Publisher Full Text \| Free Full Text 8. Soler Artigas M, Loth DW, Wain LV, et al.: Genome-wide association and large- scale follow up identifies 16 new loci influencing lung function. Nat Genet. 2011; 43(11): 1082–90. PubMed Abstract \| Publisher Full Text \| Free Full Text 9. Hancock DB, Eijgelsheim M, Wilk JB, et al.: Meta-analyses of genome-wide 1. Rabe KF, Hurd S, Anzueto A, et al.: Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD executive summary. Am J Respir Crit Care Med. 2007; 176(6): 532–55. PubMed Abstract \| Publisher Full Text 2. Palmer LJ, Knuiman MW, Divitini ML, et al.: Familial aggregation and heritability of adult lung function: results from the Busselton Health Study. Eur Respir J. 2001; 17(4): 696–702. PubMed Abstract \| Publisher Full Text 1. Rabe KF, Hurd S, Anzueto A, et al.: Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD executive summary. Am J Respir Crit Care Med. 2007; 176(6): 532–55. PubMed Abstract \| Publisher Full Text 2. Palmer LJ, Knuiman MW, Divitini ML, et al.: Familial aggregation and heritability of adult lung function: results from the Busselton Health Study. Eur Respir J. 2001; 17(4): 696–702. PubMed Abstract \| Publisher Full Text 3. Wilk JB, DeStefano AL, Joost O, et al.: Linkage and association with pulmonary function measures on chromosome 6q27 in the Framingham Heart Study. Hum Mol Genet. 2003; 12(21): 2745–51. PubMed Abstract \| Publisher Full Text 4. Klimentidis YC, Vazquez AI, de Los Campos G, et al.: Heritability of pulmonary function estimated from pedigree and whole-genome markers. Front Genet. 2013; 4: 174. PubMed Abstract \| Publisher Full Text \| Free Full Text 3. Wilk JB, DeStefano AL, Joost O, et al.: Linkage and association with pulmonary function measures on chromosome 6q27 in the Framingham Heart Study. Hum Mol Genet. 2003; 12(21): 2745–51. PubMed Abstract \| Publisher Full Text Supplementary material Supplementary material Supplementary Information: File includes Supplementary Note, Supplementary Methods, Supplementary Figures and Supplementary Tables, as detailed below. Click here to access the data. Click here to access the data. Supplementary Note includes individual study descriptions. Supplementary Note includes individual study descriptions. Supplementary Note includes individual study descriptions. Supplementary Methods includes details of study level quality control procedures and eQTL analyses. Supplementary Figures: Supplementary Methods includes details of study level quality control procedures and eQTL analyses. Supplementary Tables: Supplementary Table 1 Details of study specific genotyping platform, genotype calling procedure and software. Supplementary Table 2 - Association results for all SNPs identified in single variant association discovery analyses (P<10-4). Supplementary Table 3 - Association results for SNPs identified in single variant association discovery analyses (P<10-4), located in known lung function regions. Supplementary Table 2 Association results for all SNPs identified in single variant association discovery analyses (P<10 ). Supplementary Table 3 - Association results for SNPs identified in single variant association discovery analyses (P<10-4), located in known lung function regions. Supplementary Table 4 - Single variant association result for the seven novel signals, in smoking and ancestry subgroups. Supplementary Table 5 - Single variant association result for rs1448044 and FVC in ever smokers and never smokers separately, and in all samples combined. Supplementary Table 4 - Single variant association result for the seven novel signals, in smoking and ancestry subgroups. Supplementary Table 5 - Single variant association result for rs1448044 and FVC in ever smokers and never smokers separately, and in all samples combined. Supplementary Table 6 - Association results for all genes identified in discovery SKAT analyses (meta-analysis P<10-4). Supplementary Table 7 - Association results for all genes identified in discovery Weighted sum test (WST) test analyses (P<10-4). Supplementary Table 8 - Evidence for the role of novel variants identified in single variant association analyses as eQTLs. Supplementary Table 9 SIFT/Polyphen predictions for sentinel SNPs and proxies (r2>0 8) Supplementary Table 9 - SIFT/Polyphen predictions for sentinel SNPs and proxies (r2>0.8). Supplementary Table 10 - Protein and RNA expression results all implicated genes from the single variant association analyses. Supplementary Table 11 - Look-up of association results for SNPs at 7 of the 12 loci which showed allele frequency differences between individuals from different regions in the UK. Supplementary Table 11 - Look-up of association results for SNPs at 7 of the 12 loci which showed allele frequency differences between individuals from different regions in the UK. Supplementary Table 12 - All traits results for the seven novel lung function loci. Supplementary Table 12 - All traits results for the seven novel lung function loci. Supplementary Table 13 - Genomic Inflation Factors: consortium and meta-analysis level. Supplementary Table 13 - Genomic Inflation Factors: consortium and meta-analysis level. 1. References Supplementary Figures: Supplementary Figure 1 - Quantile-quantile (QQ) and Manhattan plots for consortium-wide analyses, and the combined meta-analysis. Supplementary Figure 2 - Region Plots for novel loci. Supplementary Figure 4 - Trait Transformation Sensitivity Analysis 4. Klimentidis YC, Vazquez AI, de Los Campos G, et al.: Heritability of pulmonary function estimated from pedigree and whole-genome markers. Front Genet. 2013; 4: 174. PubMed Abstract \| Publisher Full Text \| Free Full Text 1. Rabe KF, Hurd S, Anzueto A, et al.: Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD executive summary. Am J Respir Crit Care Med. 2007; 176(6): 532–55. PubMed Abstract \| Publisher Full Text 2. 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Jackson V: SpiroMeta-CHARGE exome chip meta-analysis sumary results. 2017. http://www.doi.org/10.17605/OSF.IO/NSDPJ Page 20 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 No competing interests were disclosed. Competing Interests: We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Current Referee Status: Current Referee Status: 03 July 2018 Referee Report , Robin Beaumont Rachel M. Freathy Institute of Biomedical and Clinical Science, University of Exeter, Exeter, UK Institute of Biomedical and Clinical Science, University of Exeter, Exeter, UK Thank you to the authors for responding to and addressing our comments. I have one further comment on the replication analysis using UK Biobank data. The sensitivity analyses which the authors carried out showed that adjusting for covariates prior to inverse-normalization does affect the results. While this does not affect the main conclusions drawn, it may affect the results of the gene-based tests, and in addition, other investigators using the methods as a guide may draw inappropriate conclusions if adjusting for principal components prior to inverse-normalizing their phenotype. Ideally, the UK Biobank analysis should be redone with the appropriate phenotype transformation, and the methods and results sections updated accordingly. However, if the authors consider that such a revision would be too extensive, given that the conclusions do not change, it would at least be helpful to note the issue as a limitation in the discussion and make it clear in the methods that adjusting for covariates (such as principal components) should be done after inverse-normalising the phenotype – so it can be used appropriately by others. No competing interests were disclosed. Competing Interests: doi:10.21956/wellcomeopenres.15514.r33384 doi:10.21956/wellcomeopenres.15514.r33384 , Robin Beaumont Rachel M. Freathy Institute of Biomedical and Clinical Science, University of Exeter, Exeter, UK , Robin Beaumont Rachel M. Freathy Institute of Biomedical and Clinical Science, University of Exeter, Exeter, UK Author Response 17 Jul 2018 , University of Leicester, UK Victoria Jackson Author Response 17 Jul 2018 , University of Leicester, UK Victoria Jackson Thank you for you approval of our article, and your additional comment. As suggested, we have added a further limitation to the discussion of the paper outlining the issue regarding the trait transformation. This has also been noted in the methods. We have also included the results of the sensitivity analyses in the supplement (Supplementary Figure 4). No competing interests were disclosed. Competing Interests: No competing interests were disclosed. Competing Interests: Version 1 Page 21 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 doi:10.21956/wellcomeopenres.13627.r30984 , Lisa Strug Naim Panjwani Research Institute, Hospital for Sick Children, Toronto, ON, Canada The Hospital for Sick Children, Toronto, ON, Canada 1 2 1 2 , Lisa Strug Naim Panjwani 1 2 Research Institute, Hospital for Sick Children, Toronto, ON, Canada 1 Research Institute, Hospital for Sick Children, Toronto, ON, Canada 1 The Hospital for Sick Children, Toronto, ON, Canada 2 The Hospital for Sick Children, Toronto, ON, Canada 2 The Hospital for Sick Children, Toronto, ON, Canada 2 The authors have performed a large genome-wide association study in subjects of European (36,998 in the discovery set and 111,556 in the replication set) and African (7,721 in the discovery set) ancestries for various lung function measures: FEV1, FVC and FEV1/FVC ratio. Both common and rare variant analyses are performed, and the effect of smoking on the associations is also assessed. The discovery set consisted of CHARGE and SpiroMeta consortia meta analysis using the Human Exome array, while the replication set consisted of genotypes on the HumanCoreExome array and the UK Biobank’s custom arrays. A total of 7 novel regions were identified by the authors that met the overall (discovery+replication) Bonferroni-adjusted P-value of 2.8x10^-7 after adjustment for various covariates such as age, sex, height, and ancestry using principal components. All identified novel SNPs are of common frequency, and two of the SNPs are in high LD with missense variants predicted to be damaging. Some areas for improvement: Two rare variant tests were chosen and applied to the data as opposed to choosing a combined test (e.g. Derkach et al 2013 Genetic Epidemiology). A combined test would be more powerful. Two rare variant tests were chosen and applied to the data as opposed to choosing a combined test (e.g. Derkach et al 2013 Genetic Epidemiology). A combined test would be more powerful. The authors should explain why there was an inverse normalization of the traits in SpiroMeta but not in CHARGE, and provide some sensitivity analysis. There appear to be very large differences in Effect Allele Frequencies between the discovery and replication samples. Do the authors have an explanation for this? This might point to local ancestry differences that could be relevant, and should be further investigated. The eQTL analysis could formally investigate colocalization as opposed to cross-referencing individual associated SNPs with public repositories, and there are several different methods that achieve this goal: e.g. COLOC, eCAVIAR, Sherlock, RTC or EnLoc. Are the conclusions drawn adequately supported by the results? Yes No competing interests were disclosed. Competing Interests: , University of Leicester, UK Victoria Jackson Thank you the second set of reviewers for your helpful comments. Again, we have addressed specific points below, and and made appropriate amendments to the manuscript. 1. Two rare variant tests were chosen and applied to the data as opposed to choosing a combined test (e.g. Derkach et al 2013 Genetic Epidemiology). A combined test would be more powerful. We agree, a combined test would have been the preferred choice for gene-based association testing. However, in this instance, the gene-based tests were chosen due to practical reasons, as SKAT and WST, the two tests utilised, were both implemented by the meta-analysis software used by the two contributing consortia (RAREMETAL and seqMeta). Since this was a meta-analysis, and only summary statistics were available for each study, the gene-based tests we were able to utilise were restricted to those implemented by these two software packages at the time of the meta-analyses. For example, the suggested method by Derkach et al. requires permutation to calculate P-values with adequately controlled type 1 errors, which would not have been possible with the summary statistics available. 2. The authors should explain why there was an inverse normalization of the traits in SpiroMeta but not in CHARGE, and provide some sensitivity analysis. No competing interests were disclosed. Competing Interests: We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. doi:10.21956/wellcomeopenres.13627.r30984 In the replication analyses section, it is stated that “Traits were adjusted for age, age^2, height, sex, ten principal components and pack-years (ever smokers only), and inverse normally ” For clarity, the authors should be specific about whether the trait (FEV1, FVC, or transformed. FEV1/FVC) was inverse normalized first and age, age^2, sex, 10 PCs were then added as covariates in the genetic association model In the methods section for the rare variant testing Skat appears to be incorrectly referred to as a Fisher’s combined method. The authors should provide the justification for their various significance criteria used in each of the analyses. The authors should list the MAF alongside the p-values reported in the text for clarity for the single variant analysis results Is the work clearly and accurately presented and does it cite the current literature? Yes Page 22 of 28 Page 22 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes No competing interests were disclosed. Competing Interests: Yes 2. The authors should explain why there was an inverse normalization of the traits in SpiroMeta but not in CHARGE, and provide some sensitivity analysis. As mentioned in response to the other reviewers’ comments, we agree that using the raw trait in CHARGE and the transformed trait in SpiroMeta was not optimal; by the time we had made the decision to combine the results from the two consortia, all studies had already completed analyses, and reanalysis across the many cohorts would not have been feasible. Page 23 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 3. There appear to be very large differences in Effect Allele Frequencies between the discovery and replication samples. Do the authors have an explanation for this? This might point to local ancestry differences that could be relevant, and should be further investigated. Thank you for highlighting this. There was an error with the effect allele frequencies for the replication samples in Supplementary Table 2; these have now been amended, and the allele frequencies are more consistent in the discovery and replication samples. Where there are still some differences between the discovery and replication allele frequencies, these are where the discovery meta-analysis included individuals of both European and African ancestry, whereas the replication dataset included individuals of European ancestry only. 4. The eQTL analysis could formally investigate colocalization as opposed to cross-referencing individual associated SNPs with public repositories, and there are several different methods that achieve this goal: e.g. COLOC, eCAVIAR, Sherlock, RTC or EnLoc. Tests of colocalisation are more usually undertaken in dense genome-wide data, whereas the (often rare) putative causal variants included on the exome array in our study were relatively sparsely distributed. Furthermore, we did not have access to the lung eQTL data required to undertake a tests of colocalisation. We now acknowledge that the eQTL analysis did not include formal tests of colocalisation in the discussion, and in the example we highlight the variants are in complete LD. 5. In the replication analyses section, it is stated that “Traits were adjusted for age, age , height, sex, ten principal components and pack-years (ever smokers only), and inverse normally transformed.” For clarity, the authors should be specific about whether the trait (FEV , FVC, or FEV /FVC) was inverse normalized first and age, age , sex, 10 PCs were then added as covariates in the genetic association model. 2. The authors should explain why there was an inverse normalization of the traits in SpiroMeta but not in CHARGE, and provide some sensitivity analysis. 2 1 1 2 We have clarified in the methods for the replication analysis that “Traits were adjusted for age, age , height, sex, ten principal components and pack-years (ever smokers only), and the adjusted traits were inverse normally transformed.” 2 6. In the methods section for the rare variant testing Skat appears to be incorrectly referred to as a Fisher’s combined method. Within each consortium we generated results for SKAT. Subsequently, we combined the SKAT results from the two consortia using Fisher’s Method for combing P-values. We have clarified this in the text as “For genes which contained at least 2 polymorphic SNPs in both consortia, we combined the results of the consortium level gene based tests using either z-score meta-analysis (for the WST analysis) or Fisher’s Method for combining P-values (in the case of SKAT).”. 7. The authors should provide the justification for their various significance criteria used in each of the analyses. “We identified SNPs of interest as those with an overall P<10 and a consistent direction of effect and P<0·05 observed in both consortia. Rather than using a strict Bonferroni correction for defining -5 Page 24 of 28 Page 24 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 the significance threshold, we adopted the more lenient P<10 threshold in order to increase the power to detect variants with modest effect in our discovery analyses, whilst the requirement for consistency in results from the two consortia aimed to limit false positives. All SNPs meeting these thresholds were followed up in independent replicatizon cohorts.” -5 “We identified genes of interest as those with P<0·05 observed in both consortia and an overall P<10 , thresholds again chosen to limit both false positive and false negative findings.” The overall thresholds for the combined discovery and replication analyses were based on Bonferroni corrected thresholds, as already stated in the text. -4 8. The authors should list the MAF alongside the p-values reported in the text for clarity for the single variant analysis results MAFs and P-values have now been added to the main text for all reported loci. No competing interests were disclosed. Competing Interests: 25 January 2018 Referee Report doi:10.21956/wellcomeopenres.13627.r29790 doi:10.21956/wellcomeopenres.13627.r29790 The authors performed GWAS of FEV1, FVC and FEV1/FVC ratio at 179,215 SNPs from exome arrays. They identified 6 common frequency SNPs associated with at least one of these traits. They also identified 1 SNP in a region with known frequency differences across European populations suggesting that population structure may not have been fully accounted for in their analyses. Strengths of the study include the large sample size and comprehensive approach to assessing associations with low frequency and rare variants. We have the following concerns. Main concerns: 1. The phenotypes seem to have been adjusted for covariates and ancestry specific principal components prior to being inverse normally transformed. This transformation has the potential to introduce correlations between principal components and the inverse normally transformed phenotype ( ). Since one of the SNPs https://www.biorxiv.org/content/early/2017/05/15/137232 identified as being associated with the phenotype is known to vary in frequency across European populations, and the authors note that they cannot rule out the effects of population structure on the identified associations this raises concerns that some of the other associations could also be artefacts driven by failure to properly account for population stratification. It should explicitly be mentioned in the methods whether adjustments were made for ancestry specific principal components prior to inverse normal transforming the phenotype in the SpiroMeta Consortium component of the meta analysis or was included as a covariate in the phenotype - SNP association analysis. 2. Indeed, in the replication analysis in UK Biobank principal components were adjusted for prior to inverse normally transforming the data. Was genotyping chip adjusted for in this cohort (which should be done in the phenotype - SNP analysis)? The UKBiLEVE chip was enriched for smokers, which could affect association analyses unless chip is included as a covariate. In addition the Page 25 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 interim data release (which seems to be what is used here - please clarify in the methods whether the data comes from the interim (2015) or full (2017) data release) featured some discrepancies between the two chips, which can introduce spurious associations especially if adjustment is not made for genotyping chip. 3. Why was raw trait used in CHARGE but inverse normalised in SpiroMeta Consortium? This seems an odd choice 3. Why was raw trait used in CHARGE but inverse normalised in SpiroMeta Consortium? doi:10.21956/wellcomeopenres.13627.r29790 This seems an odd choice Minor concerns: 1. In the discussion, the authors mention that the 6 identified SNPs not attributed to population structure passed the Bonferroni significance threshold. They then mention that the SNPs ALSO pass Bonferroni corrected significance thresholds in the replication analysis. This could be misleading, since not all SNPs passed the Bonferroni threshold in the discovery only dataset. 2. The authors mention that correction was made for genomic inflation statistic (λ), but we could not find the statistics relating to this. The figures should be given in the manuscript. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? Yes No competing interests were disclosed. Competing Interests: We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. Reader Comment 12 Jun 2018 , University of Leicester, UK Victoria Jackson We thank the reviewers for their helpful comments. We have addressed each specific comment below, and amended the manuscript correspondingly. 1. The phenotypes seem to have been adjusted for covariates and ancestry specific principal components prior to being inverse normally transformed. This transformation has the potential to Page 26 of 28 2. The authors mention that correction was made for genomic inflation statistic (λ), but we could not find the statistics relating to this. The figures should be given in the manuscript. 2. The authors mention that correction was made for genomic inflation statistic (λ), but we could not find the statistics relating to this. The figures should be given in the manuscript. Is the work clearly and accurately presented and does it cite the current literature? Yes Are all the source data underlying the results available to ensure full reproducibilit No source data required Page 26 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 components prior to being inverse normally transformed. This transformation has the potential to introduce correlations between principal components and the inverse normally transformed phenotype (https://www.biorxiv.org/content/early/2017/05/15/137232). Since one of the SNPs identified as being associated with the phenotype is known to vary in frequency across European populations, and the authors note that they cannot rule out the effects of population structure on the identified associations this raises concerns that some of the other associations could also be artefacts driven by failure to properly account for population stratification. It should explicitly be mentioned in the methods whether adjustments were made for ancestry specific principal components prior to inverse normal transforming the phenotype in the SpiroMeta Consortium component of the meta analysis or was included as a covariate in the phenotype - SNP association analysis. In the SpiroMeta Consortium component of the analyses, adjustment for ancestry principal components (PCs) was not undertaken prior to transformation, rather PCs were adjusted for when fitting the SNP-trait associations. This is ambiguous in the text, and so we have amended the methods accordingly (Statistical analyses section, new wording below). Given that the adjustment for ancestry PCs was undertaken after phenotype transformation, we don't expect there to have been an introduction of correlation between the transformed trait and population structure. "Traits were adjusted for sex, age, age and height, and inverse normally transformed prior to association testing. For studies with unrelated individuals, SNP-trait associations were tested using linear models, with adjustments made for the first 10 ancestry principal components, whilst studies with related individuals utilised linear mixed models to account for familial relationships and underlying population structure." 2 2. Indeed, in the replication analysis in UK Biobank principal components were adjusted for prior to inverse normally transforming the data. Was genotyping chip adjusted for in this cohort (which should be done in the phenotype - SNP analysis)? The UKBiLEVE chip was enriched for smokers, which could affect association analyses unless chip is included as a covariate. In addition the interim data release (which seems to be what is used here - please clarify in the methods whether the data comes from the interim (2015) or full (2017) data release) featured some discrepancies between the two chips, which can introduce spurious associations especially if adjustment is not made for genotyping chip. Are all the source data underlying the results available to ensure full reproducibilit No source data required In the UK Biobank data, principal components (PCs) were adjusted for prior to transformation. As a sensitivity analysis, we have repeated the analysis for the six reported SNPs (the LCT SNP was not available in UK Biobank), transforming the phenotypes, and then adjusting for all covariates (including PCs) during the SNP-trait association test. For comparison, we have done this for all six SNPs with all three traits. Comparisons of these two analyses (not adjusted prior to transformation vs with adjustment prior to transformation) are shown here: j p ) https://doi.org/10.6084/m9.figshare.5959906. For each SNP, the P-value comparison is highlighted for the trait we report the association with, and the dashed lines indicate the Bonferroni corrected significance threshold for independent replication (P<1·47×10 ). Whilst there is a difference in the P-values for some SNP-trait combinations, (more significant P-values in the analysis with covariate adjustment prior to transformation for 5 of the 6 SNPs), the SNPs all meet the replication P-value threshold in both analyses. -3 We have clarified in the methods (Study design, cohorts and genotyping section) that the UK Biobank data used was from the 2015 interim release. The UK Biobank analysis was stratified by Page 27 of 28 Wellcome Open Research 2018, 3:4 Last updated: 31 AUG 2018 smoking status (ever and never) and also chip (UK BiLEVE array and UK Biobank array). It was not clear from the methods previously that the analysis was stratified for chip, so we have now made this clear in the methods. We have also tested whether any of the six reported SNPs available in UK Biobank had different MAFs in the UK BiLEVE and UK Biobank samples (suggestive of a chip effect); however none showed evidence of this: https://doi.org/10.6084/m9.figshare.5959927. 3. Why was raw trait used in CHARGE but inverse normalised in SpiroMeta Consortium? This seems an odd choice We agree that using the raw trait in CHARGE and the transformed trait in SpiroMeta was not ideal; however it was not planned to combine the results of these consortia from the outset. By the time we had made the decision to combine the results from the two consortia, all studies had already completed analyses and it was not feasible for contributing studies to repeat the analyses with/out the transformation, as this would have involved a substantial amount of reanalysis from contributing studies. Are all the source data underlying the results available to ensure full reproducibilit No source data required Since the effect estimates were not on the same scale we could not do an inverse variance weighted meta-analysis; therefore we did a P-value based meta-analysis. This analysis should be valid given that appropriate analyses were done within each consortium. o co ce s 1. In the discussion, the authors mention that the 6 identified SNPs not attributed to population structure passed the Bonferroni significance threshold. They then mention that the SNPs ALSO pass Bonferroni corrected significance thresholds in the replication analysis. This could be misleading, since not all SNPs passed the Bonferroni threshold in the discovery only dataset. We have reworded this section of the discussion as follows: "There were six SNPs which reached P<10 in the discovery stage meta-analysis of single variant associations, and subsequently met the Bonferroni corrected significance threshold for independent replication (P<1·47×10 , corrected for 34 SNPs being tested). In the combined analyses of our discovery and replication analyses, these six SNPs met the exome chip-wide significance threshold (P<2·8×10 )." -5 3 -7 2. The authors mention that correction was made for genomic inflation statistic (λ), but we could not find the statistics relating to this. The figures should be given in the manuscript. We have added Supplementary table 13 to the supplement. No competing interests were disclosed. Competing Interests: Page 28 of 28
W4382363565.txt	https://www.researchsquare.com/article/rs-3085244/latest.pdf	en		Ecotype maintenance in Callicarpa subpubescens via local environmental adaptation amidst frequent hybridization and low pre- and post-mating barriers	Research Square (Research Square)	2,023	cc-by	9,534	Ecotype maintenance in Callicarpa subpubescens via local environmental adaptation amidst frequent hybridization and low preand post-mating barriers Suzuki Setsuko Forestry and Forest Products Research Institute https://orcid.org/0000-0002-0612-1853 Kyoko Sugai Shimane University https://orcid.org/0000-0003-2426-6156 Ichiro Tamaki Gifu Academy of Forest Science and Culture https://orcid.org/0000-0003-2315-243X Kayo Hayama Ogasawara Environmental Planning Laboratory Hidetoshi Kato Makino Herbarium Article Keywords: adaptive introgression, adaptive radiation, Callicarpa subpubescens, cryptic species, hybrid zone, ongoing speciation, the Bonin Islands Posted Date: June 28th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3085244/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: There is no duality of interest Version of Record: A version of this preprint was published at Heredity on May 7th, 2024. See the published version at https://doi.org/10.1038/s41437-024-00684-3. Page 1/18 Abstract Adaptive radiations frequently occur on oceanic islands, but the mechanisms behind the maintenance of species and ecotypes after such radiations remain poorly understood, especially regarding potential gene flow among sympatric congeners. Callicarpa subpubescens is endemic to the oceanic Ogasawara Islands, and it has been suggested that multiple ecotypes exist within the southern part of this group, the Hahajima Islands, each associated with a unique localized habitat. Here, we determined the habitat characteristics of each ecotype and the degree of pre- and post-mating isolation by investigating flowering time overlap cross compatibility, and gene flow among ecotypes using an EST-SSR marker analysis of adult trees and naturally pollinated seeds. Our study confirmed the presence of four distinct ecotypes on the Hahajima Islands, with one likely arising from hybridization. Each ecotype was confined to specific island habitats, and distinct leaf morphologies and plant heights suggested local adaptation. However, overlapping flowering times and successful artificial cross pollination indicated little pre- and post-mating isolation. Hybridization rates in adult trees and naturally pollinated seeds were 37.2% and 26.4%, respectively, and most hybrids were backcrosses with few first- or second-generation hybrids. The hybridization rates of each ecotype and paternal correlation indicated that the flowering synchrony and close spatial distribution of ecotypes contributed to hybridization among ecotypes. Overall, C. subpubescens likely maintains four distinct ecotypes in spite of frequent hybridization probably due to selection pressures that result in reversion to the original ecotype via backcrossing, since this is well-adapted to the local environment. Introduction Adaptive radiations are a pattern of ecological speciation in which a single species, over a relatively short period of time, differentiates into multiple closely related sympatric species showing morphological and physiological differentiation resulting from adaptation to contrasting environments or ecological niches (Gillespieet al. 2001; Givnish 1997; Schluter 2000). Adaptive radiations can involve ecological speciation, in which adaptation to different environments or ecological niches leads to the development of isolation barriers and reproductive isolation (Rundle and Nosil 2005; Schluter 2001). Reproductive isolation involves two broad types of isolation mechanism: pre- and post-mating barriers. The former prevents gene flow between different species or populations, for example by changing flower color (Bradshaw and Schemske 2003; Hoballahet al. 2007), morphology (Yanget al. 2007), odor (Okamoto et al. 2015), and/or flowering phenology (Martin et al. 2007) etc. In contrast, the latter prevent fertilization or the production of viable or fertile hybrid offspring after pollination (Case and Willis 2008; Sandstedt et al. 2021). Adaptive radiations are well suited for studying environmental adaptation during ecological speciation because they are characterized by the rapid emergence of many species that exhibit diverse environmental adaptations. Adaptive radiations have been shown to occur in many plants and animals, especially on oceanic islands (Baldwin 1997; Chiba and Cowie 2016; Grant and Grant 1996), probably due to the small number of species available to occupy diverse ecological niches. Many papers have reported when adaptive radiations occur, however, few studies have examined how species and ecotypes are maintained after diversification by directly measuring the degree of preand post-mating isolation (Christie and Strauss 2019), or by quantifying gene flow in naturally pollinated seeds (Goulson and Jerrim 1997). It is therefore important to clarify the adaptive evolutionary processes of island organisms to understand how biodiversity is maintained on oceanic islands. In recent years, it has become clear that hybridization between different evolutionary lineages or taxa has caused the rapid diversification of ecological traits and promoted adaptive radiations (Meier et al. 2017). It has also been shown that introgression, i.e., the transfer of genes from one taxon to another via hybridization and recurrent backcrossing, is potentially advantageous during the colonization of new niches because it can add novel genes and improves the fitness of the recipient taxon (Chhatre et al. 2018; Suarez-Gonzalez et al. 2016). Thus, introgression facilitates the rapid colonization of recipient taxon to a new niche that the donor taxon has inhabited (Arnold and Kunte 2017). In addition, hybrid zones are areas in which genetically distinct taxa come into contact to form hybrids that exhibit traits intermediate to those of the parent species (Barton and Hewitt 1985). Studies of hybrid zones suggest that most hybrids are less fit than their parents in their parents’ niches but are more fit in novel niches (Arnold and Hodges 1995; Barton 2001; Burke and Arnold, 2001; Lexer et al. 2003). Moreover, the ecological conditions that facilitate the establishment of hybrid zones may also likely to promote adaptive radiation since this both require new and previously unused niches (Seehausen 2004). The Ogasawara Islands are oceanic islands located in the northwest Pacific Ocean off the coast of Japan, approximately 1,000 km south of Tokyo. This island chain comprises four distinct groups: the Mukojima, Chichijima, and Hahajima Islands (collectively called the Bonin Islands) and the Volcano Islands. Their total land area is small (~80 km2), but their endemic species rates are as high as 40% for vascular flora (Onoet al. 1986) and more than 90% for land snails (Tomiyama and Kurozumi 1991). The elevation of the Hahajima Islands is the highest among the Bonin Islands, and their topography is also more varied relative to the others. Furthermore, cloud cover and fog frequently occur at high elevations, which allow the area to develop endemic mesic scrub that reach 1–2 m in height (Shimizu 1992). The genus Callicarpa (Lamiaceae) in the Ogasawara Islands includes three recognized endemic species, Callicarpa parvifolia, C. glabra, and C. subpubescens, which are considered to represent an adaptive radiation (Ono 1991). All three are dioecious, despite most Callicarpa species being hermaphroditic (Kawakubo 1990). Callicarpa parvifolia and C. glabra are distributed only in the Chichijima Islands, while C. Page 2/18 subpubescens is distributed widely across the Ogasawara Islands, including on the isolated Volcano Islands. Pollen dispersal occurs via insects, such as endemic small bees, introduced honey bees (Abe 2006), and endemic Xylocopa bees (SS personal observation). The sizes of fruits and seeds are approximately 3 mm and 2 mm, respectively, and small birds, such as the brown-eared bulbul (Hypsipetes amaurotis) are known to disperse their seeds (Sugai et al. 2019). Sugai et al. (2019) investigated the population genetic structure and phylogenetic analyses of these three Callicarpa species throughout the Ogasawara Islands using 14 microsatellite (SSR) markers. They found that the three species were clearly genetically distinct in the Chichijima Islands, while in the Hahajima Islands C. subpubescens had differentiated into three genetic groups, the spatial distribution of which appeared to be related to habitat differences rather than geographic gradients. Kawakubo (1986) also showed that variation in the leaf morphology of C. subpubescensin the Hahajima Islands was much higher than in the Chichijima Islands. These facts suggest that genetically distinct groups of C. subpubescens in the Hahajima Islands may form distinct ecotypes that represent ongoing adaptive radiation. Sugai et al. (2019) also uncovered significant admixture of genetic groups in some populations, which suggests that gene flow and hybridization occurred among genetic groups. Information on the frequency of hybridization, which genetic group pairs had higher hybridization rates, and whether different life history stages (e.g., adult trees vs. seeds) have different hybridization rates, is necessary to understand the mechanisms maintaining the distinctness of genetic groups. In this study, we conducted a detailed genetic analysis of C. subpubescens throughout the Hahajima and satellite Imoutojima Islands, which is located approximately 5.6 km south-southeast of the southern tip of Hahajima Island (Fig. S1). To do so, we analyzed samples from populations and isolated trees using 14 expressed sequence tag (EST)–based SSR markers. We aimed to answer the following questions: 1) Are there associations between genetic group and spatial distribution, habitat, leaf morphology, and the size structure of each genetic group. That is, are these genetic groups really ecotypes? If so, 2) Are there pre- and post-mating reproductive isolation mechanisms between ecotypes? 3) What are the hybridization rates of each ecotype in adult trees and naturally pollinated seeds? 4) How are ecotypes maintained? Materials and Methods Sample collection We comprehensively sampled leaves of 581 and 114 trees from the Hahajima and Imoutojima Islands (hereafter collectively referred to as the Hahajima Islands), respectively (Fig. 1, Fig. S1). These leaf samples were used to provide data for adult trees. Adult trees included nine populations taken from the Hahajima Islands that were sampled by Sugai et al. (2019) (i.e., SHHA, SHHB, SHHC, SHHD, SHHE, SHHF, SHHG, SHIA, and SHIB). Tree locations were recorded using a GPS receiver (Garmin GPSmap 60CSx). Upon harvest, leaf samples were desiccated using silica gel for DNA extraction. During this study, we identified four ecotypes in the Hahajima Islands by their distinctive morphological and genetic features. These ecotypes were named “G: Glabrescent,” “T: Tall,” “D: Dwarf,” and “M: Middle” (Table 1). To investigate the current gene flow among ecotypes at the seed stage, in 2013 naturally pollinated seeds were sampled from 76 maternal trees from five sites (i.e., 12–19 trees per site) dominated by each ecotype (Fig. 1, Table S1). For ecotype G, we sampled seeds from two sites, Gn and Gs. Seeds of C. subpubescens are small (i.e., ~2 mm) and it is therefore difficult to extract DNA directly from seeds; we therefore extracted DNA from germinated seedlings. Cold stratification for seeds were performed at 4°C in wet moss for six months and were then germinated at room temperature. We obtained a total of 1,260 seedlings from five sites (201–304 seeds per site; Table S1). These were preserved at −20°C until DNA extraction. DNA extraction and genotyping Genomic DNA was extracted from sampled leaves and seedlings using a modified CTAB method. Genotypes of each sample were characterized by the 17 EST-SSR markers listed in Table S2, which were developed for C. subpubescens (Setsukoet al. 2018). PCR was carried out in 6 µl reaction mixtures containing ca. 1 ng genomic DNA, 2.5 µl Type-it Multiplex PCR Master Mix (Qiagen, Hilden, Germany), and 0.2 µM of each primer. PCR conditions were as follows: 95°C for 5 min, then 35 or 38 cycles of 94°C for 30 s, 55°C or 60°C for 90 s, 72°C for 90 s, followed by final extension at 60°C for 30 min. PCR fragments were then separated using a 3130 Genetic Analyzer (Applied Biosystems, CA, USA) and genotyped using GeneMarker software (SoftGenetics, PA, USA). Characteristics of EST-SSR markers To check whether each EST-SSR locus met the requirements for population genetic analyses, we used BayeScan 2.1 (1,000,000 simulations) (Foll 2012) to identify outlier loci, which we defined as those with excessively high or low FST compared to neutral expectations. The existence of null alleles was checked using Micro-Checker version 2.2.3 (Van Oosterhoutet al. 2004) and linkage disequilibrium between loci in each population was tested using GENEPOP version 4.7 (Raymond and Rousset 1995; Rousset 2008). For these analyses, seven populations in the Hahajima Islands that did not have a pattern of admixture in Sugai et al. (2019), listed in Table S2, were used because the adult trees we sampled did not always aggregate as a population (Fig. 1) and population admixture might bias these results. Genetic analysis Page 3/18 We used the Bayesian clustering program STRUCTURE version 2.3.4 (Falushet al. 2007; Pritchard et al. 2000) to identify genetic groups of adult C. subpubescens trees in the Hahajima Islands, then checked whether these genetic groups corresponded to morphological ecotypes. This program assigns individuals to K subpopulations (clusters) based on an admixture model and a correlated allele frequencies model. We used runs involving 100,000 Markov chain Monte Carlo (MCMC) iterations after a burn-in period of 50,000 iterations. The analysis was repeated 30 times for each value of K from 1 to 10. The optimal value of K was selected by assessing the likelihood distribution (mean Ln P(K)) and ΔK values (Evannoet al. 2005). Next, we checked for the existence of minor clusters using the online version of CLUMPAK (Kopelmanet al. 2015). For this analysis, we defined the genetic cluster with the largest Q value as the ecotype of adult trees. Trees assigned at Q ≥ 0.9 to each cluster were then considered to be pure adult trees, and trees with Q < 0.9 were considered to be hybrid adult trees (Kato et al. 2014; Li et al. 2021). The hybridization rate of each ecotype was calculated as the percentage of hybridized trees relative to the total number of trees of that ecotype. For hybrid adult trees, largest and second largest Q values for each type of tree were used to estimate which ecotypes interbreed and formed the hybrid. Hybrid adult trees with 0.4 ≤ largest Q < 0.6 was defined as first or second filial generation hybrids (i.e., F1 or F2), and hybrids with 0.6 ≤ largest Q < 0.9 were considered to be backcross hybrids (Liet al. 2021). The results of our STRUCTURE analysis suggested that ecotype M resulted from hybridization between ecotypes G and T (see results). Therefore, we also conducted a principal coordinate analysis (PCoA) and a HIest (Fitzpatrick 2012) analysis using pure adults of ecotypes G, T, and M to test hybridization origin possibilities for ecotype M. PCoA was conducted using GenAlEx version 6.501 (Peakall and Smouse 2012). HIest is a program that estimates ancestry (S) and interclass heterozygosity (HI), where both S and HI range from 0 to 1. S is similar to the Q value estimated by STRUCURE and was defined in terms of ancestry of ecotype T—i.e., values of 0 and 1 indicate that a putative hybrid is actually a pure ecotype G or T, respectively. Expected values of HI for pure ecotypes, first filial generation hybrids (F1), second filial generation hybrids (F2), and first-generation backcross hybrids (BC1) were 0, 1, 0.5, and 0.5, respectively. By plotting S and HI on a triangular plot, we can see the hybrid status of the two ecotypes. The reference allele frequencies of ecotypes G and T were calculated using pure adult ecotypes G and T. When calculating allele frequencies for the alleles detected only in ecotype M, these alleles were assumed to be detected only once in both ecotypes G and T. Parameters were estimated using the maximum likelihood method with a simulated annealing algorithm of 10,000 iterations, surf options, and a start-grid value of 100. To evaluate the power of the genetic markers used, we simulated 1,000 individuals each of pure ecotype G, pure ecotype T, F1, F2, BC1 to G (BC1G), and BC1 to T (BC1T) using a sample function of R and assuming an infinite population size. Current hybridization rates among ecotypes on the seed level were estimated using naturally pollinated seeds sampled at five sites (Fig. 1). We assessed the Q values of each seed using the USEPOPINFO option in STRUCTURE. This analysis was performed using pure adult trees (POPFLAG = 1) and seeds sampled from pure adult trees (POPFLAG = 0). Allele frequencies were updated using only the reference data with POPFLAG = 1. K was fixed at four and we ran models 30 times with 100,000 MCMC iterations after a burn-in period of 50,000 iterations. STRUCTURE analysis of adult trees revealed that 27 of 76 maternal trees were hybrids (Table S1), and therefore we eliminated seeds of hybrid trees from this analysis. We therefore used a total of 847 seeds (i.e., 85–272 seeds per site) from 49 pure maternal trees (i.e., 6–15 trees per site). We defined seeds with an assigned Q ≥ 0.9 for each cluster as pure seeds, and those with Q < 0.9 as hybrid seeds. Hybridization rates for naturally pollinated seeds were calculated as the percentage of the number of hybridized seeds relative to the total number of seeds. For hybrid seeds, the ecotype showing the largest Q value other than that of the ecotype of the maternal tree was defined as the ecotype of the pollen parent. Paternal correlation was estimated among all seed-parent pairs from the obtained genotypes of maternal trees and seeds using POLDISP (Robledo-Arnuncioet al, 2007). Then, we compared the paternal correlation within sites and between sites. For this analysis, we used all 1,260 seeds genotyped from all 76 maternal trees, including seeds from hybrid trees (Table S1), since paternal correlation is the probability of seeds sired by the same paternal tree in different maternal trees, thus paternal correlation is unaffected by whether the maternal tree is pure or hybrid. Ecotype characteristics To investigate the habitat of each ecotype, we extracted the forest type of adult trees that were categorized as pure ecotypes from a vegetation map (Fig. 1) sourced from the Biodiversity Center of Japan (1999-), and obtained elevation and slope data for all pure adult trees using ArcGIS Desktop version 10.8.2 (ESRI Japan, Tokyo, Japan). Elevation and slope values were extracted from a 10 m mesh digital elevation model provided by the Geospatial Information Authority of Japan. We used the medium and fine categories of the vegetation map, which indicated the dominant species, physiognomy, and geographical conditions. For the extracted vegetation categories of C. subpubescens on Hahajima Island, mesic scrub (i.e., a forest height of 1–2 m), dry scrub (1–6 m), and mesic forest (4–20 m) accounted for 69% of all total categories. The rest included 19% that was plantation forest and a remaining 10% that was Freycinetia formosana scrub and alien grassland of Kalanchoe pinnata and other species. We classified these into three major forest types: mesic forest, mesic scrub, and dry scrub (Shimizu, 1992). In this scheme alien grasslands were classified as dry scrub and F. formosana scrub was included in the mesic scrub category based on the ecological characteristics and habitats of each species. Plantation forests were excluded from our analyses since the original forest Page 4/18 type was unknown. In addition, we also investigated the forest height, the presence or absence of overstory trees of each adult C. subpubescens pure ecotype, and the species of overstory tree, if any. The relative photosynthetic photon flux density (rPPFD) was calculated from the following equation by simultaneously measuring the photosynthetic photon flux density (PPFD) above the canopy of each adult C. subpubescens pure ecotypeand at a nearby open site: rPPFD = (PPFD above the canopy / PPFD at open site) × 100. To characterize the leaf morphology of each ecotype, we sampled two to five intact leaves from a total of 28 pure adult trees (i.e., seven trees per ecotype). Moreover, we also examined the following 11 leaf traits: total length, blade length, width of leaf blade, hair density on the upper and lower surface of the leaf (i.e., number of hairs per 4 mm2), number of serrations per 30 mm, thickness of leaf blade, leaf area (LA), leaf mass per area (LMA), ratio of blade length to total leaf length, and the ratio of leaf blade width to length; where applicable, these measurements were taken as described by Kawakubo (1986). These characteristics were subjected to principal component analysis (PCA) to test the morphological aggregation of leaves of each ecotype. To characterize size distribution of adult trees of each ecotype, we measured the maximum stem length, the maximum diameter at breast height (DBH), and counted the number of stems per tree. Our samples included a total of 81 trees (i.e., 13–24 trees per ecotype) that were categorized as pure ecotypes. Pre- and post-mating reproductive barriers To determine whether pre-mating isolation exists among ecotypes, the flowering phenology of 57 trees (i.e., 9–18 trees per ecotype) that were categorized as a pure ecotype were investigated. The number of flowing cymes was counted for each tree once a month for eight months (i.e., May 2014 to January 2015). To determine whether post-mating isolation exists among ecotypes, we conducted artificial inter-crossings via pollination between different ecotypes, and intra-crossing via pollination within the same ecotypes. These experiments used plants derived from cutting seedlings raised in a greenhouse. For inter-cross pollination, a total 16 cymes from five maternal plants of ecotype D were crossed with two paternal plants of ecotype G (G × D). For intra-cross pollination, a total of 18 cymes from five maternal plants of ecotype D were crossed with eight paternal plants of ecotype D (D × D, Table S3). Fruit set rates were calculated for each cyme using the following equation: (number of fruits / number of flowers in the cyme) × 100. Next a total of 240 seeds from 10 pairs of inter-crosses and 384 seeds from 16 pairs of intra-crosses were sown. Their germination was monitored for six months (Table S3). The germination rate for each crossing pair was calculated using the following equation: number of germinated seedlings / number of sown seeds) × 100. Ninety-six germinated seedlings from each cross type were then transferred to pots and their mortality was tracked for one year in a laboratory environment with LED lighting and regular watering (Table S3). The mortality rate for each crossing pair was calculated using the following equation: (number of dead seedlings / number of seedlings transferred to pots) × 100. During the course of mortality tracking, we compared differences in soil moisture requirements among ecotypes. In addition to seedlings derived from artificial crossings (i.e., G × D and D × D), natural pollinated seeds from site Gn were sown and grown under the same conditions. Subsequently, an EST-SSR analysis was performed for seedlings from site Gn using the same method as for the adult trees, and only pure ecotype G seedlings were used for this experiment. Three months after transferred to pots, watering was temporarily stopped and the soil moisture content at the moment when seedlings began to wilt was measured using a soil moisture sensor (SM300, Delta-T Devices Ltd, Cambridge, UK). The volumetric soil water content (q% vol.) was calculated using the following equation: q = −27.8V 5 + 30.3V 4 − 0.7V 3 − 9.0V 2 + 3.8V. Here, V is the measured voltage value. This equation was obtained from the relationship between V and the volumetric water content of soil used for cultivation of seedlings. Results Characteristics of microsatellite markers We conducted an outlier test using BayeScan, and no outliers were detected for any of the 17 EST-SSR markers at a false discovery rate of 0.05 (Table S2). Three (Cal_0219, Cal_0351 and Cal_1632) of 17 markers may have null alleles since estimated their null allele frequency was significant in more than two out of the seven populations (Table S2). No significant linkage disequilibrium was observed between loci in any population for the 17 markers. Thus, we excluded three loci that might have null alleles and used 14 markers for further analyses. Ecotypes of Callicarpa subpubescens in the Hahajima Islands The STRUCTURE analysis showed that the log-likelihoods converged and reached a plateau at K = 6 (Fig. S2a). At K = 2, ∆K was highest, with smaller peaks at K = 4 and 6 (Fig. S2b). As we explain below, four phenotypical ecotypes of C. subpubescens were recognized in the Hahajima Islands; K = 2 could not differentiate these ecotypes (Fig. S2c), while K = 4 corresponded to the phenotypical ecotypes. K = 6 differentiated between three subtypes of ecotype G, which were found in the northern and southern parts of the Hahajima Islands and in the Imoutojima Page 5/18 Islands, and this was therefore probably caused by isolation by distance. Thus, we determined that K = 4 could represent the phenotypical ecotypes of C. subpubescens in the Hahajima Islands. In a STRUCTURE analysis at K = 3, we observed a genetic cluster that corresponded to ecotype M at K = 4 that showed an admixed pattern of ecotypes G and T (Fig. 2a). In this group, most trees had higher Q values of ecotype T than those of ecotype G. A PCoA analysis using pure adults from ecotypes G, T, and M revealed two axes (1 and 2) that explained 27.6% of the variation and clearly separated the three ecotypes (Fig. S3a). Moreover, axes 1 and 3, which collectively explain 21.8% of the variation, showed an overlap between ecotypes G and M, while ecotype T was separated (Fig. S3b). The PCoA suggested ecotype M was closer to ecotype G than T. In the HIest analysis, triangular plots between S and HI for the observed data showed that most points of ecotypes G and T were close to each pure ecotype, although the ecotype G datapoints were more scattered than the ecotype T datapoints (Fig. S4a). Specifically, we observed that datapoints for ecotype M were located in the left half of the plot area with located on the base and left-side lines of the triangle. The observed distribution of datapoints of ecotype M was therefore rather similar to a simulation of first-generation backcross hybrids to ecotype G (BC1G) (Fig. S4b). Spatial distribution, habitat, and morphological traits of ecotypes Pure ecotype D had the narrowest distribution range and was allopatrically distributed with other ecotypes (Fig. 1). We found that 78% inhabited the dry scrub with a mean height of 2.1 m on steep cliffs at elevations of 186.4 ± 37.6 m (Fig S5). No overstory trees existed in 11% of the locations of pure ecotype D, while 82% had alien Leucaena leucocephala trees in the overstory; regardless, the mean rPPFD was as high as 88%. On the other hand, pure ecotype G showed the widest distribution range, being present over all of Hahajima Island from low to high elevation (i.e., elevation: 158.5 ± 105.1 m). Moreover, 93% of the trees of this ecotype inhabited mesic forests that had a mean height of 7.8 m. All pure ecotype G individuals examined were present in the understory of mesic forests, with a mean rPPFD of 22%. Next, pure ecotypes T and M were both distributed on the ridges of the central mountain, but pure ecotype T (el. 302.2 ± 58.3 m) was distributed at a lower elevation than pure ecotype M (el. 394.0 ± 24.3 m). 88% of ecotype T trees inhabited mesic forests with a mean height of 7.9 m, while pure ecotype M was more concentrated in higher elevation areas and inhabited both mesic scrub (31%) and mesic forests (69%) with a mean forest height 3.9 m. Pure ecotype T had no overstory trees in 69% of the locations and constituted the forest canopy, while 30% had native trees in the overstory. On the other hand, 71% of pure ecotype M trees also had no overstory trees and therefore constituted the forest canopy, while 29% had alien Bischofia javanica in the overstory and showed a forest height of more than 5 m. The mean rPPFD was therefore as high as 88% for ecotype T and 86% for ecotype M. Next, PCA was conducted using eleven leaf morphological traits of pure adult trees for each ecotype. This analysis revealed that the first and second principal components explained 71.3% and 23.5% of the variation, respectively, and accounted for 94.8% of the total variation (Fig. 3). The distribution of each plot was not clearly separated, but different ecotypes did not overlap with each other. Ecotype M was located between ecotypes G and T. The leaf morphology of ecotype G was characterized by large leaf area, few hairs on either side of the leaf, and few leaf serrations (Fig. S6). The leaf morphology of ecotype D featured small, rounded, and thick leaves with many hairs. Ecotype T was characterized by short petioles and many hairs. Finally, ecotype M had moderate-sized leaves and a moderate number of hairs compared to the other ecotypes. The maximum stem length of pure adult trees significantly differed among all ecotype pairs except between ecotypes G and M (p < 0.05, Fig. 4a). In addition, DBH was significantly different among ecotypes except between ecotypes G and D, and G and M (p < 0.05, Fig. 4b). The largest maximum stem length and DBH value was found in ecotype T (mean: 7.1 m and 9.8 cm, respectively), followed by ecotypes M (mean: 3.5 m and 5.5 cm, respectively), G (mean: 3.0 m and 2.7 cm, respectively), and D (mean: 1.5 m and 0.7 cm, respectively). The number of stems within individual trees was significantly larger in ecotype D (mean: 7.7) than other ecotypes (mean: 1.1–1.5; p < 0.05, Fig. 4c). Pre- and post-mating reproductive isolation mechanisms The main flowering times of pure ecotypes G and M were almost the same from June to July and showed the same peak in July. The flowering time of pure ecotype T was from July to December with a peak in October (Fig. 5). The flowering time of pure ecotype D was long, lasting from July to January (except in September), with two peaks in August and November. This period overlapped with most trees of the other ecotypes. Flowering patterns were similar to those reported by Sugai et al. (2019), except that ecotype D showed a higher number of flowering trees in June and August. Although we found differences in peak flowering among all ecotype pairs except ecotypes G and M, the flowering periods more or less overlapped among all ecotypes. This suggests that all ecotypes have the potential to hybridize with others and thus that premating isolation among the ecotypes is not perfect. We found no significant difference in fruit set rate between inter-cross G × D and intra-cross D × D (p = 0.63, Fig. S7a). Moreover, with respect to germination rate, we found that seeds from inter-cross G × D were significantly more likely to germinate than intra-cross D × D (p < 0.05, Fig. S7b), and that the seedling mortality rate was not significantly different between inter- and intra-cross seedlings (p = 0.68, Fig. S7c). We note that the genetic distance between ecotypes G and D is rather large (Fig. 2a). Although we could not conduct artificial crossings for all ecotype Page 6/18 pairs, we found no evidence of mechanisms of post-mating isolation among ecotypes. Volumetric soil water content when seedlings began to wilt was found to be significantly lower in intra-cross D × D seedlings than in pure ecotype G, while inter-cross G × D seedlings showed intermediate values that did not significantly different from those of pure ecotype G and intra-cross D × D (Fig. S7d). Hybridization rate and paternal correlation We identified 365 pure adult trees on Hahajima Island, of which 154, 67, 53, and 91 were ecotypes G, T, D, and M, respectively. In contrast, we also found 216 adult trees on Hahajima Island that were hybrids, of which 184 were backcrosses and 32 were F1 or F2. The total hybridization rate of adult trees on Hahajima Island was therefore 37.2%. The hybridization rate of adult trees of each ecotype was ~20% for ecotypes T and M and ~40% for ecotypes G and M (Fig. 2a, Table 2a). Among the 216 hybrid adult trees, the ecotype pair G-M accounted for the highest percentage at 52.3%, while the ecotype pairs T-D and D-M both accounted for the lowest percentage at 6.0% (Table S4). Next, we found that the total hybridization rate of naturally pollinated seeds was 26.4%. The hybridization rate of naturally pollinated seeds for each ecotype was lowest at site T (7.7%) and highest at site Gs (48.2%; Fig. 2b, Table 2b). Among 224 hybrid seeds identified, the G-M ecotype pair was the most abundant at 27.2%, while the T-D ecotype pair was the least abundant at 8.0% (Table S4). The ecotype of the pollen parents of the hybrid seeds varied by site, with ecotype M being the most common at Gn, while ecotype T was the most common at Gs (Table 2). At site M, ecotype T was the most common pollen parent of hybrid seeds. Paternal correlations were always positive among maternal trees at the same sites and were particularly high at sites T and D (Fig. 6). On the other hand, paternal correlations among maternal trees of different sites were negative except for the Gn-Gs and Gn-M site pairs and were especially low for the Gn-T and Gs-T site pairs. Discussion Ecotypes of Callicarpa subpubescens in the Hahajima Islands Phenotypic and genetic clustering analysis revealed the existence of four ecotypes of C. subpubescens in the Hahajima Islands. Sugai et al. (2019) reported the existence of three genetic groups in the Hahajima Islands, and the population SHHE, which in that study showed an admixture pattern, was identified here as ecotype M. A genetic cluster with relatively few samples can be difficult to detect by STRUCTURE analysis (Meirmans 2019). Thus, the greater number of adult trees collected in this study likely allowed us to clearly identify ecotype M as a separate genetic cluster. The results of STRUCTURE, PCoA, and HIest analyses suggest that ecotype M may have resulted from a hybridization event between ecotypes G and T. An admixture analysis using more than 2,000 SNPs obtained by restriction site associated DNA sequencing (RAD-Seq) also showed that ecotype M is an admixture of ecotypes T and G (Setsuko et al. unpublished). A PCA analysis of leaf morphology revealed that ecotype M showed a distribution intermediate between ecotypes G and T. Moreover, the maximum stem length and DBH of ecotype M was also intermediate between ecotypes G and T, although we found no significant differences between ecotypes G and M. The flowering time of ecotype M was similar to that of ecotype G. In many plants and animals, speciation has been observed to accelerate via hybridization (Abbottet al. 2010; Mallet 2007). The presence of four ecotypes within a single tree species on small islands of approximately 20 km2 in size may be attributed not only to adaptive radiation, but also to hybridization among ecotypes via secondary contact. Local adaptation of ecotypes Ecotype D is mainly distributed in dry scrub locations on steep cliffs, while the other ecotypes are distributed in mesic forests and mesic scrub. Seedlings of intra-cross D × D wilted at lower soil moisture content than pure ecotype G seedlings. The maximum stem length and DBH of ecotype D were low and it had smaller, thicker leaves that contained many hairs. Previous studies have identified a negative correlation between drought tolerance and tree height (McGregor et al. 2021), and small, thick, trichome-rich leaves are also known to be an adaptation to dry areas (Ilyas et al, 2021; Tsujii et al. 2016). Taken together, these facts suggest that ecotype D is adapted to lower soil moisture and this may be why it inhabits soil types that the other ecotypes do not. In the Bonin Islands, several endemic tree species are known to exhibit different genetic groups within a single species that are distributed in mesic forest and dry scrub (Sugaiet al. 2022; Tsuneki et al. 2014). This suggests that genetic differentiation by soil moisture conditions is probably a common pattern of differentiation in the Bonin Islands. Ecotype G, which is distributed throughout the understory of the mesic forests, has almost no leaf hairs, whereas ecotypes T and M, which grow in bright areas and constitute the forest canopy, have hairs on their leaves. Ecotype D, that also have hairs on their leaves, may have originally constituted the canopy of dry scrub, although at present the upper layer is covered by alien trees. Leaf hairs are known to reduce photoinhibition caused by strong sunlight (Ripleyet al. 1999). Growing ecotype G in a sunny location causes leaf burn and atrophy, while no such phenomenon occurs in the other three hairy ecotypes (SS personal observation). Taken together, these findings suggest that ecotype G is clearly not adapted to full sun exposure. Thus, ecotype G and other hairy ecotypes are considered to have undergone adaptation to the contrasting light intensity regimes that characterize the canopy and understory environments of mesic forests. Page 7/18 Ecotypes M and T mostly occur in separate habitats, although a few ecotype T plants are distributed within the distribution area of ecotype M. Ecotype M was predominantly distributed along high-elevation mountain ridges in mesic scrub, which is characterized by lower forest height, or at the edge of mesic forests. Ecotype M constitutes the forest canopy of mesic scrub or mesic forests with low tree height (i.e., at most 4 m excluding alien trees), while ecotype T constitutes the forest canopy of mesic forests (which can be as high as 8 m or more). Since ecotype M is assumed to be derived from the hybridization of ecotypes G and T, the distribution of ecotype M can therefore be considered as a hybrid zone. This hybrid zone likely formed because ecotype M is adaptive in a new niche, mesic scrub, in which the other ecotypes had not previously dominated. Conversely, ecotype M is not adapted to the habitats of its parents (i.e., ecotypes G and T). Mesic scrub is distributed in areas with frequent cloud cover in areas above 350 m elevation on Hahajima Island (Shimizu 2001), and frequent cloud cover tends to reduce the amount of sunlight (Loope and Giambelluca 1998). Ecotype M may therefore be able to dominate in the mesic scrub because ecotype M is more shade-tolerant than ecotype T due to its parentage from ecotype G, which is distributed in the mesic forest understory. If this is the case, ecotype M may be an example of adaptive introgression (Suarez-Gonzalezet al. 2018). On the other hand, ecotype M inhabited mesic scrub and mesic forests with low forest height, and did not inhabit mesic forests with high forest height, where ecotype T inhabited. The reason why ecotype M cannot survive in those areas may be because ecotype M, which has ecotype G as a parent, cannot grow as tall as ecotype T. To test these possibilities, future studies are needed. Specifically, common garden experiments are needed to determine whether differences in shade tolerance and growth rates are between ecotypes T and M, as well as which genes ecotype M has acquired from its parental ecotypes. Hybridization rates among ecotypes and contributing factors Paternal correlations were generally positive within the same site and negative between different sites. Moreover, we observed substantial negative values for site pairs Gn-T and Gs-T, which differ in flowering peaks, and exceptionally positive correlations for site pairs Gn-Gs and GnM, which showed synchronized flowering peaks. This is consistent with the results that the most common hybrid mating ecotype pair was GM, both in adult trees and in naturally pollinated seeds. These results indicate that the degree of overlap in flowering time contributes to the degree of hybridization between ecotypes, as has been shown by other studies (Campbellet al. 2016; Field et al. 2011). On the other hand, ecotype D had the longest flowering time (i.e., from summer to early winter) and therefore had potential to hybridize with all other ecotypes. Interestingly, the hybridization rates of adult trees and natural pollinated seeds of ecotype D were not very high, and paternal correlations were negative for pairs that included D and any of the other sites. This was probably because ecotype D is locally distributed on dry scrub on steep cliffs. Pollen dispersal is therefore limited by distance (Adams 1992), and hybridization rates are therefore expected to lower for locally isolated ecotypes (Lagache et al. 2013). Ecotype maintenance mechanism The total hybridization rates were about 30%–40% at both the adult tree and natural pollinated seed stages. This result suggests that ecotypes of C. subpubescens on Hahajima Island are maintained despite relatively high gene flow between ecotypes. This could be explained as follows: The results of the STRUCTURE analysis of adult trees (K = 4) suggested that most hybrid individuals are backcrosses. Even when F1 trees were produced by hybridization between ecotypes, backcrossing pollination would be more frequently occur than F2 because F1 trees are rarer than pure trees (Tochigiet al. 2021). As backcrosses are then iteratively repeated, the genetic composition of the offspring derived from hybridization would revert to the genetic composition of the pure ecotypes. Next, we note that there may be a reason for the small number of F1 or F2. Soil moisture when the seedlings began to wilt was lowest in intracross D × D, intermediate in inter-cross G × D, and was the highest for pure ecotype G. This suggests that fitness of hybrids in the dry habitat is inferior to pure ecotype D. In Machilus, a tree species endemic to the Bonin Islands, hybrids with high genetic composition of mesic ecotypes that inhabit dry habitats are removed from dry habitat alongside pure mesic ecotype individuals as they mature (Tsuneki 2012). This result indicates that the fitness of F1 or F2 hybrids of mesic and dry ecotypes in dry habitats is lower than in backcrosses to dry ecotypes, which results in a lower number of F1 or F2 trees. In fact, in this study, we found very few F1 or F2 relative to backcrosses. These facts suggest that selection against hybrids, especially F1 or F2, is occurring. The lower fitness of hybrids would also contribute the maintenance of parental ecotypes (Lepais and Gerber 2011; Twyfordet al. 2015), and reversion to the original ecotype, which is adapted to a specific environment, through backcrossing would contribute to ecotype maintenance. However, in the present study, we compared only the fitness of ecotypes of G, D, and F1 hybrids, so it remains necessary to elucidate the relationship between adaptive traits and survival rates among other ecotype pairs and hybrids between them. Declarations Acknowledgements Page 8/18 The authors are grateful to Y. Nakamura for providing the location of the adult trees; Dr. C. Migita, A. Hisamatsu, M. Yokoya and Y. Yoshii for their experimental support; Drs. T. Nagamitsu, N. Nakanishi, and J.R.P. Worth for their valuable advice. We also thank Metropolis of Tokyo, the Ministry of the Environmental Government of Japan, and Forestry Agency of Japan for allowing this study. This research was conducted using the Ogasawara Field Research Station of Tokyo Metropolitan University. This work was funded by Grants-in-Aid for Science Research from the Japanese Society for Promotion of Science (JP26290073, JP15K07203, JP21K05694), the Environment Research and Technology Development Fund of the Ministry of the Environment, Japan (4-1402). Author Contributions SS, KS, KH, and HK designed the research. SS, KS, KH, and HK sampled materials. SS performed all the laboratory work. SS, KS, and IT performed data analysis. All co-authors discussed the results. SS and IT wrote the paper. 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Name, ID, and characteristics of each ecotype Page 11/18 Ecotype Glabrescent Tall Dwarf Middle Ecotype ID G T D M Forest type and habitat Elevation Slope Forest height rPPFD Leaf hair density Flowering time Tree size Growth form (Range, average (m)) (Range, average (°)) (Average (m)) (Average (%)) (Average density of the upper and lower surfaces) (Range, peak) (Average max. stem length (m), Average max. stem DBH (cm)) (Average no. stems) Understory of mesic forest (13–424, 158.5) (3–30, 17.8) High Dark Glabrescent Summer Middle Tree (7.8) (21.7) (0.5, 0.4) (Jun.– Jul., Jul.) (3.0 m, 2.72 cm) (1.3) Crown of mesic forest (215– 427, 302.2) (3–41, 17.3) High Bright Many Autumn Tall Tree (7.9) (88.0) (50.8, 52.6) (Jul.– Dec., Oct.) (7.1 m, 9.97 cm) (1.1) Crown of dry scrub (101– 279, 186.4) (4–37, 25.1) Low Bright Many SummerWinter Dwarf Bush (2.1) (88.1) (32.3, 42.1) (Jul.– Jan., Aug & Nov.) (1.5 m, 0.68 cm) (7.7) (321– 456, 394.0) (4–41, 19.4) Low Bright Middle Summer Middle Tree (3.9) (85.7) (16.9, 13.1) (Jun.– Jul., Jul) (3.5 m, 5.52 cm) (1.5) Crown of mesic forest and mesic scrub Table 2. Purity and hybridization rates (%) of adult trees for each ecotype (a) and naturally pollinated seeds for each seed sampling site (b) a) Ecotype Purity rate (%) Pairwise hybridization rate (%) Total hybridization rate (%) (2nd largest Q) (Largest Q) G T D M G 58.3 - 8.7 4.9 28 41.7 T 79.8 4.8 - 4.8 10.7 20.2 D 77.9 4.4 13.2 - 4.4 22.1 M 55.2 23.6 15.2 6.1 - 44.8 b) Seed sampling site Purity rate (%) Pairwise hybridization rate (%) Total hybridization rate (%) (Pollen parent) G T D M Gn 67.9 - 5.7 7.9 18.6 32.1 Gs 51.8 - 27.1 12.9 8.2 48.2 T 92.3 4.8 - 2.6 0.4 7.7 D 72.7 10.6 5.6 - 11.1 27.3 M 58.6 16.4 24.3 0.7 - 41.4 Page 12/18 Figures Figure 1 Spatial distribution of adult trees of pure ecotypes and hybrids plotted on a topographic map of Hahajima Island (left) and pure ecotypes on a vegetation map of Hahajima and Imoutojima Islands (right). Enlarged distribution maps in the dashed square show five seed collection sites and indicate the spatial distribution of maternal trees. Letters indicated by arrows and letters in parentheses to the right of the seed collection site name are the population names and locations reported in Sugai et al. (2019). Page 13/18 Figure 2 Results of STRUCTURE analysis. (a) Bar plots of adult trees of each ecotype on Hahajima Island for K = 3 and 4. Neighbor-joining trees to the right of the bar plot show relationships among each cluster, and numbers beside the circles indicate the FST values of each cluster. (b) Bar plots of naturally pollinated seeds for each site at K = 4. Vertical columns represent individuals and bar height are proportional to the posterior mean of the estimated admixture proportion. Individuals were ordered by Q value at K = 4 for each ecotype. Horizontal red bars above the bar plot indicate individuals with Q < 0.9 (i.e., hybrids), and the numbers above indicate the hybridization rate for each category. Page 14/18 Figure 3 Distributions of the first and second principal components (i.e., PC1 and PC2) for eleven leaf morphological traits. Hair_U; leaf hair density on the upper surface, Hair_L; leaf hair density on the lower surface, Serration; number of serrations, LA; leaf area, LMA; leaf mass per area. Page 15/18 Figure 4 Size distribution of pure adult trees of each ecotype. Different letters indicate significant differences among ecotypes (p< 0.05, pairwise t-test with Bonferroni correction). Page 16/18 Figure 5 Temporal changes in the number of flowering cymes of each pure adult tree. Each horizontal line indicates an individual. Darker colors indicate a greater number of flowering cymes. Page 17/18 Figure 6 Paternal correlation within (black bars) and between sites (gray bars). Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTablesXXXFigsHDY23A0142.pdf Page 18/18
https://openalex.org/W2376802192	https://www.beilstein-journals.org/bjoc/content/pdf/1860-5397-12-92.pdf	English	null	Stimuli-responsive HBPS-<i>g</i>-PDMAEMA and its application as nanocarrier in loading hydrophobic molecules	Beilstein journal of organic chemistry	2,016	cc-by	6,395	Full Research Paper Beilstein J. Org. Chem. 2016, 12, 939–949. doi:10.3762/bjoc.12.92 Received: 18 December 2015 Accepted: 20 April 2016 Published: 10 May 2016 Associate Editor: H. Ritter © 2016 Chen et al; licensee Beilstein-Institut. License and terms: see end of document. Abstract The topic of stimuli-responsive nanocarriers for loading guest molecules is dynamic. It has been widely studied in applications in- cluding drug controlled release, smart sensing, catalysis, and modeling. In this paper, a graft copolymer (hyperbranched poly- styrene)-g-poly[2-(dimethylamino)ethyl methacrylate] (HBPS-g-PDMAEMA) was synthesized and characterized by 1H NMR and GPC. It was observed that the star-like HBPS-g-PDMAEMA formed aggregates in aqueous solution. The influence of polymer con- centration, ionic strength and pH value on the aggregates in aqueous solution was investigated by using UV–vis spectroscopy and DLS analysis. The results showed that size of aggregates was affected by a corresponding stimulus. In addition, the loading ability of HBPS-g-PDMAEMA aggregates was investigated by using pyrene or Nile red as the model guest molecules by using UV–vis and fluorescence spectroscopy. The results showed that HBPS-g-PDMAEMA aggregates were capable to encapsulate small hydro- phobic molecules. These newly prepared HBPS-g-PDMAEMA nanocarriers might be used in, e.g., medicine or catalysis. Stimuli-responsive HBPS-g-PDMAEMA and its application as nanocarrier in loading hydrophobic molecules Yongsheng Chen, Li Wang, Haojie Yu, Zain-Ul-Abdin, Ruoli Sun, Guanghui Jing, Rongbai Tong and Zheng Deng Full Research Paper Open Access Address: State Key Laboratory of Chemical Engineering College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China Email: Li Wang* - opl_wl@dial.zju.edu.cn; Haojie Yu* - hjyu@zju.edu.cn * Corresponding author Keywords: amphiphilic polymer; hyperbranched polystyrene; phase transition; poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA); stimuli-responsive nanocarriers Beilstein J. Org. Chem. 2016, 12, 939–949. doi:10.3762/bjoc.12.92 Received: 18 December 2015 Accepted: 20 April 2016 Published: 10 May 2016 Associate Editor: H. Ritter © 2016 Chen et al; licensee Beilstein-Institut. License and terms: see end of document. Full Research Paper Open Access Address: State Key Laboratory of Chemical Engineering College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China Email: Li Wang* - opl_wl@dial.zju.edu.cn; Haojie Yu* - hjyu@zju.edu.cn * Corresponding author Keywords: amphiphilic polymer; hyperbranched polystyrene; phase transition; poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA); stimuli-responsive nanocarriers Beilstein J. Org. Chem. 2016, 12, 939–949. doi:10.3762/bjoc.12.92 Received: 18 December 2015 Accepted: 20 April 2016 Published: 10 May 2016 Associate Editor: H. Ritter © 2016 Chen et al; licensee Beilstein-Institut. License and terms: see end of document. Open Access Beilstein J. Org. Chem. 2016, 12, 939–949. doi:10.3762/bjoc.12.92 Received: 18 December 2015 Accepted: 20 April 2016 Published: 10 May 2016 Associate Editor: H. Ritter © 2016 Chen et al; licensee Beilstein-Institut. License and terms: see end of document. Results and Discussion Synthesis and characterization of HBPS and HBPE-g-PDMAEMA The target polymer HBPS-g-PDMAEMA was prepared through AT-SCVP and following ATRP strategies as shown in Figure 1A. HBPS with hyperbranched topology was prepared through atom transfer radical self-condensing vinyl polymeriza- tion (AT-SCVP) by using vinylbenzyl chloride (VBC), a com- mercially available monomer, as monomer and self-initiator. The feeding ratio of VBC/CuCl/2,2-bipyrene (bpy) was 10:1:2. The 1H NMR spectrum of HBPS showed peaks around 5.7 and 5.2 ppm attributed to double bonds [26,27] as shown in Figure 1B. For AT-SCVP, the polymerization was initiated by benzyl chloride of VBC. It was found that each polymer chain was tethered with a double bond and chloride groups. Each monomer inserted into the polymer chain has one initiate site to start propagation of the side chains, which leads to the forma- tion of hyperbranched topology. The broad peak from 4.1 to 4.9 ppm was attributed to the methylene and methine protons adjacent to chlorine atoms, in which the split peak around 4.6 ppm was attributed to methylene and the split peak around 4.8 ppm was attributed to methine. The broad peak from 6.0 ppm to 7.5 ppm was attributed to phenyl groups. The ob- tained HBPS was used as macro initiator to polymerize DMAEMA through ATRP. The comparison of GPC curves in Figure 1C showed that the curve of the graft copolymer was shifted to less elution time. The GPC results show Mn of HBPS to be 4.4 kg/mol with a molecular weight distribution of 1.85 and Mn of HBPS-g-PDMAEMA is 151.3 kg/mol with a molec- ular weight distribution of 2.84. The molecular weight of HBPS-g-PDMAEMA is bigger than HBPS, which demon- strated that HBPS-containing chlorine atoms successfully initi- ated the ATRP of DMAEMA, which resulted in HBPS-g- PDMAEMA. Figure 1B shows the 1H NMR spectrum of HBPS-g-PDMAEMA. The structure of PDMAEMA was clearly confirmed according to literature [28] and the signal of HBPS segment was weak and appeared between 6.0 and 7.5 ppm [29]. The results of 1H NMR and GPC measurements showed that the target polymer HBPS-g-PDMAEMA was suc- cessfully prepared. Hyperbranched or star-like polymers have become attractive to academia and industry in recent years due to their special chem- ical and physical properties. In comparison with their linear counterpart, these polymers afford promising applications such as drug delivery [15-17], gene delivery [18-21] and catalysis [22,23]. Polystyrene is a biocompatible material with low polarity. Introduction ising candidates for transporting hydrophobic guest molecules such as anticancer or antitumor drugs. In the cases of polymeric nanocarriers with responsive shell and stable core, the external hydrophilic shell makes these nanocarriers soluble in water and Stimuli-responsive polymers have attracted much attention due to their broad applications including drug controlled release [1-5], sensing [6,7] and 4D printing [8,9]. Nanocarriers pre- pared from amphiphilic stimuli-responsive polymers are prom- 939 939 Beilstein J. Org. Chem. 2016, 12, 939–949. affords stimuli-responsive properties. The interior hydrophobic core acts as a container for guest molecules and affords protec- tion [10-12]. It is urgent to study their stimuli-responsive properties and to develop suitable nanocarriers for certain applications. HBPS-g-PDMAEMA aggregates stability in aqueous solution. The PDMAEMA segments behaved as functional chains to afford stimuli-responsive properties. The phase-transition be- havior of the polymer aggregates in aqueous solution was studied. The size of the polymer aggregates under different conditions (polymer concentration, ionic strength and pH value) was investigated. The loading ability for small hydrophobic molecules was studied. To the best of our knowledge, this is the first report to prepare HBPS-containing stimuli-responsive ma- terials for loading small molecules. Poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) is a well-known stimuli-responsive polymer that responds to changes in temperature, pH and ionic strength [13,14]. Among temperature-responsive polymers, PDMAEMA is an important material, as its low critical solution temperature (LCST) is close to the human body temperature. The stimuli-responsive poly- mers can be prepared by grafting or blocking hydrophilic PDMAEMA chains on a hydrophobic chain. These amphiphilic polymers may aggregate in water to form nanoparticles having stimuli-responsive properties. Different hydrophobic segments with different microstructures give the aggregates adjustable properties that can be used for various applications under differ- ent conditions. The study of the encapsulation of hydrophobic molecules into amphiphilic nanocarriers is important for cancer or tumor therapies because many of the newly synthesized effi- cient and useful drugs are insoluble in water. The influence of temperature on HBPE-g- PDMAEMA aggregates PDMAEMA in water at a concentration of 10 mg/mL. Figure 2B shows the phase transition of HBPS-g-PDMAEMA in water. When the temperature is lower than the LCST, the solution is clear, which demonstrates that the PDMAEMA seg- ment is soluble in water. When the temperature is higher than the LCST, the solution becomes opaque, which demonstrates that the PDMAEMA chains become partially insoluble. In aqueous solution, the phase inversion depends on the formation or breakdown of hydrogen bonds between polymer chains and water molecules as well as entropically driven effects. Figure 2C shows TEM images of HBPS-g-PDMAEMA aggre- gates. It was observed that the morphology of aggregates is inhomogeneous and the aggregates were composed of clusters of smaller units. These aggregates were soluble in water due to water soluble PDMAEMA chains. The aggregates were also stable enough because the incorporated HBPS segment is an excellent hydrophobic polymeric chain. PDMAEMA in water at a concentration of 10 mg/mL. Figure 2B shows the phase transition of HBPS-g-PDMAEMA in water. When the temperature is lower than the LCST, the solution is clear, which demonstrates that the PDMAEMA seg- ment is soluble in water. When the temperature is higher than the LCST, the solution becomes opaque, which demonstrates that the PDMAEMA chains become partially insoluble. In aqueous solution, the phase inversion depends on the formation or breakdown of hydrogen bonds between polymer chains and water molecules as well as entropically driven effects. Figure 2C shows TEM images of HBPS-g-PDMAEMA aggre- gates. It was observed that the morphology of aggregates is inhomogeneous and the aggregates were composed of clusters of smaller units. These aggregates were soluble in water due to water soluble PDMAEMA chains. The aggregates were also stable enough because the incorporated HBPS segment is an excellent hydrophobic polymeric chain. The PDMAEMA polycation becomes soluble in water due to the protonation of amine groups and hydrogen bonding with water molecules. Furthermore, PDMAEMA is sensitive to tem- perature, pH and ionic strength. The influence of temperature on PDMAEMA is important, because the LCST of PDMAEMA in water is near the human body temperature; a fact that might be exploited in drug delivery systems. We checked the light transmittance of the HBPS-g-PDMAEMA aqueous solution at a wavelength of 500 nm and a concentration of 10 mg/mL by using a UV–vis spectrophotometer equipped with a water bath, as shown in Figure 2A. Results and Discussion Synthesis and characterization of HBPS and HBPE-g-PDMAEMA It has been found that a hydrophobic polymer segment with low polarity was helpful in drug delivery systems and in the protection of drugs from degradation [12,24]. Star-like amphiphilic polymers can be prepared by grafting hydrophilic stimuli-responsive chains on hyperbranched polystyrene. Chen reported a star-like polymer HBPS-g-PNIPAM in which HBPS was prepared by click chemistry to load small hydrophobic molecules [25]. However, its preparation needs specially de- signed monomers, the preparation of which is tedious and time- comsuming. Hyperbranched polystyrene (HBPS) prepared through atom transfer radical self-condensing vinyl polymeriza- tion (AT-SCVP) was firstly reported in 1996 [26]. It used vinylbenzyl chloride (VBC), which is an important commercial- ly available monomer and its industrial synthesis was started from 1957, as monomer with a self-initiation site [27]. Produced HBPS is tethered with lot of peripheral chlorine atoms available for further modification or functional chain extension. Here, we prepared a hyperbranched graft copolymer HBPS-g- PDMAEMA. HBPS segments behaved as hyperbranched topology support and also as hydrophobic segment to give 940 Beilstein J. Org. Chem. 2016, 12, 939–949. Figure 1: (A) The synthetic routes to HBPS and HBPS-g-PDMAEMA; (B) 1H NMR spectra of HBPS and HBPS-g-PDMAEMA and (C) GPC curves of HBPS and HBPS-g-PDMAEMA. Figure 1: (A) The synthetic routes to HBPS and HBPS-g-PDMAEMA; (B) 1H NMR spectra of HBPS and HBPS-g-PDMAEMA and (C) GPC curves of HBPS and HBPS-g-PDMAEMA. The influence of temperature on HBPE-g- PDMAEMA aggregates In this case, pH value and ionic strength were not adjusted and deionized water was directly used to prepare the samples. The transmittance was constant around 0.95 from 25 to 30 °C. As the temperature was increased, the transmittance was decreased and reached a stable value at 40 °C. The curve of the first deriv- ative showed that the transmittance decreased dramatically at a temperature of about 39 °C, which is the LCST of HBPS-g- To further investigate the behavior of HBPS-g-PDMAEMA aggregates in water, DLS measurements were performed and the change in aggregate size as a function of temperature and 941 Beilstein J. Org. Chem. 2016, 12, 939–949. Figure 2: (A) The transmittance of HBPS-g-PDMAEMA aqueous solution of 10 mg/mL (pH 7); (B) pictures of aqueous solution of HBPS-g-PDMAEMA at 10 mg/mL below and above the LCST and (C) TEM images of HBPS-g-PDMAEMA aggregates. The transmittance of HBPS-g-PDMAEMA aqueous solution of 10 mg/mL (pH 7); (B) pictures of aqueous solution of HBPS-g-PDMAEMA below and above the LCST and (C) TEM images of HBPS-g-PDMAEMA aggregates. ature was increased further, PDMAEMA chains started to shrink and the size of the aggregates decreased. concentration were recorded. The solutions were prepared by dissolving HBPS-g-PDMAEMA in water. The results of the DLS measurements are shown in Figure 3A. Higher polymer concentrations led to larger aggregate sizes at each temperature. The HBPS-g-PDMAEMA aggregates at higher concentrations, such as 2 and 5 mg/mL, showed the maximum size at tempera- tures of 35–40 °C, close to the LCST of 10 mg/mL HBPS-g- PDMAEMA aggregates in water at 39 °C. Above 40 °C, the size of HBPS-g-PDMAEMA aggregates decreased with in- creasing temperature. The solution with concentration 1 mg/mL showed no obvious increase close to the LCST, but at higher temperatures the aggregate sizes become smaller. Figure 3B shows that these three samples all exhibit phase-inversion be- havior. When the temperature was higher than the LCST, the solution became opaque. Figure 3C showed the proposed mech- anism to explain the size change with temperature. At a temper- ature below LCST, the aggregates were formed from self- assembly of amphiphilic graft copolymer HBPS-g-PDMAEMA. When the temperature reached the LCST, the PDMAEMA chains became insoluble in water and the relatively higher con- centration made the PDMAEMA chains of two aggregates coil with each other, which led to bigger aggregates. As the temper- The effect of ionic strength on the aggregation behavior of HBPS-g-PDMAEMA The electrostatic interaction is a major factor to influence the behavior of PDMAEMA in aqueous solution. So in this work, the influence of ionic strength, which affects the electrostatic interactions, was studied. In this case, pH was not adjusted and deionized water was used to prepare buffer solutions of differ- ent ionic strengths. Figure 4A shows the size of 1 mg/mL HBPS-g-PDMAEMA aggregates at NaCl concentrations of 0, 5 and 20 mM as a function of the temperature. The size of polymer aggregates decreased with increasing temperature either in deionized water or aqueous solution with relatively low concentrations of NaCl, and there was no maximum point. This is because higher concentrations of NaCl lowered the repulsive electrostatic interactions between PDMAEMA chains. So, higher concentrations of NaCl led to smaller aggregation sizes. The change of size was caused from the tendency to insolubility of PDMAEMA segments at higher temperature, but for the sample with 154 mM NaCl, the trend was very different. 942 Beilstein J. Org. Chem. 2016, 12, 939–949. Figure 3: (A) The effect of HBPS-g-PDMAEMA concentration on the size of aggregates (pH 7); (B) photographs of phase inversion of HBPS-g- PDMAEMA at different concentrations of 1, 2 and 5 mg/mL in aqueous solutions and (C) the illustrated mechanism of changed sizes of HBPS-g- PDMAEMA aggregates in aqueous solution at different concentrations. Figure 3: (A) The effect of HBPS-g-PDMAEMA concentration on the size of aggregates (pH 7); (B) photographs of phase inversion of HBPS-g- PDMAEMA at different concentrations of 1, 2 and 5 mg/mL in aqueous solutions and (C) the illustrated mechanism of changed sizes of HBPS-g- PDMAEMA aggregates in aqueous solution at different concentrations. The size increased with heating and reached above 500 nm at 40 °C to even 2500 nm at 55 °C. The concentration of NaCl was sufficiently high to break the electrostatic interaction of PDMAEMA segments, so aggregation occurred and large sized aggregates were formed. also investigated. In this case, the change in ionic strength was ignored and it was not adjusted by using NaCl due to the small amounts of added NaOH or HCl. The solution samples were prepared by the addition of certain amounts of polymer in aqueous solutions with certain pH values of 5.9, 7.4 and 8.5. The concentrations of all the aqueous HBPS-g-PDMAEMA solutions were 1 mg/mL. Figure 5A shows photographs of each sample solution before and after heating. The effect of ionic strength on the aggregation behavior of HBPS-g-PDMAEMA The solution with pH 5.9 showed no obvious change before and after heating. The solutions with pH 7.4 and 8.5 showed phase-inversion and become turbid after heating. To further demonstrate phase- inversion behavior, transmittance of the solution was recorded at 500 nm and at different temperatures by using UV–vis mea- phase-transition of HBPS-g-PDMAEMA The phase-inversion behavior of HBPS-g-PDMAEMA depends on the hydrophobicity of PDMAEMA chains. The protonation of the tethered amine groups and the hydrogen bonding with water molecules are the major factors. In this work, the effect of pH on the phase-inversion behavior of HBPS-g-PDMAEMA is 943 Beilstein J. Org. Chem. 2016, 12, 939–949. Figure 4: The sizes of 1 mg/mL HBPE-g-PDMAEMA aggregates in aqueous solutions with different concentration of NaCl (A) 0, 5 and 20 mM; and (B) 154 mM (pH 7). Figure 5: (A) Photographs of phase inversion of 1 mg/mL HBPS-g-PDMAEMA in aqueous solution with different pH values and (B) the transmittance at 500 nm of HBPS-g-PDMAEMA in aqueous solution with different pH values. Figure 4: The sizes of 1 mg/mL HBPE-g-PDMAEMA aggregates in aqueous solutions with different concentration of NaCl (A) 0, 5 and 20 mM; and (B) 154 mM (pH 7). Figure 4: The sizes of 1 mg/mL HBPE-g-PDMAEMA aggregates in aqueous solutions with different concentration of NaCl (A) 0, 5 and 20 mM; and (B) 154 mM (pH 7). Figure 5: (A) Photographs of phase inversion of 1 mg/mL HBPS-g-PDMAEMA in aqueous solution with different pH values and (B) the transmittance at 500 nm of HBPS-g-PDMAEMA in aqueous solution with different pH values. Figure 5: (A) Photographs of phase inversion of 1 mg/mL HBPS-g-PDMAEMA in aqueous solution with different pH values and (B) the transmittance at 500 nm of HBPS-g-PDMAEMA in aqueous solution with different pH values. shown in Figure 6A. At higher pH, a lower degree of proton- ation occurred, which led to a smaller size of HBPS-g- PDMAEMA aggregates in aqueous solution. Figure 6B shows DLS results of HBPS-g-PDMAEMA aggregates in aqueous solution at pH 7.0, 7.4 and 8.5 at 30 and 50 °C. In deionized water, a broad size distribution of HBPS-g-PDMAEMA aggre- gates was obtained. The size of many aggregates in deionized water was higher than 500 nm. It is very interesting that in basic environment, the aggregates showed a lower distribution than in deionized water. The effect of temperature and pH on polydis- persity index (PDI) of HBPS-g-PDMAEMA aggregates was studied, as shown in Figure 6C. As temperature was increased from 35 °C, the PDI of these two samples sharply decreased below 0.1, which means that the aggregates under these condi- tions may be uniform. The PDI and the size of nanoparticles surements as shown in Figure 5B. phase-transition of HBPS-g-PDMAEMA The transmittance of the solution with pH 5.9 showed no turning-point, but the transmit- tance of the solutions with pH 7.4 and 8.5 showed obvious turning points with increasing in temperatures. The acidic envi- ronment could protonate the amine groups and make the PDMAEMA molecules soluble in water even at temperature above its LCST. Furthermore, the LCST of the solution with pH 8.5 was lower than that of the solution with pH 7.4. This may be caused by a high degree of amine deprotonation. The high pH value led to the insolubility of PDMAEMA chains, which resulted in the phase inversion of HBPS-g-PDMAEMA aggregates. The effect of pH on the size of HBPS-g-PDMAEMA aggre- gates was investigated by DLS analysis, the results of which are 944 Beilstein J. Org. Chem. 2016, 12, 939–949. Figure 6: The effect of pH on: (A) the size of 1 mg/mL HBPS-g-PDMAEMA aggregates; (B) the DLS results of 1 mg/mL HBPS-g-PDMAEMA aggre- gates in aqueous solution with pH 7.0, 7.4 and 8.5 at 30 and 50 °C; and (C) PDI of HBPS-g-PDMAEMA as function of the temperature in aqueous solutions with pH 7.4 and 8.5. Figure 6: The effect of pH on: (A) the size of 1 mg/mL HBPS-g-PDMAEMA aggregates; (B) the DLS results of 1 mg/mL HBPS-g-PDMAEMA aggre- gates in aqueous solution with pH 7.0, 7.4 and 8.5 at 30 and 50 °C; and (C) PDI of HBPS-g-PDMAEMA as function of the temperature in aqueous solutions with pH 7.4 and 8.5. the peak attributed to pyrene around 337 nm was increased with the increase in the concentration of HBPS-g-PDMAEMA, which indicated that pyrene was transported to the aqueous solution by using HBPS-g-PDMAEMA. used for biomedicine are important. For the human body, the size of these particles should be about 50–200 nm which could be excreted without any toxic effects. Larger nanoparticles may eliminate quickly from human body and smaller nanoparticles may block blood capillaries. In this work, pH 7.4 was similar to the pH of human blood and pH 8.5 was in the range of some secretion. In these pH, HBPS-g-PDMAEMA aggregates showed a narrow distribution which means that these stimuli- responsive polymers may be suitable for medicinal use. The graft copolymer HBPE-g-PDMAEMA is an amphiphilic polymer containing a hydrophobic HBPS segment and hydro- philic PDMAEMA segments. The amphiphilic structure of this polymer affords the capability to disperse small hydrophobic molecules in aqueous solution. phase-transition of HBPS-g-PDMAEMA The ratio of I3/I1 from fluores- cence spectroscopy, the intensity ratio between the peak around 382 nm and the peak around 373 nm, showed a relatively polar environment, with higher values meaning lower polarity. In this work, the pyrene/HBPS-g-PDMAEMA aqueous sample solu- tions were used for detecting the environmentally located polarity of encapsulated pyrene. Figure 7C,D showed that the intensity of I3 (normalized at I1) was increased to 0.68, as con- centration of HBPE-g-PDMAEMA was increased to 2.0 mg/mL. Although this value is much lower than pure ali- phatic hydrocarbons (1.65–1.8) [24], the improvement com- pared to water is a strong evidence for the loading capacity of HBPS-g-PDMAEMA for small hydrophobic molecules. The encapsulation of hydrophobic molecules in HBPS-g-PDMAEMA aggregates To confirm the loading capability of HBPS-g-PDMAEMA aggregates for hydrophobic molecules, pyrene was used as a model molecule. Pyrene, a fluorescence-sensitive molecule, is often used for detecting the local polarity of amphiphiles. To prepare the pyrene/HBPS-g-PDMAEMA aqueous sample solu- tions, 5 to 6 mg pyrene was added in 5 mL HBPS-g- PDMAEMA solution with concentrations from 0 to 10.0 mg/mL followed by agitation for 2 h and filtration through a 0.45 μm micro-filtration membrane. Figure 7A,B shows UV–vis data below 25 °C. It was observed that the intensity of 945 Beilstein J. Org. Chem. 2016, 12, 939–949. Figure 7: (A) The UV–vis spectra of HBPS-g-PDMAEMA/pyrene solution with different polymer concentrations; (B) the absorption at 337 nm of HBPS-g-PDMAEMA/pyrene solution with different polymer concentrations. (C)The fluorescence spectra normalized at 373 nm of HBPS-g- PDMAEMA/pyrene solution with different polymer concentrations and (D) the I3/I1 values of HBPS-g-PDMAEMA/pyrene solution with different polymer concentration. Figure 7: (A) The UV–vis spectra of HBPS-g-PDMAEMA/pyrene solution with different polymer concentrations; (B) the absorption at 337 nm of HBPS-g-PDMAEMA/pyrene solution with different polymer concentrations. (C)The fluorescence spectra normalized at 373 nm of HBPS-g- PDMAEMA/pyrene solution with different polymer concentrations and (D) the I3/I1 values of HBPS-g-PDMAEMA/pyrene solution with different polymer concentration. To further confirm the capability of HBPE-g-PDMAEMA aggregates to encapsulate small hydrophobic molecules, Nile red (NR), a commonly used fluorescence dye was used as a model molecule. The preparation of HBPS-g-PDMAEMA/NR solutions was similar to HBPS-g-PDMAEMA/pyrene solutions. As shown in Figure 8A, the encapsulation of NR by HBPE-g- PDMAEMA aggregates can be observed by the naked eye. The solution containing these aggregates showed the pink color of NR in hydrophobic environment, and solutions without aggre- gates were colorless. These results indicated the successful en- capsulation of NR in HBPE-g-PDMAEMA aggregates. The fluorescence spectra of HBPE-g-PDMAEMA/NR in aqueous solution shows peaks around 625 nm which were attributed to NR, as shown in Figure 8B. Figure 8C shows the fluorescence intensity of the peaks around 625 nm as a function of the con- centration of HBPE-g-PDMAEMA aggregates, and it was found that a large amount of NR was dispersed in water with more HBPE-g-PDMAEMA aggregates. The trend was non- linear unlike the linear trend of NR encapsulation in micelles, as the size and aggregation of HBPE-g-PDMAEMA are influ- enced by the polymer concentration as shown in Figure 3. The encapsulation of hydrophobic molecules in HBPS-g-PDMAEMA aggregates To further confirm the capability of HBPE-g-PDMAEMA aggregates to encapsulate small hydrophobic molecules, Nile red (NR), a commonly used fluorescence dye was used as a model molecule. The preparation of HBPS-g-PDMAEMA/NR solutions was similar to HBPS-g-PDMAEMA/pyrene solutions. As shown in Figure 8A, the encapsulation of NR by HBPE-g- PDMAEMA aggregates can be observed by the naked eye. The solution containing these aggregates showed the pink color of NR in hydrophobic environment, and solutions without aggre- gates were colorless. These results indicated the successful en- capsulation of NR in HBPE-g-PDMAEMA aggregates. The fluorescence spectra of HBPE-g-PDMAEMA/NR in aqueous solution shows peaks around 625 nm which were attributed to NR, as shown in Figure 8B. Figure 8C shows the fluorescence intensity of the peaks around 625 nm as a function of the con- centration of HBPE-g-PDMAEMA aggregates, and it was found that a large amount of NR was dispersed in water with more HBPE-g-PDMAEMA aggregates. The trend was non- linear unlike the linear trend of NR encapsulation in micelles, as the size and aggregation of HBPE-g-PDMAEMA are influ- enced by the polymer concentration as shown in Figure 3. was drawn as shown in Figure 9A and the above mentioned HBPS-g-PDMAEMA/NR solutions were studied by UV–vis spectroscopy as shown in Figure 9B. The standard curve of ab- sorbance at 520 nm of NR in dioxane exhibits an excellent linear relationship of which R2 is 0.99. By using the standard curve, the concentration of NR in each sample solution can be calculated. From calculation and fitting line, the result shows that 1 mg of HBPS-g-PDMAEMA could load about 28 μg NR, which is limited. It is maybe a result of low ratio of HBPS seg- ments and this inefficient loading method. The R2 of the best linear fit line is 0.96 and it shows that the concentration of NR increasing linearly with concentration of HBPS-g-PDMAEMA. It demonstrates that HBPS-g-PDMAEMA is still intact up to a concentration of 0.1 mg/mL. Therefore, the lower critical aggre- gation concentration in water is beyond 0.1 mg/mL. Conclusion In this paper, the graft copolymer HBPS-g-PDMAEMA was synthesized by ATRP and characterized by 1H NMR and GPC. The phase-inversion behavior of HBPS-g-PDMAEMA in aqueous solution was studied. Obvious optical changes were observed when the temperature reached the LCST. The influ- ence of polymer concentration, ionic strength and pH value was also investigated. The size of the aggregates was affected by the To further investigate the loading capability for NR of HBPE-g- PDMAEMA aggregates in aqueous solution, a standard curve 946 Beilstein J. Org. Chem. 2016, 12, 939–949. Figure 8: (A) Photographs of HBPS-g-PDMAEMA/NR in aqueous solution with different polymer concentrations; (B) the fluorescence spectra of aqueous HBPS-g-PDMAEMA/NR solutions with different polymer concentrations; (C) the fluorescence intensity of HBPS-g-PDMAEMA/NR in aqueous solutions with different polymer concentrations at peak around 627 nm. Figure 9: (A) Plot of the concentration of NR as a function of the UV–vis absorbance at a wavelength of 520 nm in dioxane. (B) Plot of the concentra- tion of NR as a function the concentration of HBPS-g-PDMAEMA. (Red line is the best linear fit line used for studying loading capacity.) Figure 8: (A) Photographs of HBPS-g-PDMAEMA/NR in aqueous solution with different polymer concentrations; (B) the fluorescence spectra of aqueous HBPS-g-PDMAEMA/NR solutions with different polymer concentrations; (C) the fluorescence intensity of HBPS-g-PDMAEMA/NR in aqueous solutions with different polymer concentrations at peak around 627 nm. Figure 8: (A) Photographs of HBPS-g-PDMAEMA/NR in aqueous solution with different polymer concentrations; (B) the fluorescence spectra of aqueous HBPS-g-PDMAEMA/NR solutions with different polymer concentrations; (C) the fluorescence intensity of HBPS-g-PDMAEMA/NR in aqueous solutions with different polymer concentrations at peak around 627 nm. Figure 9: (A) Plot of the concentration of NR as a function of the UV–vis absorbance at a wavelength of 520 nm in dioxane. (B) Plot of the concentra- tion of NR as a function the concentration of HBPS-g-PDMAEMA. (Red line is the best linear fit line used for studying loading capacity.) Figure 9: (A) Plot of the concentration of NR as a function of the UV–vis absorbance at a wavelength of 520 nm in dioxane. (B) Plot of th tion of NR as a function the concentration of HBPS-g-PDMAEMA. (Red line is the best linear fit line used for studying loading capacity.) Figure 9: (A) Plot of the concentration of NR as a function of the UV–vis absorbance at a wavelength of 520 nm in dioxane. Characterizations and measurements The proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker 400M spectrometer. The molecular weight of polymer was determined by gel permeation chroma- tography (GPC) (Waters-Wyatt) equipped with RI, UV, viscosity and LS detectors using tetrahydrofuran (THF) as eluent. The UV–vis spectra were recorded on a UV3802 (UNICO) ultraviolet spectrophotometer. The size and zeta potential of the polymer in aqueous solution at different temper- ature were measured on a Zetasizer 3000HSA dynamic light scattering (DLS)/zeta potential analyzer. The fluorescence spec- tra were recorded on an F-280 (Tianjin Gangdong) fluores- cence spectrophotometer. Conclusion (B) Plot of the concentra- tion of NR as a function the concentration of HBPS-g-PDMAEMA. (Red line is the best linear fit line used for studying loading capacity.) showed no phase inversion. The HBPS-g-PDMAEMA aggre- gates/pyrene solutions were used to study the loading ability for small hydrophobic molecules. The results demonstrated that the HBPS-g-PDMAEMA aggregates were capable of encapsulating small hydrophobic molecules. The I3/I1 value reached 0.7 when the polymer concentration was above 2.0 mg/mL. The encapsulation of Nile red showed that HBPS- corresponding stimulus. A higher temperature led to the shrinkage of PDMAEMA chains and resulted in the decrease in aggregates size. The polymer tends to further aggregation near the LCST at higher polymer concentrations. It was found that the basic environment results in narrow PDI of HBPS-g- PDMAEMA aggregates at a temperature above the LCST. In acidic environment, the HBPS-g-PDMAEMA aggregates corresponding stimulus. A higher temperature led to the shrinkage of PDMAEMA chains and resulted in the decrease in aggregates size. The polymer tends to further aggregation near the LCST at higher polymer concentrations. It was found that the basic environment results in narrow PDI of HBPS-g- PDMAEMA aggregates at a temperature above the LCST. In acidic environment, the HBPS-g-PDMAEMA aggregates 947 Beilstein J. Org. Chem. 2016, 12, 939–949. (82 μmol) were added into monomer solution. The reaction mixture was heated to 90 °C in an oil bath under constant stir- ring. After 3 h, the polymerization was stopped by immersing the reaction mixture in liquid nitrogen and putting it under ventilation. The product was precipitated by dropwise addition of the reaction solution in a large amount of hexane. After filtra- tion, the white product was collected and then redissolved in THF. In order to remove the Cu complex, the diluted solution was passed through a neutral alumina column. The eluted solu- tion was concentrated and dropwise added in large amount of hexane to precipitate the product. This process was repeated and then the obtained product was dried under vacuum at 40 °C. g-PDMAEMA aggregates are good containers for small hydro- phobic molecules. Preparation of HBPS and HBPS-g-PDMAEMA [26] The synthetic routes to HBPS and HBPS-g-PDMAEMA are shown in Figure 1A. The synthesis of HBPS was done by using a Schlenk line system under argon atmosphere. The solution of monomer was prepared by the addition of 3.0 mL VBC (21.1 mmol) in 8.4 mL benzyl chloride followed by deoxygena- tion by three cycles of freezing–thawing–freezing. The Cu com- plex solution was prepared by dissolving 0.86 g CuCl and 2.72 g bpy in 8 mL BC in the absence of oxygen. Then 2.0 mL of Cu complex solution containing 0.21 g CuCl (2.1 mmol) and 0.68 g bpy (4.2 mmol) was added into monomer solution. The resulting reaction mixture was heated to 120 °C in oil bath with constant stirring. After 4 h, the polymerization was stopped by placing the resulting reaction mixture into liquid nitrogen and under ventilation. The solution was diluted with THF. To remove Cu complex, the diluted solution was passed through neutral alumina column. The eluted solution was concentrated and dropwise added in large amount of methanol. After filtra- tion, white solid product was collected and redissolved in THF. The purified polymer was precipitated by adding resulting solu- tion in large amount of methanol. This process was repeated and the obtained product was dried under vacuum at 40 °C. 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https://openalex.org/W2069438954	https://www.frontiersin.org/articles/10.3389/fmicb.2012.00339/pdf	English	null	Use of functional gene arrays for elucidating in situ biodegradation	Frontiers in microbiology	2,012	cc-by	12,420	REVIEW ARTICLE published: 21 September 2012 doi: 10.3389/fmicb.2012.00339 REVIEW ARTICLE published: 21 September 2012 doi: 10.3389/fmicb.2012.00339 REVIEW ARTICLE REVIEW ARTICLE published: 21 September 2012 doi: 10.3389/fmicb.2012.00339 INTRODUCTION microbial communities (Guschin et al., 1997), several types have been designed to examine microbial communities. These include (i) phylogenetic oligonucleotide arrays (POA), designed to exam- ine phylogenetic relatedness or community composition using 16S rRNA or other conserved phylogenetic genes (Small et al., 2001; Loy et al., 2002; Wilson et al., 2002; Brodie et al., 2006); (ii) com- munity genome arrays (CGA),designed to examine the relatedness of microbial species or strains or to identify community mem- bers using whole-genomic DNA probes (Wu et al., 2004; Zhang et al., 2004); (iii) metagenomic arrays (MGA), designed as a high- throughput screening method using environmental clone library inserts as probes (Sebat et al., 2003; Mockler and Ecker, 2005; Gresham et al., 2008); (iv) whole-genome ORF arrays (WGA), designed to examine gene expression of individual microorgan- isms using probes for all ORFs in one or more genomes (Wilson et al., 1999), but can also be used for comparative genomics (Mur- ray et al., 2001); and (v) functional gene arrays (FGAs), designed to examine multiple functional genes at one time using probes for key genes involved in microbial functional processes of inter- est (Wu et al., 2001; He et al., 2007, 2010a). This review will focus on FGAs. As the most phylogenetically and functionally diverse group of organisms on the planet (estimated 2000–50,000 microbial species per gram of soil; Torsvik et al., 1990; Hong et al., 2006; Schloss and Handelsman, 2006; Roesch et al., 2007), microorganisms are critical to ecosystem functioning and are involved in the bio- geochemical cycling of carbon, nitrogen, sulfur, phosphorus, and metals, as well as degradation or stabilization of contam- inants in the environment. However, because a vast majority (>99%) of microorganisms remain uncultured (Amann et al., 1995; Fuhrman and Campbell, 1998; Whitman et al., 1998), culture-independent approaches must be used to gain a com- prehensive picture of microbial communities. However, many of the culture-independent methods, such as 16S rRNA gene- based cloning or quantitative PCR, require a PCR ampliﬁcation step, which introduces well-known biases (Suzuki and Giovan- noni, 1996; Warnecke et al., 1997; Lueders and Friedrich, 2003). In addition, since many functional genes have too much variance or too few sequences available, conserved PCR primers cannot be designed for many functional genes. Even if primers could be designed for many functional genes, performing PCR with many different primer sets would be cost- and time-prohibitive. Joy D. Van Nostrand1,2, Zhili He1,2 and Jizhong Zhou1,2* 1 Institute for Environmental Genomics, University of Oklahoma, Norman, OK, USA 2 Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, USA Joy D. Van Nostrand1,2, Zhili He1,2 and Jizhong Zhou1,2* 1 Institute for Environmental Genomics, University of Oklahoma, Norman, OK, USA 2 Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, USA Microarrays have revolutionized the study of microbiology by providing a high-throughput method for examining thousands of genes with a single test and overcome the limita- tions of many culture-independent approaches. Functional gene arrays (FGA) probe a wide range of genes involved in a variety of functions of interest to microbial ecology (e.g., carbon degradation, N ﬁxation, metal resistance) from many different microorganisms, cultured and uncultured.The most comprehensive FGA to date is the GeoChip array, which targets tens of thousands of genes involved in the geochemical cycling of carbon, nitro- gen, phosphorus, and sulfur, metal resistance and reduction, energy processing, antibiotic resistance and contaminant degradation as well as phylogenetic information (gyrB). Since the development of GeoChips, many studies have been performed using this FGA and have shown it to be a powerful tool for rapid, sensitive, and speciﬁc examination of microbial communities in a high-throughput manner. As such, the GeoChip is well-suited for linking geochemical processes with microbial community function and structure. This technology has been used successfully to examine microbial communities before, during, and after in situ bioremediation at a variety of contaminated sites.These studies have expanded our understanding of biodegradation and bioremediation processes and the associated microor- ganisms and environmental conditions responsible. This review provides an overview of FGA development with a focus on the GeoChip and highlights speciﬁc GeoChip studies involving in situ bioremediation. Reviewed by: Steven Ripp, University of Tennessee, USA Dwayne A. Elias, Oak Ridge National Laboratory, USA Correspondence: Jizhong Zhou, Institute for Environmental Genomics, University of Oklahoma, 101 David L. Boren Boulevard, Norman, OK 73019, USA; Department of Microbiology and Plant Biology, University of Oklahoma, 101 David L. Boren Boulevard, Norman, OK 73019, USA. e-mail: jzhou@ou.edu Keywords: functional gene microarray, GeoChip, microbial communities, bioremediation, biodegradation www.frontiersin.org Edited by: Matthew Fields, Montana State University, USA Matthew Fields, Montana State University, USA Matthew Fields, Montana State University, USA Reviewed by: Steven Ripp, University of Tennessee, USA Dwayne A. Elias, Oak Ridge National Laboratory USA Use of functional gene arrays for elucidating in situ biodegradation Joy D. Van Nostrand1,2, Zhili He1,2 and Jizhong Zhou1,2 1 Institute for Environmental Genomics, University of Oklahoma, Norman, OK, USA 2 Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, USA MICROARRAYS VS. HIGH-THROUGHPUT SEQUENCING Cap- ture microarrays have been developed, which use lower stringency conditions to hybridize or “capture” sequence variants (Albert et al., 2007; Okou et al., 2007). These captured sequences are then washed off and sequenced. An array with probes speciﬁc for viral families has been developed that uses the hybridization pattern to classify novel viruses (Wang et al., 2002; Ksiazek et al., 2003). As such, microarray and sequencing approaches could be used to maximize the beneﬁts and minimize the deﬁciencies of each. focus on speciﬁc functional groups or locations. For example, FGAs have been designed to examine methanotrophs (Bodrossy et al., 2003, 2006; Stralis-Pavese et al., 2004), N-cycling genes (Taroncher-Oldenburg et al., 2003; Jenkins et al., 2004; Steward et al., 2004; Zhang et al., 2007a), pathogens and virulence factors (Call et al.,2003; Kosti´c et al.,2005; Cleven et al.,2006; Miller et al., 2008; Palka-Santiniet al.,2009),rhizobialisolates(Bontempset al., 2005), and acid mine drainage (AMD) and bioleaching systems (Yin et al., 2007). The most comprehensive FGAs reported to date are the GeoChip arrays. The GeoChip 1.0 had 2006 oligonucleotide probes (50-mers) for genes involved in nitriﬁcation, denitriﬁ- cation, nitrogen ﬁxation, methane oxidation, sulfate reduction (Tiquia et al., 2004), organic contaminant degradation, and metal resistance (Rhee et al., 2004). This array was used in several studies examining microbial communities at uranium (U)-contaminated sites (Wu et al., 2006a; Waldron et al., 2009), in the Gulf of Mexico (Wu et al., 2008), and under different land use strategies (Zhang et al., 2007b) and showed FGAs to be useful for microbial com- munity studies. GeoChip 2.0 was developed to provide a truly comprehensive probe set for multiple functional gene categories and to provide increased speciﬁcity for highly homologous gene variants (He et al., 2007). GeoChip 2.0 contains 24,243 (50-mer) oligonucleotide probes targeting ∼10,000 functional genes from 150 gene families involved in the geochemical cycling of C, N, and P cycling, sulfate reduction, metal reduction and resistance, and organic contaminant degradation. MICROARRAYS VS. HIGH-THROUGHPUT SEQUENCING Microarrays allow the examination of thousands of genes at one time without the need for PCR ampliﬁcation of each gene. Since microarrays were ﬁrst shown to be valuable for the study of While this review focuses on functional gene microarrays, high- throughput sequencing has become an increasingly popular choice September 2012 \| Volume 3 \| Article 339 \| 1 FGAs for elucidation of bioremediation Van Nostrand et al. for examining and monitoring microbial communities and is even being used for metatranscriptome analysis (vanVliet,2010). How- ever, while many of the technical challenges of microarrays and high-throughput sequencing have been overcome, each still has some distinct advantages and disadvantages, which make them ideal as complementary approaches: (i) Random sampling errors. In most sequencing studies, only a small proportion of the micro- bial community is actually sampled (McKenna et al., 2008) and while theoretically with true random sampling the probability of sampling the same fraction of the community multiple times is low (Zhou et al., 2008), one would expect that dominant popula- tions would have a greater chance of being sampled multiple times. These sampling errors can result in low reproducibility between technical replicates (17.2 ± 2.3% for two replicates; 8.2 ± 2.3% for three; Zhou et al., 2011). Microarrays, in contrast, interro- gate all samples against the same set of sequences (probes), so that the same population is sampled each time. (ii) Relative abun- dance. Abundance of individual species will vary greatly within microbial communities. With sequencing-based approaches there will be a bias toward the most abundant sequences in the envi- ronment so that many of the obtained sequences will represent the most abundant species/sequences while possibly missing lesser abundant species/sequences. Microarrays are not affected in the same way since lesser abundance sequences will still hybridize to their corresponding probe and as long as it is above the detection limit, it will be detected. (iii) New sequence detec- tion. One of the greatest advantages of sequencing is that new sequences are easily detected since any sequences in the sam- ple can be sequenced (open system). Microarrays, in contrast, can detect only the limited number of sequences covered by the probe set on the array (closed system), as such, it is not able to detect new sequences. Some new microarray techniques have been developed to allow the discovery of new sequences. MICROARRAYS VS. HIGH-THROUGHPUT SEQUENCING This array has been used in numerous studies to examine microbial communities at met- als contaminated sites (Gao et al., 2007; Van Nostrand et al., 2009, 2011), oil or diesel-contaminated sites (Rodríguez-Martínez et al., 2006; Liang et al., 2009a,b), coral mucus (Kimes et al., 2009), lake or river samples (Ta¸s et al., 2009; Parnell et al., 2010), deep sea samples (Mason et al., 2009; Wang et al., 2009), Antarctic soils (Yergeau et al., 2007) and to examine the taxa–area relationship (Zhou et al., 2008). GeoChip 3.0 covers 56,990 sequences from 292 gene families, greatly increasing the number of genes and categories covered compared to GeoChip 2.0 and added new control features (He et al., 2010a). New gene categories include antibiotic resistance, energy processing, and phylogenetic markers (i.e., gyrB). A set of 16S rRNA gene probes were added as positive controls, human, plant, or hyperthermophile gene probes were added as nega- tive controls, and a common oligo reference standard (CORS) was added for data normalization and comparison. The CORS is composed of an artiﬁcial sequence probe that is co-spotted with each gene probe and the complementary CORS target, labeled with a contrasting ﬂuorescent dye to the sample, which is then spiked into each sample prior to hybridization (Liang et al., 2010). The signal intensity of the CORS probe can then used to nor- malize the signal intensity of the sample and allows comparison of samples hybridized at different times. In addition, a compu- tational pipeline has been developed for GeoChip probe design and data analysis. The GeoChip 3.0 has been used to examine microbial communities associated with elevated CO2 (He et al., 2010b), to examine communities within coal formation produc- tion waters (Wawrik et al., 2012b) or rhizosphere communities in As-contaminated sites (Xiong et al., 2010). GeoChip DESIGN PROBE DESIGN AND SELECTION GeoChip covers a wide range of functional genes and currently includes sequences from bacteria, Achaea, fungi, and viruses. The ﬁrst step in designing new probes for the array is deciding which processes should be included. Then genes for enzymes or proteins that are key to the process of interest are selected. These could be catalytic subunits or proteins with recognition sites or that pro- vide functional speciﬁcity. Next, keywords are selected to search public sequence databases (e.g., GenBank). The keywords should be as broad as possible since proteins from different microorgan- isms may be annotated differently or have more general or speciﬁc annotations. Once the sequences are downloaded, they are con- ﬁrmed by HMMER alignment1 with preselected seed sequences. The seed sequences are those sequences for which the protein iden- tity and function have been experimentally conﬁrmed. This is a critical step in the design process and these sequences should be selected with care. The HMMER conﬁrmed sequences are then used to design gene- or group-speciﬁc 50-mer oligonucleotide probes using new versions of the CommOligo software (Li et al., 2005) and experimentally determined criteria based on sequence homology (≤90% identity for gene-speciﬁc probes, and ≥96% for group-speciﬁc probes), continuous stretch length (≤20 bases for gene-speciﬁc probes, and ≥35 for group-speciﬁc probes), and free energy (≥35 kJ mol−1 for gene-speciﬁc probes, and ≤60 kJ mol−1 for group-speciﬁc probes; He et al., 2005b; Liebich et al., 2006). TheprobesarethenBLASTedagainsttheGenBankdatabase to conﬁrm speciﬁcity. Keywords, downloaded sequences, seed sequences, HMMER conﬁrmed sequences, and designed probes are stored in corresponding databases for use in future array updates. Nucleic acid quality is of great importance for microarray anal- ysis. DNA and RNA should have an A260 to A280 ratio ∼1.8 and >1.9, respectively and an A260 to A320 ≥1.7. The A260 to A320 ratio is most important in determining microarray success (Ning et al., 2009). Some environmental samples, especially those with high humics, can be difﬁcult to purify up to the necessary level. A gel puriﬁcation strategy followed by a phenol–chloroform–butanol extraction (Xie et al., 2007; Liang et al., 2011) has been successful with a wide range of soil and sediment samples. g p Large amounts of DNA (e.g., 1 μg) or RNA (e.g., 5 μg) are needed for GeoChip hybridization. DEVELOPMENT OF FUNCTIONAL GENE ARRAYS The ﬁrst FGA developed used PCR-amplicon probes and targeted fourN-cyclinggenes(nirS,nirK,amoA,andpmoA;Wuet al.,2001). However, since PCR-amplicons were used, only a limited num- ber of genes could be included because conserved primers can only be designed for a few functional genes. In addition, it would be cost- and time-prohibitive to amplify genes from hundreds of microorganisms or clones in order to achieve a truly diverse probe set. Most microarrays now use oligonucleotide probes, which are more speciﬁc (Zhou, 2003), can be easily customized (Denef et al., 2003; Zhou, 2003; Gentry et al., 2006), and are relatively inexpensive. Since then several different FGAs have been developed, although most of these cover only a limited number of genes and September 2012 \| Volume 3 \| Article 339 \| 2 Frontiers in Microbiology \| Microbiotechnology, Ecotoxicology and Bioremediation 2 FGAs for elucidation of bioremediation Van Nostrand et al. well-established freeze-grind method with detergent lysis (Zhou et al., 1996; Hurt et al., 2001) since it provides high molecular weight DNA, important for later ampliﬁcation steps. The use of RNA presents some challenges as mRNA is unstable and has a low abundance in environmental samples. Several papers have described methods for extracting environmental RNA,including a protocol for the dual extraction of both DNA and RNA (Hurt et al., 2001; Burgmann et al., 2003) or RNA alone (McGrath et al., 2008; Poretsky et al., 2009a). Methods for mRNA enrichment include size separation by gel electrophoresis (McGrath et al., 2008) or use of commercial kits [MICROBExpress (Ambion) and/or mRNA- ONLY (Epicentre Biotechnologies); Poretsky et al., 2009b; Mettel et al., 2010]. Size separation obtained 115–155 ng mRNA from 4.6–5.3 μg total RNA (McGrath et al., 2008). Using commercial kits, Mettel et al. (2010) were able to obtain 140–530 ng of mRNA from 0.4–2.0 μg total RNA per 0.5 g soil. GeoChip 4.0, the newest version, is synthesized by Nimble- gen (Madison, WI, USA) in their 12-plex format and contains 83,992 probes targeting 152,414 genes in 410 gene categories (Lu et al., 2012a). In addition to added genes in most categories, new categories added include stress response, antibiotic resistance, and bacteriophage genes. It has been used to examine microbial communities during the 2010 Gulf oil spill (Lu et al., 2012a), GeoChip DESIGN PROBE DESIGN AND SELECTION However, it can be difﬁcult to get sufﬁcient quantities of nucleic acid from some types of samples (e.g., water) or the sample is too difﬁcult or impossible to replace to use such large quantities of nucleic acid. In this case, ampliﬁcation of DNA or RNA can be done using either whole community genome ampliﬁcation (WCGA; Wu et al., 2006a) or whole community RNA ampliﬁcation (WCRA; Gao et al., 2007). WCGA uses the Templiphi 500 ampliﬁcation kit (phi 29 DNA polymerase, GE Healthcare, Piscataway, NJ, USA) with a modiﬁed ampliﬁcation buffer and using 1–100 ng DNA provides a sensitive (10 fg detection limit) and representative ampliﬁcation (<0.5% of ampliﬁed genes showed >2-fold difference from unampliﬁed; Wu et al., 2006a). WCRA provides a representative ampliﬁcation with 50–100 ng of starting material. The newly designed probe sets can then be commercially synthesized. Several options are available for producing arrays. Synthesized oligonucleotide probes can be spotted onto nylon membranes (Steward et al., 2004) or glass slides (Taroncher- Oldenburg et al., 2003; Rhee et al., 2004; Tiquia et al., 2004). Glass slides are more frequently used since they have less background ﬂuorescence (Schena et al., 1995, 1996) and allow higher probe density (Ehrenreich, 2006). Probes can also be added to slides usingbubbleJetprinting(Okamotoet al.,2000),laser-inducedfor- ward transfer (Serra et al., 2004), or photolithography (Chen et al., 2009). In addition, a few companies, such as Agilent or Affymetrix, synthesize custom microarrays using a customer’s probe set. There are commercial kits available for microbial RNA ampliﬁ- cation such as the MessageAmpTM II-Bacteria RNA Ampliﬁcation Kit (Life Technologies, Grand Island, NY, USA). There are also other commercially available methods for WCGA. Wang et al. (2011) compared two of these (Bacillus stearothermophilus DNA polymerase (Bst) and REPLI-g; Qiagen, Valencia, CA, USA) with the modiﬁed Templiphi kit (Wu et al., 2006a). The ampliﬁcation bias for all methods was relatively low (<3-fold). Less bias was observed with REPLI-g and Templiphi for pure culture DNA and with REPLI-g for community DNA while Bst showed the least inhibition by lesser quality DNA. The ampliﬁed (or unampliﬁed) nucleic acids are directly labeled with a ﬂuorescent dye (Cy3 or Cy5) using random priming with the Klenow fragment of DNA polymerase for DNA (Wu et al., 2006a) or SuperscriptTM II/III RNase H-reverse transcriptase for RNA (He et al., 2005b). The labeled DNA/RNA is then puriﬁed and dried for hybridization. 1http://hmmer.wustl.edu/ TARGET PREPARATION GeoChip can be hybridized with either DNA or RNA. Most DNA samples used for GeoChip analysis are extracted using a September 2012 \| Volume 3 \| Article 339 \| 3 www.frontiersin.org \| 3 FGAs for elucidation of bioremediation Van Nostrand et al. SEQUENCE COVERAGE f h b One of the main objectives in developing FGAs was to provide a truly comprehensive probe set (He et al.,2007). Each new GeoChip version has expanded the coverage of gene variants and expands the number of genes covered (He et al., 2007, 2010a; Lu et al., 2012a). This continued expansion is challenging as the number of gene sequences available is constantly increasing as new sequences are being submitted to public databases. While the GeoChip design pipeline2 has an automated update feature which uses the previ- ously selected key words and seed sequences to search the NCBI database, downloading new sequences and designing probes is still time consuming due to the sheer volume of sequences avail- able. As such, better and faster computation systems are needed. In addition, available microarray probe density limits are rapidly being approached as the number of GeoChip probes increases. So, new methods of array construction to increase probe density are needed. DATA ANALYSIS Due to the large volume of data obtained from GeoChip, data analysis can be very challenging. The data has a multivariate structure and the number of variables is much larger than the number of observations (p ≫n). To assist users with data analysis steps, a pipeline is available which performs many of the com- mon analyses3. Some common descriptive statistics used include relative abundance of gene categories or subcategories, richness and diversity (α and β) indices, and percentages of gene overlap between samples. To compare the overall community structure, unconstrained ordination [principal component analysis (PCA) and correspondence analysis (CA)] to reduce the dimensionality of variables in order to maximize the visible variability of the data or hierarchical cluster analysis (HCA), which groups communi- ties based on the similarity of their gene proﬁles, can be used. To compare communities, response ratios, which compare the signal intensity of genes between conditions (Luo et al.,2006; Liang et al., 2009a), t-tests,ANOVA,and dissimilarity tests can be used. Several methods can be used to examine the relationship between commu- nities and environmental parameters. These include constrained ordination, such as canonical correspondence analysis (CCA; ter Braak, 1986), distance-based redundancy analysis (db-RDA; Leg- endre and Anderson, 1999), variation partitioning analysis (VPA; ØklandandEilertsen,1994; RametteandTiedje,2007),andMantel test. A relatively new analysis method is the random matrix theory- based (Mehta, 1990) neural network analysis (NNA) used to examine gene relationships within microbial ecological networks (Zhou et al., 2010). 2http://ieg.ou.edu/ 3ieg.ou.edu/microarray/ HYBRIDIZATION AND IMAGE ANALYSIS The labeled nucleic acids are then hybridized to the microarray at 42–50◦C with 40–50% formamide (He et al., 2007, 2010a,b; Lu et al., 2012a). Hybridization speciﬁcity can be adjusted by varying the temperature or the formamide concentration (the effective hybridization temperature increases by 0.6◦C for every 1% of formamide). Hybridized slides are then scanned and analyzed by quantifying the pixel density (intensity) of each spot using image analysis software. Commercial manufacturers often have their own analysis software or other microarray analysis software can be used, such as GenePix Pro (Molecular Devices, Sunny- vale, CA, USA), GeneSpotter (MicroDiscovery, San Diego, CA, USA), or ImaGene (BioDiscovery, El Segundo, CA, USA). For GeoChip data, there is a data analysis pipeline2 for rapid prepro- cessing and data analysis. Poor and low quality spots and outliers, based on Grubbs’ test of outliers (Grubbs, 1969), are removed and then the signal intensities of all spots are normalized. Positive spots can be determined using signal-to-noise ratio [SNR = (sig- nal mean −background mean)/background standard devia- tion], signal-to-both-standard-deviations ratio [SSDR = (signal mean −background mean)/(signal standard deviation −back- ground standard deviation)] (He and Zhou, 2008), or signal- to-background ratio (SBR = signal mean/background mean) (Loy et al., 2002). Having high-quality nucleic acids (non-degraded, large fragments to improve ampliﬁcation yields, absence of inhibitors or contam- inants which may impede subsequent ampliﬁcation and labeling steps) is the most important criterion for successful microarray experiments. Nucleic acids can be puriﬁed using commercial kits although the presence of humic acids and other contaminants can still be a problem. If large amounts of DNA are present, an agarose gel puriﬁcation followed by phenol–chloroform–butanol extrac- tion (Xie et al., 2007; Liang et al., 2009b) can be used, but large amounts of DNA are lost with this method so it is not practical for low abundance samples. So, better puriﬁcation methods with high recovery yields are needed. Frontiers in Microbiology \| Microbiotechnology, Ecotoxicology and Bioremediation 3ieg.ou.edu/microarray/ SPECIFICITY AND SENSITIVITY Two key issues for microarray hybridization of microbial com- munities are speciﬁcity and sensitivity since environmental com- munities can have such vast diversities. Both of these conditions can be improved at various stages of microarray design, construc- tion, target preparation, or hybridization. During probe design, determining the best criteria for probe design, such as using exper- imentally determined design criteria (He et al., 2005b; Liebich et al.,2006) can improve speciﬁcity, thus decreasing the number of false positives (He et al., 2007). Probe length also affects speciﬁcity and sensitivity; longer probes are more sensitive, but less speciﬁc (Denef et al., 2003; He et al., 2005a). The method of array synthesis can also affect sensitivity and speciﬁcity. Increasing the probe concentration per spot can increase sensitivity (Cho and Tiedje, 2002; Relógio et al., 2002; Zhou and Thompson, 2002). However, this may also decrease speciﬁcity by decreasing the overall probe signal intensity (Denef et al., 2003). The choice of array surface can also be important as use of unmodiﬁed array slides can decrease background ﬂuo- rescence thus requiring a lower signal ﬂuorescence for detection (Kumar et al., 2000; Gudnason et al., 2008). Target preparation strategies can also affect these parameters. Ampliﬁcation of community DNA can increase sensitivity. WCGA was able to representatively amplify 1–250 ng of community DNA September 2012 \| Volume 3 \| Article 339 \| 4 Frontiers in Microbiology \| Microbiotechnology, Ecotoxicology and Bioremediation FGAs for elucidation of bioremediation Van Nostrand et al. (Wu et al., 2006a), increasing the detection limit from 25 ng to 10 pg (2 bacterial cells); however, using such small quantities of starting material greatly increases the ampliﬁcation bias compared to the bias observed with 1 ng of DNA. Labeling methods can also affect sensitivity. For example, cyanine dye-doped nanoparti- cles or tyramide signal ampliﬁcation labeling are able to increase sensitivity 10-fold (Denef et al., 2003; Zhou and Zhou, 2004). site, genes for denitriﬁcation, organic contaminant degradation, metal resistance, and sulﬁte reduction (dsr) were detected. A simi- lar sample set using the same array was examined in greater detail in a later study (Waldron et al., 2009). In this study, sulfate, pH, U, andTcwerefoundtobethemostimportantdriversindetermining the microbial community structure, with pH and the combination of U and Tc explaining ∼21% of the variance observed or 29–40% when all four variables were included. Hybridization conditions can also be used to increase speciﬁcity and sensitivity. MONITORING GENE ACTIVITY Most GeoChip analysis has involved the use of DNA, so that only gene abundance can be determined. These changes can be used to infer microbial activity, but cannot provide direct proof of activity. mRNA can be used for FGA analysis to monitor activity (Dennis et al., 2003; Bodrossy et al., 2006; Gao et al., 2007; Wawrik et al.,2012a),althoughasmentionedabove,workingwithenviron- mental RNA can be challenging. Stable isotope probing (SIP) has also been used with GeoChip to monitor microbial activity (Leigh et al., 2007). Gaoet al.(2007)usedampliﬁedcommunitymRNAfromaden- itrifying ﬂuidized bed reactor to examine microbial activity. Genes for nitrate and nitrite reduction, organic contaminant degrada- tion, sulﬁte reduction, and polyphosphate kinase were detected, consistent with reactor operation (Gao et al.,2007). Another study usedampliﬁedcommunitymRNAtoexaminenitrateutilizationin marine bacterial communities (Wawrik et al., 2012a). Hybridiza- tion results indicated activity by ureC,nirS,nirK,narG,nosZ,napA, nrfA, amoA, and nifH genes, indicating that urea cycling, denitriﬁ- cation, dissimilatory nitrate, nitrite reduction, and N ﬁxation were occurring (Wawrik et al., 2012a). In this same remediation system, the sediment microbial com- munity was examined with GeoChip 2.0 after 2 years of operation (Xu et al., 2010). Sediment samples were collected from 11 wells, 5 from the outer loop and 6 from the inner loop. Results revealed signiﬁcant differences between the microbial communities in the inner and outer loops. The inner loop communities had higher gene numbers and greater diversity than those in the outer loop and inner and outer loop samples were grouped separately based on hierarchical clustering and principle component analy- sis, indicating that the ethanol injections stimulated the microbial communities in the inner loop. In addition, genes important for U(VI) reduction such as cytochrome c, dsr, and denitriﬁcation as well as genes involved in metal resistance and organic contaminant degradation were enriched in the inner loop where electron donor was added. This study demonstrated the importance of U(VI)- reducing populations for the maintenance of reducing U(IV) in this bioremediation system. Another method of monitoring microbial activity with GeoChip is to combine it with SIP (Leigh et al., 2007). Micro- cosms were set up from soil samples collected from the root zone of a tree growing in a PCB-contaminated site and fed 13C-labeled or unlabeled biphenyl. Genes involved in biphenyl degradation were detected as were other organic contaminant degradation genes including those for degradation of benzoate, catechol, naphthalene, and phenol. SPECIFICITY AND SENSITIVITY Temperature and formamide concentration can be modiﬁed to adjust stringency thus altering speciﬁcity (Wu et al., 2001). A lower hybridization solution volume (Shalon et al., 1996) and mixing during hybridization (Adey et al., 2002) have both been shown to increase sensitivity. Decreasing ozone levels, which can degrade cy-dye signal (Branham et al.,2007), can also improve sensitivity. Another study at this site examined a pilot-scale ﬁeld biore- mediation system which used ethanol as an electron donor to stimulate microbial communities and immobilize U(VI) by reduc- tion to U(IV) (Luo et al., 2006; Wu et al., 2006b,c). GeoChip 2.0 was used to examine the microbial communities during different phases of operation. A period of active U(VI) reduction occurred after initial start-up (days 137–304). During this period U(VI) reduction was relatively rapid and genes associated with deni- triﬁcation, sulfate reduction, and Fe(III) reduction increased in abundance, suggesting that these populations were involved in U(VI) reduction (Van Nostrand et al., 2011). This active reduc- tion was followed by a maintenance period during which the low level of U(VI) was maintained, and the denitrifying, sulfate- and Fe(III)-reducing communities remained in higher abundance. Next, the stability of the bioreduced U(IV) was examined by allow- ing the system to enter periods of starvation (ethanol injections were halted) and reoxidation (dissolved O2 entered the system). The functional communities showed distinct clustering patterns based on whether the system received ethanol or not, indicat- ing a shift in community structure with the return of ethanol injections (Van Nostrand et al., 2009). While total gene numbers increased once ethanol injection was restarted, the relative abun- danceof eachgenegroupchangedlittleduringandafterstarvation, indicating a functionally diverse community which could be stim- ulated after adverse conditions. Chemical oxygen demand (COD, i.e., ethanol) was the most important driver in determining com- munity structure, but temperature, sulfate, and U(VI) were also important. OIL-CONTAMINATED SITES The microbial community associated with a bioremediation system comprised of a ﬂuidized bed reactor to clean diesel- contaminated groundwater in Vega Baja, Puerto Rico was exam- ined with the GeoChip 1.0 (Rodríguez-Martínez et al., 2006). Genes involved in the degradation of diesel fuel and other organic contaminants (acetylene, aniline, benzoate, biphenyl, cyclohex- anol, methyl tert-butyl ether, naphthalene, phthalate, protocate- chuate, and toluene) were detected. Increased signal intensities for genes involved in anaerobic benzoate degradation indicated a shift toward anaerobiosis over time, a conclusion supported by other experimental evidence. Xie et al. (2011) examined ﬁve AMD sites in China using GeoChip 2.0 to determine the functional diversity and metabolic potential of microbial communities in these sites and to determine how the communities responded to environmental conditions. The sites showed a great deal of variability in regards to the micro- bial communities with ∼150–1000 functional genes detected in each sample. Most of the genes represented on the GeoChip that were involved in C, N, S cycling and metal resistance were detected in all of the AMD sites. Results indicated that the immediate envi- ronmental conditions were important in forming the variations in the functional structure of microbial communities as opposed to spatial distance. There was a positive correlation between Zn resistance gene abundance and Zn concentration but not for other metals. However, the concentrations of B, Co, Cu, La, Mg, and S were signiﬁcantly correlated with the community structure in these communities. Overall, results suggested that AMD microbial communities may not be as simple as previously thought. Liang et al. (2009b) examined the effect of different bioreme- diation treatments on microbial communities using laboratory scale bioremediation systems with sediment from contaminated oil ﬁelds and inoculated with oil degrading enrichment cultures. The systems were incubated 242 days, treated with ozone, and incubated an additional 125 days. Many oil degradation genes (benzene, benzoate, catechol, polyaromatic hydrocarbon aromat- ics, protocatechuate, phthalate) were detected with GeoChip 2.0. Ozonation treatment resulted in an almost 50% reduction in the number of functional genes detected. Gene numbers increased again after a recovery period and the community retained the ability to degrade oil. GeoChip 2.0 has also been used to probe pure culture isolates for the presence of speciﬁc genes. Four Ni-resistant Gram-positive actinomycetes were hybridized to GeoChip to get a better idea of what metal resistance genes were present (Van Nostrand et al., 2007). APPLICATION OF GeoChip TO BIOREMEDIATION STUDIES METALS CONTAMINATED SITES Several GeoChip-related studies have examined microbial com- munities from U-contaminated groundwater at the U.S. Depart- ment of Energy (DOE) Oak Ridge Integrated Field Research Challenge (OR-IFRC) site. Groundwater samples covering a range of contamination levels and an uncontaminated background sam- ple were compared using GeoChip 1.0 (Wu et al., 2006a). Samples from the uncontaminated site and those with lower levels of con- taminants had higher functional gene diversity and gene numbers. In addition, as expected based on the contaminants present at this Another GeoChip 2.0 study examined groundwater microbial communities at a ﬁeld site examining the use of acetate to stimu- late U(VI)-reducing microorganisms in the subsurface at the Old Riﬂe site, a former U ore processing facility in Riﬂe CO (Liang September 2012 \| Volume 3 \| Article 339 \| 5 www.frontiersin.org 5 FGAs for elucidation of bioremediation Van Nostrand et al. the contaminated, planted samples while only 7 were detected in the contaminated/unplanted samples. Substrate-induced respira- tion, K concentration, and nitrate concentration were the most important environmental variables in determining the functional community structure. et al., 2012). The study compared communities taken during a shift from sulfate to Fe(III)-reducing conditions. The over- all community structure changed with the switch from Fe(III)- to sulfate-reducing conditions and were reﬂective of the redox conditions at the site. Sulfate-reducing and methane-generating microorganisms increased in abundance under sulfate-reducing conditions. Acetate, U(VI) and redox potential were important environmental variables in determining the microbial community structure. Frontiers in Microbiology \| Microbiotechnology, Ecotoxicology and Bioremediation OIL-CONTAMINATED SITES Genes associated with resistance to Al, As, Cd, Cr, Cu, Hg, Ni, Te, and Zn were detected. AnotherstudyusedGeoChip2.0tocharacterizemicrobialcom- munities along an oil contaminant gradient and found a decreased number of functional genes as the contaminant levels increased although genes involved in the degradation of biphenyl, cate- chol, and protocatechuate increased in the more contaminated samples (Liang et al., 2009a). The most important environmental factors in determining the microbial community structure were oil concentration and soil available nitrogen. MICROBIAL COMMUNITIES ASSOCIATED WITH PHYTOREMEDIATION Microbial communities from the rhizosphere of the arsenic- hyperaccumulating plant Pteris vittata and non-rhizosphere sam- ples were examined using GeoChip 3.0 (Xiong et al., 2010). The functional gene diversity was signiﬁcantly correlated with As concentration. Interestingly, As contaminated rhizosphere sam- ples had higher functional gene diversity than non-rhizosphere samples even though the non-rhizosphere samples had a lower level of As. In addition, greater numbers of As resistance genes, with higher signal intensities, were detected in rhizosphere sam- ples compared to non-rhizosphere samples and very few genes were detected in both environments, suggesting that the rhizo- sphere and non-rhizosphere microbial communities were distinct. Results suggested that the P. vittata rhizosphere may protect the microbial communities from As contamination. methanotrophs. Environ. Microbiol. 5, 566–582. MICROBIAL COMMUNITIES ASSOCIATED WITH PHYTOREMEDIATION Liang et al. (2011) collected contaminated and uncontaminated soils from ﬁve oil ﬁelds across China in order to determine whether oil contamination or geographic location played a larger role in determining the microbial community structure. Results from GeoChip 2.0 indicated that communities from uncontaminated sites had higher functional gene diversity than those from contam- inated sites in the same geographical area. Overall, the microbial communities clustered based on geographic location; however, when only organic contaminant degradation genes were exam- ined, the contaminated samples clustered together. Geographic location was able to explain ∼33% of the microbial community variation observed, oil explained ∼10% of the variation, and soil geochemistry explained another 12%, while the remainder (∼41%) was unexplained. Another study used GeoChip 2.0 to examine microbial com- munities in Zn- and Cd-contaminated soil microcosms with or without Thlaspi caerulescens, a Cd and Zn hyperaccumulator plant (Epelde et al., 2010). Higher numbers of functional genes were detected in the contaminated samples than in uncontam- inated samples and in planted samples compared to unplanted. Thirty-ﬁve to forty-seven percent of the variation in commu- nity structure observed was explained by metal concentrations. All of the Cd and/or Zn resistance genes (12) were detected in GeoChip 4.0 was used to compare microbial communities in oil-contaminated water to those from uncontaminated water in order to understand the effects of the 2010 Gulf of Mexico oil spill (Hazen et al., 2010; Lu et al., 2012a). Results indicated that after only 40 days the presence of the hydrocarbon plume September 2012 \| Volume 3 \| Article 339 \| 6 Frontiers in Microbiology \| Microbiotechnology, Ecotoxicology and Bioremediation 6 FGAs for elucidation of bioremediation Van Nostrand et al. (1100 m depth) caused a signiﬁcant shift in the microbial com- munity functional structure and composition and that indigenous microorganisms, similar to known petroleum degraders, were stimulated by the hydrocarbon plume. Many genes associated with hydrocarbon degradation were signiﬁcantly enriched in plume samples (Hazen et al., 2010; Lu et al., 2012a). Genes that were enriched in plume samples included those for naphthalene 1,2-dioxygenase, β-oxidation of benzylsuccinate, cyclohexanone 1,2-monooxygenase,and alkene monooxygenase (Lu et al.,2012a). These ﬁndings suggest that the microbial communities in the Gulf of Mexico were capable of intrinsic bioremediation and that the presence of the oil stimulated the oil-degrading community mem- bers and were important in determining the fate of the deep-sea oil spill. Song, X., Richmond, T. A., Middle, C. M., Rodesch, M. J., Packard, C. J., Weinstock, G. M., and Gibbs, R. A. (2007). Direct selection of human genomic loci by microar- ray hybridization. Nat. Methods 4, 903–905. MICROBIAL COMMUNITIES ASSOCIATED WITH PHYTOREMEDIATION (nahA, rhda, nahQ, narR), and genes involved in the degrada- tion of one-ring aromatic compounds. In addition, an over- all shift in community composition and structure was noted in spiked soils as determined by non-metric multidimensional scaling. Another study examined microbial communities associated with a leachate-contaminated landﬁll using GeoChip 3.0 (Lu et al., 2012b). Groundwater samples were collected from wells along a ﬂowpath of the landﬁll. Communities directly under the land- ﬁll and in the closest well had signiﬁcantly lower functional gene diversity and richness. Genes involved in the anaerobic degrada- tion of organic contaminates such as aromatic acids (bclA, bbs, tutFDG), phenoxyacetic acid herbicides (ftdA) atrazine (atzABC, trzN, trzA, trzE) were detected in all wells. Based on canonical cor- respondence analysis, the environmental variables (pH, sulfate, ammonia, and dissolved organic carbon) had signiﬁcant effects on the community structure. ACKNOWLEDGMENTS The effort for preparing this review was supported by the Ofﬁce of Science, Ofﬁce of Biological and Environmental Research, of the U. S. Department of Energy under Con- tract No. DE-AC02-05CH11231 through ENIGMA – Ecosystems and Networks Integrated with Genes and Molecular Assem- blies (http://enigma.lbl.gov), a Scientiﬁc Focus Area Program at Lawrence Berkeley National Laboratory and the Oklahoma Applied Research Support (OARS), Oklahoma Center for the Advancement of Science and Technology (OCAST), the State of Oklahoma through the Project AR062-034. Bodrossy, L., Stralis-Pavese, N., Mur- rell, J. C., Radajewski, S., Weilhar- ter, A., and Sessitsch, A. (2003). Development and validation of a diagnostic microbial microarray for PESTICIDE CONTAMINATION In a study using GeoChip 2.0 to examine three atrazine- contaminated aquifers and a background site, Liebich et al. (2009) detected more genes in the background site compared to the contaminated sites. The aquifer with the highest level of con- tamination had the highest number of genes, most involved in contaminant degradation, compared to the other contaminated samples. Atrazine-degradation genes were detected in all contam- inated samples and veriﬁed by PCR. These results indicated that even small amounts of contaminant were enough to select for speciﬁc degrading populations. OTHER CONTAMINANTS GeoChip 2.0 was used to examine phenanthrene-spiked soil microcosms to examine the effect of phenanthrene on micro- bial communities (Ding et al.,2012). Communities were examined after a 21-day incubation and compared with communities from day 0. A larger number of genes were detected in spiked soils compared to the control soils. Genes showing an increase in the spiked soils included dioxygenases involved in aromatic com- pound degradation, genes involved in the degradation of PAHs SUMMARY h h The GeoChip arrays have been shown to be powerful tools in linking microbial function to ecosystem processes and are able to provide sensitive, speciﬁc, and potentially quantitative informa- tion. Use of this array in bioremediation studies have expanded our understanding of the microbial processes and communities at work in these sites and provide information necessary for the successful improvement and application of bioremediation strate- gies. Over the past decade, great improvements have been made in regards to microarray technology, design, and application. How- ever, there are still technical hurdles that need to be overcome to further improve sensitivity and speciﬁcity in addition to bet- ter methods of nucleic acid extraction and puriﬁcation. Improved bioinformatics tools are also needed to assist with data processing and analysis. River sediments from industrial pollutant and pesticide- contaminated sites were examined with GeoChip 2.0 and the results indicated that contaminant level was not a major driver in these systems (Ta¸s et al., 2009). Instead, C/N ratio, depth, total Kjeldahl N, and location were the strongest drivers in determining the community structure. 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Received: 25 July 2012; paper pending published: 20 August 2012; accepted: 03 September 2012; published online: 21 September 2012. Citation: Van Nostrand JD, He Z and Zhou J (2012) Use of functional gene arrays for elucidating in situ biodegra- dation. Front. Microbio. 3:339. doi: 10.3389/fmicb.2012.00339 REFERENCES Citation: Van Nostrand JD, He Z and Zhou J (2012) Use of functional gene arrays for elucidating in situ biodegra- dation. Front. Microbio. 3:339. doi: 10.3389/fmicb.2012.00339 Received: 25 July 2012; paper pending published: 20 August 2012; accepted: 03 September 2012; published online: 21 September 2012. Copyright © 2012 Van Nostrand, He and Zhou. This is an open-access article dis- tributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduc- tion in other forums, provided the origi- nal authors and source are credited and subject to any copyright notices concern- ing any third-party graphics etc. Zhou, J., and Thompson, D. K. (2002). Challenges in applying microarrays to environmental studies. Curr. Opin. Biotechnol. 13, 204–207. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any Zhou, J., Wu, L., Deng, Y., Zhi, X., Jiang, Y., Tu, Q., Xie, J., Van Nostrand, J. D., He, Z., and Zhou, J. September 2012 \| Volume 3 \| Article 339 \| 11 www.frontiersin.org
https://openalex.org/W2151866902	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-015-2188-7	English	null	Integrative enrichment analysis: a new computational method to detect dysregulated pathways in heterogeneous samples	BMC genomics	2,015	cc-by	14,744	METHODOLOGY ARTICLE Open Access Integrative enrichment analysis: a new computational method to detect dysregulated pathways in heterogeneous samples 1 2* 1* Yu et al. BMC Genomics (2015) 16:918 DOI 10.1186/s12864-015-2188-7 METHODOLOGY ARTICLE Open Access Integrative enrichment analysis: a new computational method to detect dysregulated pathways in heterogeneous samples 1 2* 1* Yu et al. BMC Genomics (2015) 16:918 DOI 10.1186/s12864-015-2188-7 Yu et al. BMC Genomics (2015) 16:918 DOI 10.1186/s12864-015-2188-7 Integrative enrichment analysis: a new computational method to detect dysregulated pathways in heterogeneous samples Xiangtian Yu1, Tao Zeng2* and Guojun Li1* Xiangtian Yu1, Tao Zeng2* and Guojun Li1* Abstract Background: Pathway enrichment analysis is a useful tool to study biology and biomedicine, due to its functional screening on well-defined biological procedures rather than separate molecules. The measurement of malfunctions of pathways with a phenotype change, e.g., from normal to diseased, is the key issue when applying enrichment analysis on a pathway. The differentially expressed genes (DEGs) are widely focused in conventional analysis, which is based on the great purity of samples. However, the disease samples are usually heterogeneous, so that, the genes with great differential expression variance (DEVGs) are becoming attractive and important to indicate the specific state of a biological system. In the context of differential expression variance, it is still a challenge to measure the enrichment or status of a pathway. To address this issue, we proposed Integrative Enrichment Analysis (IEA) based on a novel enrichment measurement. Results: The main competitive ability of IEA is to identify dysregulated pathways containing DEGs and DEVGs simultaneously, which are usually under-scored by other methods. Next, IEA provides two additional assistant approaches to investigate such dysregulated pathways. One is to infer the association among identified dysregulated pathways and expected target pathways by estimating pathway crosstalks. The other one is to recognize subtype-factors as dysregulated pathways associated to particular clinical indices according to the DEVGs’ relative expressions rather than conventional raw expressions. Based on a previously established evaluation scheme, we found that, in particular cohorts (i.e., a group of real gene expression datasets from human patients), a few target disease pathways can be significantly high-ranked by IEA, which is more effective than other state-of-the-art methods. Furthermore, we present a proof-of-concept study on Diabetes to indicate: IEA rather than conventional ORA or GSEA can capture the under-estimated dysregulated pathways full of DEVGs and DEGs; these newly identified pathways could be significantly linked to prior-known disease pathways by estimated crosstalks; and many candidate subtype-factors recognized by IEA also have significant relation with the risk of subtypes of genotype-phenotype associations. Conclusions: Totally, IEA supplies a new tool to carry on enrichment analysis in the complicate context of clinical application (i.e., heterogeneity of disease), as a necessary complementary and cooperative approach to conventional ones. * Correspondence: zengtao@sibs.ac.cn; guojunsdu@gmail.com 2Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Cell Building Level 3, YueYang Road 320, Shanghai 200031, China 1School of Mathematics, Shandong University, Jinan 250100, China Background enrichment or status of a pathway. A solution to this problem can promote the efficiency of pathway enrich- ment analysis on genotype-phenotype association be- cause it will consider more complete information about the expression changes of pathway genes. It can also provide new insights on the biological pathways by inte- grating additional expression and network features. In this work, we propose a multiple-label based enrichment analysis to detect such dysregulated pathways, which simultaneously takes into account the genes with differ- ential expression (a label as DEGs) and genes with differ- ential expression variance (the other label as DEVGs) together (Fig. 1). Being a computational approach based on the prior knowledge, pathway enrichment analysis is widely used in the study of genotype-phenotype associations [1]. Bio- logical pathway as a set of interactive genes (and a few of their interactions with biomolecules) produces par- ticular cellular response/outcome by executing a series of functional cascades. It is curated by experts from wide range of science fields [2, 3] so that can supply more creditable functional details than general GO module or network module. Different from exploring the unknown or indeterminate functions by network module, pathway-centered analysis always makes an effort to cap- ture the permutation of established functions (e.g., KEGG pathways [2, 3]) in the change of phenotypes (e.g., from normal to diseased). As a key approach of pathway-centered analysis, the pathway enrichment ana- lysis or well-known gene set enrichment analysis (GSEA) [1] can identify dysregulated pathway by qualitatively measuring the changed status of a pathway [4]. Obviously, the hypothesis underlying IEA is that the dysregulated pathways involved in disease heterogeneity would be full of DEGs and/or DEVGs. That means the identified pathways by IEA would be disease pathways or their up-streams/down-streams (e.g., heterogeneity- relevant pathways or subtype-relevant pathways). How- ever, current methods in pathway enrichment analysis only expect to give high-rank to disease pathways (e.g., target pathways in approach evaluation). When IEA identifies up-streams/down-streams of disease pathways, it further assistantly supplies a network of pathways to recover a global functional map and infer the associa- tions among disease pathways and subtype-relevant pathways. Noted, the biological meaning of the edge in such network of pathways is the pathway crosstalk, which is just an important biological mechanism or functional relationship among pathways [23–26]. Background Con- ventional researches tend to simply determine a pathway crosstalk by the overlapped genes in two pathways [27], which disregard the statistical significance of the genes and interactions involved in the pathway crosstalk. By contrast, DEGs and DEVGs in one pathway can be used as seeds, and further detected their interactive genes in the candidate crosstalking pathways by a random walk restart algorithm [28]. The significance of a pathway crosstalk can be finally evaluated by the genes involved in this crosstalk as their enrichments in two pathways (i.e., the proposed multiple-label based enrichment). In the pathway enrichment analysis, the dysregula- tion of a pathway is the most important issue [5], and should be mathematically defined and measured well [6]. It can estimate the conditional enrichment or status of a pathway, which is assumed to be asso- ciated with particular phenotypes. Current researches generally use genes with significantly differential ex- pressions or differential correlations to evaluate the extent of the dysregulation of a pathway. One kind of conventional method is evaluating the dysfunction of pathways in different conditions [7–9], such as FiDePa (Finding Deregulated Paths Algorithm) [10], SPIA (Signaling Pathway Impact Analysis) [11] and iPEAP (Integrative Pathway Enrichment Analysis Platform) [12]. The other kind is using pathways to characterize individual samples [13, 14], like CORGs [15] and Pathifier [16]. Generally, all these methods focus on the genes with differential expression and their enrichments in pathways (i.e., the analysis in the context of differential expression) [17, 18], which as- sume the samples are of good purity in genotype- phenotype association study. However, in the study of complicated phenotypes, e.g., cancer study, a relevant problem is the samples with the same disease pheno- type might be full of different unknown subtypes due to disease heterogeneity [19]. It is necessary to detect genes with new features observable in the compli- cated disease samples, and enhance the pathway en- richment analysis to be applicable in such previously unexpected situation [20]. Based on the above concepts and mathematical models, a new pathway-centered analysis framework, the integrative enrichment analysis (IEA), is imple- mented as (i) pathway enrichment score calculated by the hypergeometric test on differential genes (DEGs and DEVGs); (ii) pathway crosstalk ranked by the random walk and hypergeometric test on rewired molecule networks; (iii) pathway-phenotype associ- ation and subtype-factors determined by DEVGs in pathways. Abstract * Correspondence: zengtao@sibs.ac.cn; guojunsdu@gmail.com 2Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Cell Building Level 3, YueYang Road 320, Shanghai 200031, China 1School of Mathematics, Shandong University, Jinan 250100, China © 2015 Yu et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yu et al. BMC Genomics (2015) 16:918 Page 2 of 19 Background According to a previously established evalu- ation scheme [29], we found that, in particular co- horts (i.e., a group of real gene expression datasets from human patients), a few target disease pathways Actually, there are new expression features extracted in recent studies, e.g., genes with differential expression variances [21, 22]. In the context of differential expres- sion variance, it is still a challenge to measure the Yu et al. BMC Genomics (2015) 16:918 Page 3 of 19 Page 3 of 19 Fig. 1 Major differences between the measurements of dysregulated pathways used in conventional enrichment analysis and integrative enrichment analysis (IEA) rences between the measurements of dysregulated pathways used in conventional enrichment analysis and integrative i (IEA) Fig. 1 Major differences between the measurements of dysregulated pathways used in conventional enrichment analysis and integrative enrichment analysis (IEA) can be significantly high-ranked by IEA, which sup- plied the evidences of the deviation-based disease characteristics (i.e., disease subtypes), and IEA is more effective than other state-of-the-art methods in this condition. Furthermore, by a proof-of-concept study, we shows the details of IEA on analyzing real tran- scriptional data related to complex diseases, e.g., Dia- betes and Colorectal cancer. IEA indeed captures the previously under-estimated pathways full of DEVGs and DEGs. These newly identified dysregulated path- ways would be heterogeneity-relevant pathways and are found to be significantly linked to disease path- ways (i.e., target pathways in conventional analysis) by estimated crosstalks. Many candidate subtype-factors are also recognized as DEVGs or pathways associated with the risk of subtypes of genotype-phenotype asso- ciations. Totally, IEA supplies a new way of over- representation approach [30] to carry on enrichment analysis in the complicate context of clinical applica- tion (i.e., differential expression and differential ex- pression variance), and could be easily expanded to functional class scoring or pathway topology based approaches [31–34], which will be a necessary com- plementary and cooperative approach to conventional ones [35]. The Matlab scripts of the software named IEApackage and some alternative R scripts have been deposited in GitHub and accessed in https://github.- com/bluesky2009/integrative-enrichment-analysis. This software has been developed and tested in Windows 7 or Windows 8, and Matlab 2010 or Matlab 2012. Integrative enrichment analysis in the context of differential expression variance Ob l h l h l H0 : E X ð Þ ¼ E X’ ð Þ; H0 rejected; ð1Þ ð1Þ Obviously, the conventional enrichment analysis limits to estimate the extent of differential expression rather than differential expression variance. When considering the contribution of DEVGs on pathway’s dysregulation, it is necessary to refine the conventional approach to take into account the DEGs and DEVGs together. Natur- ally, an easiest strategy is to put DEGs and DEVGs to- gether as the same dysregulated genes and use conventional hypergeometric test to obtain the P-value. However, this will disregard the respective distribution of DEGs and DEVGs in a target pathway and in the whole transcriptome. Thus, we extended the hypergeo- metric test on two kinds of enriched genes simultan- eously as bellows. Our approach, noted as HT2 (hypergeometric test on the model of the drawn of two group balls), still depends on the hypergeometric distri- bution and uses P-value to measure the dysregulation of a pathway in the context of differential expression variance. where X or X’ are the original/raw expression levels. Noted, the differential expression includes up-regulation (the expressions of genes in case samples are larger than those in control samples) and down-regulation (the ex- pressions of genes in case samples are less than those in control samples). Except for these DEGs (e.g., genes rejected by Student’s T-test in significance test), the genes with dif- ferential expression variance are also discriminative fea- tures [21, 36]. The expression variance concerned features, e.g., bimodal gene expression, is already known as an important expression pattern in the control of a transition of biological systems [37], such as: disease de- velopment, cellular differentiation, and phase transition. However, the differential expression variance of genes has not been studied in a systematic way to the best of our knowledge, especially for its usage in the pathway enrichment analysis. The differential expression of genes, used in conventional enrichment analysis, requires the gene’s expressions under different conditions to distrib- ute around different mean expression levels (seeing above formula 1). By contrast, differential expression variance of genes (named as DEVGs) can be defined as the genes’ deviations being significantly different under dissimilar conditions (deviation means the distances Briefly seen in Table 1, given there are expression data on total N genes, and x1 DEGs and x2 DEVGs selected respectively. Methods Generally, enrichment analysis includes three categor- ies of methods: over-representation approach, func- tional class scoring and pathway topology based approaches. Although these methods are all focusing Yu et al. BMC Genomics (2015) 16:918 Page 4 of 19 Page 4 of 19 Page 4 of 19 between a gene’s original expression levels and its mean expression level), such as: on evaluating the phenotype-associated pathway, they would be based on different hypothesis. This work and the proof-of-concept study are based on the over-representation approach, which measures the dysregulation extent of a pathway according to the number of dysregulated genes in this pathway. Trad- itional methods only evaluated the DEGs in a path- way; by contrast, IEA evaluates the DEGs and DEVGs in a pathway. Thus, the meaning of the statistic for the integration of IEA is as completely as possible to measure the dysregulation extent of a pathway ac- cording to the number of dysregulated genes (DEGs & DEVGs) in this pathway, which have been well de- fined and introduced in follows. H0 : E X‐u j j ð Þ ¼ E X’‐u’ j j ð Þ; H0 rejected; and H0 : E X ð Þ ¼ E X’ ð Þ; H0 not rejected ð2Þ ð2Þ where X or X’ is the original expression level, \|X-u\| or \|X’-u’\| is the relative expression level. Noted, the differential expression variance includes tight-regulation (the expression variances of genes in case samples are less than those in control samples) and relax-regulation (the expression variances of genes in case samples are larger than those in control samples). And importantly, as defined above, the DEVGs have ex- cluded DEGs, or there is no overlap between DEVGs and DEGs in this work. That means, when one gene has both differential expression and differential expression variance, this gene is thought as DEG in priority in order to be consistent with conventional analysis; and, of course, this kind of genes are worthy of deep research in future work. Differential gene expression and differential expression variance Given a gene x has expression profiles in control and case samples as X and X’ respectively, the expression variance of this gene in control and case condition are E((X-u)2) and E((X’-u’)2) respectively. Here, u and u’ are average expressions of gene x in control and case sam- ples respectively. Then, the conventional criterion and measurement of genes with differential expression (named as DEGs) are: Actually, given X or X’ satisfy normal distribution, \|X- u\| or \|X’-u’\| will be folded normal distribution, then the Wilcoxon rank sum test instead of Student’s T-test is used in the significance test of DEVGs. Integrative enrichment analysis in the context of differential expression variance Ob l h l h l For some pathway, k1 and k2 genes from pathway members (totally y genes) have differential ex- pression and differential expression variance respect- ively. Then the significance of deregulated genes as DEGs or DEVGs enriched in this pathway can be Page 5 of 19 Yu et al. BMC Genomics (2015) 16:918 Table 1 The statistic of DEGs and DEVGs for pathway enrichment analysis in the context of differential expression variance Pathway Others All DEG k1 x1-k1 x1 DEVG k2 x2-k2 x2 Others y-k1-k2 N + k1 + k2-x1-x2-y N-x1-x2 All y N-y N Table 1 The statistic of DEGs and DEVGs for pathway enrichment analysis in the context of differential expression variance well-known ranking algorithm for candidate gene prioritization [28]. It supplies the probability of search- ing the random walker at nodes in the steady state, so that, it can give a measure of proximity between source nodes (e.g., genes as seeds in a pathway) and other nodes in molecule network (e.g., genes in the candidate path- way with crosstalk). Let N be the adjacency matrix of a gene network with node set V and edge set E, in which the element Nij equals one if e(i, j) ∈E (where e(i, j) represents the inter- action between genes/nodes i and j), or zero otherwise. Based on the topological structure of the gene network, the transition matrix T can be calculated. Each element in the transition matrix is denoted as Tij and represents the probability of transition from node i to node j. The value of Tij can be given by one of two ways as follows, the first one is topology-weighted and the second one is correlation-weighted. estimated by formula 3. This P-value also ranges from zero to one. The less the P-value is, the larger dysregula- tion extent the pathway has, when the significantly lar- ger number of genes in this pathway show differential expression or differential expression variance. Integrative enrichment analysis in the context of differential expression variance Ob l h l h l P X1 ¼ k1; X2 ¼ k2 ð Þ ¼ x1 k1 x2 k2 N−x1−x2 y−k1−k2 N y P X1 > k1; X2 > k2 ð Þ ¼ 1− X <i1;i2>∈0;x1 ½ 0;x2 ½ −k1;x1 ð k2;x2 ð P X1 ¼ i1; X2 ¼ i2 ð Þ ¼ 1− X <i1;i2>∈0;x1 ½ 0;x2 ½ −k1;x1 ð k2;x2 ð x1 i1 x2 i2 N−x1−x2 y−i1−i2 N y Tij ¼ Nij di ; if e i; j ð Þ∈E 0; otherwise ; where di ¼ X j∈VNij ( Tij ¼ wijNij wi ; if e i; j ð Þ∈E 0; otherwise ; where wi ¼ X j∈VwijNij ( The RWR algorithm [28] updates the probability vectors by The RWR algorithm [28] updates the probability vectors by ð3Þ by Pkþ1 ¼ 1−λ ð ÞTPk þ λP0; k > 0 Screening subtype-factor of genotype-phenotype associations based on DEVGs and dysregulated pathways supplied by IEA The second assistant down-stream analysis method of IEA is to screen subtype-factors according to the avail- able clinical indices. As stated above, IEA focus on the DEVGs and their involved pathways, and these genes and pathways are thought as signatures of potential sub- types of heterogeneous samples. However, these hidden subtypes might have not been identified or formalized in clinics. To evaluate such new signatures or subtypes, one direct strategy is to measure the correlation between genetic signatures (e.g., DEVGs or dysregulated path- ways) and clinical indices (e.g., age or bmi). If one signa- ture is significantly related to some clinical index, the subtype represented by such signature would be medical meaningful as to be observable in clinics and this signa- ture is also called as subtype-factor related to particular clinical index. The approach to identify such subtype- factors is described in bellows. Practically, the SLC algorithm on a pathway is imple- mented as bellows: (i) Discrete the expressions of DEVGs of case samples into binary vector based on the values of controls: for a DEVG, if its expression value is larger than the mean of controls, it is one in the binary vector; otherwise, it is zero. (ii) Clustering case samples based on the binary vectors by conventional methods as hierarchical clustering or K-means, which obtains two sample clusters. (iii) Calculating the significance of difference between clinical indices among above two sample clusters. If the difference is significant, this pathway is identified as a subtype-factor of the association between the given pathway and clinical index. For each pathway, its DEVGs are used to group case (or control) samples into two clusters, when the case (or control) samples have high varying expression compared to control (or case) samples. That means these genes have over-expression in one group of samples and under-expression in the other group of samples. This pathway would be a candidate subtype-factor when these two sample clusters are discriminative on some clinical index. On this condition, a clinical subtype of samples is thought to be related to a given clinical index, which is represented by a subtype-factor (e.g., a DEVG or a dys- regulated pathway from IEA). Obviously, the clinical subtype of a particular sample might be contributed by many subtype-factors (i.e., many pathways). Estimating pathway crosstalks to link the dysregulated pathways identified by IEA and prior-known disease pathways Then, given a pathway pair (u,v), it is a pathway crosstalk only when Euv and Evu are both significant. Finally, the map of pathways consist of those selected pathway crosstalks, where a node represents a pathway and an edge represents a pathway crosstalk. controls meanwhile the other group of samples have less values than the same controls, or vice versa. That means, a hyperplane determined by a few control sam- ples could separate the samples space into two sub- spaces, and case samples in each of two sub-spaces are grouped into one cluster. Secondly, some clinical infor- mation of samples can be used to evaluate the potential subtype represented by such two clusters of case sam- ples. If the clinical values of these two groups of samples have significant difference, a clinical subtype of genotype-phenotype association (e.g., the correlation be- tween clinical indices and pathway DEVGs) is identified and the corresponding pathway is a subtype-factor cor- responding to the given clinical index. Estimating pathway crosstalks to link the dysregulated pathways identified by IEA and prior-known disease pathways Estimating pathway crosstalks to link the dysregulated pathways identified by IEA and prior-known disease pathways where T is the transition matrix and p0 is the initial probability vector with the sum of the probabilities as one. In p0, all the source nodes are assigned equal prob- abilities and other nodes are given zero. P∞is obtained when the algorithm is convergent. If P∞(i) > P∞(j), node i is thought to be more proximate to source nodes than node j does. The first assistant down-stream analysis method of IEA is to link the dysregulated pathways identified by IEA and some prior-known disease pathways. Obviously, IEA tends to detect the dysregulated pathways related to disease sub- types. These pathways would be disease pathways as cur- rently known, or the up-stream/down-stream of the disease pathways. Conventional pathway enrichment usu- ally analyses single pathway rather than multiple ones. But, the pathway crosstalk, as a pair of pathways, also plays important roles in the change of phenotypes [25]. An enrichment analysis of such pathway crosstalk requires evaluating the enrichment of interactive genes from two pathways correspondingly. And the pathway map based on such estimated pathway crosstalks is just an additional computational method to assistantly supply a bridge be- tween subtype-relevant pathways (i.e., IEA recognized pathways) and disease-relevant pathways (i.e., Target path- ways from disease database KEGG). Thus, a two-way RWR approach (twRWR) is proposed to search the genes involved in two interactive pathways and estimate their enrichment for evaluating the path- way crosstalk. The steps of two-way RWR include: (i) For each pathway u, its DEGs and DEVGs are used as source nodes/genes, and RWR is used to rank the genes in known molecule network, e.g., protein association network collected from STRING database [38]. (ii) In the high-ranked genes from above RWR analysis, the genes belonging to pathway v are the partner genes interactive with source genes. Based on the sources genes and their partner genes, the enrichment of those interactive genes (Euv) in pathways u and v can be evaluated by our HT2 approach, i.e., P-value in formula 3. Given several genes in a pathway as seeds, IEA uses random walk to find their partner genes in the other pathway. In fact, random walk with restart (RWR) is a Yu et al. BMC Genomics (2015) 16:918 Page 6 of 19 (iii) For every pathway, the analysis in steps (i) and (ii) is repeated. Screening subtype-factor of genotype-phenotype associations based on DEVGs and dysregulated pathways supplied by IEA Given a known phenotype (e.g., a clinical index), a few subtype- factors correlated with this phenotype can be found, al- though which just reveals only the tip of the iceberg for the subtypes of genotype-phenotype associations. The evaluation of biological meaning of IEA by method comparison IEA is proposed to evaluate dysregulated pathways by differential gene expression and differential expression variance together. Differential expression variance has been reported as a new and important expression change during a phenotype change [36], e.g., diseases. In this work, the biological hypotheses underlying IEA is that, the dysregulated pathways full of genes with differ- ential expression variance would be subtype-relevant pathways. Although subtype-relevant pathways for par- ticular complex disease are unclear in current pathway databases, e.g., KEGG, it is still able to investigate if prior-known disease pathways in KEGG would be subtype-relevant and if IEA can identify them. In the previous study of gene-set analysis [29], a comparison scheme has been built to evaluate the performances of different enrichment analysis methods (e.g., ORA or GSEA) based on multiple expression datasets about complex diseases. Different from previous general com- parison, we focus on the comparisons by approach- Particularly, different from conventional un-supervised clustering for subtype identification, a supervised-like clustering approach (SLC) is proposed to identify subtype-factors on the level of pathways. Firstly, the case samples can be grouped into two clusters according to their features’ values (i.e., DEVGs’ expressions) com- pared to those values of control samples: on each feature (DEVG), one group of samples have larger values than Yu et al. BMC Genomics (2015) 16:918 Page 7 of 19 Page 7 of 19 specific datasets, in order to mainly evaluate the bio- logical meaning of IEA. patterns of such gene group must differ for dis- similar clinical outcomes; thus, it uses generalized linear model to give one P-value for a group of genes, not a P-value for each gene, which can be applied to estimate the enrichment of a given pathway [42]. According to the comparison protocol [29], we ran total eight representative enrichment analysis methods on 36 GEO datasets with target pathways in KEGG, and obtained the rank of target pathway estimated by each method on each dataset; then, for each dataset, we rank the eight methods according to their prioritization per- formance or sensitivity performance [29], and this data- set is assigned as a specific-data for the Top-K methods (K is set 3); thus, all specific-data for one method can consist of K-order approach-specific dataset. The evaluation of biological meaning of IEA by method comparison Generally, on one method’s approach-specific dataset, this method should have best or comparable performances than other methods, so that, the biological characteristics as- sumed by this given method would significantly dis- played on these datasets. Therefore, we can use this strategy to investigate the biological meaning of IEA in real datasets. In bellows, we firstly summarize the bio- logical hypothesis hold by different state-of-the-art en- richment analysis methods and their respective quantitative measurements, and then discuss the com- parison between IEA and others. (v) (v) MRGSE: it proposes that the high ranks of expression changes (e.g., fold-change) of genes can indicate the differential expression of a set of genes (e.g., pathway genes); and the enrichment score or the test statistic of a pathway is the mean rank of this gene set, i.e., the average of the ranks of t-statistics of pathway genes [43]. (vi) GSA: it is similar to GSEA, and proposes two improvements as the maximal average statistic for summarizing gene-sets, and restandardization for accurate enrichment inferences [44]. (vii) ORA: it takes into account the number of differen- tially expressed genes observed in a pathway as indi- cators of pathway states; generally, it uses a basic contingency table to test the association between the differential expression status of a gene (e.g., differentially expressed gene, or not) and its mem- bership in a given gene set (e.g., pathway gene, or not), which can be measured by the P-value of a hypergeometric test [45]. (i) PLAGE: it assumes the activity of pathway rather than the expression of pathway genes determines the activated or inhibited status of pathways under different conditions; and the pathway activity is measured by an activity score as the weights of a metagene extracted from all pathway genes by SVD (singular value decomposition) [39]. (viii)IEA: it is proposed in this work to generally con- sider the contribution of expression variance in a dysregulated pathway; as one implementation, this work takes into account the number of DEGs and DEVGs observed in a pathway as indicators of pathway states; it is designed to test the association between the differential expression/differential ex- pression variance status of a gene and their member- ships in a given gene set, which can be measured by the P-value from proposed HT2 approach in this work. The evaluation of biological meaning of IEA by method comparison (ii) GSVA: it proposes the change of pathway activity between control and case should be evaluated at the level of samples, e.g., considering the variation of pathway activity over a sample population; and the pathway activity is measured by so-called GSVA score as a function of the expressions of genes inside and outside the pathway, and these scores are assessed similarly as GSEA by using the Kolmogorov-Smirnov (KS) like random walk statistic [40]. First of all, we can cluster the above eight approaches by their performances on all datasets to investigate the general association among different methods. As shown in Figs. 2 and 3, the similarity among any two methods is measured by four kinds of criterion: the first one is whether the ranks given by two methods on the same dataset are also the same (i.e., Euclidean distance on ranks in Fig. 2a); the second one is whether the ranks given by two methods have the same change tendency among different datasets (i.e., Correlation distance on ranks in Fig. 2b); the third one is whether the P-values given by two methods on the same dataset are also the same (i.e., Euclidean distance on P-values in Fig. 3a); and the last one is whether the P-values given by two methods have the same change tendency among differ- ent datasets (i.e., Correlation distance on P-values in (iii) PADOG: it assumes that, if the genes highly specific to a given pathway occur differential expressions, the respective pathway would be truly relevant in that condition; thus, a new gene set score is calculated as the mean of absolute values of weighted moderated gene t-scores where the gene weights are designed to be large for the genes appearing in few pathways and small for genes that appear in many pathways [41]. (iii) PADOG: it assumes that, if the genes highly specific to a given pathway occur differential expressions, the respective pathway would be truly relevant in that condition; thus, a new gene set score is calculated as the mean of absolute values of weighted moderated gene t-scores where the gene weights are designed to be large for the genes appearing in few pathways and small for genes that appear in many pathways [41]. The evaluation of biological meaning of IEA by method comparison (iv) GLOBALTEST: it holds an assumption that, if a group of genes (e.g., pathway genes) can be used to predict the clinical outcome, the expression (iv) GLOBALTEST: it holds an assumption that, if a group of genes (e.g., pathway genes) can be used to predict the clinical outcome, the expression Yu et al. BMC Genomics (2015) 16:918 Page 8 of 19 Fig. 2 Category of representative gene set analysis approaches based on clustering of prioritization performance. a Method clustering based on Euclidean distance of ranks of all pathways. b Method clustering based on Correlation distance of ranks of all pathways Fig. 2 Category of representative gene set analysis approaches based on clustering of prioritization performance. a Method clustering based on Euclidean distance of ranks of all pathways. b Method clustering based on Correlation distance of ranks of all pathways Fig. 3b). Obviously, GSA and PADOG are both based on conventional GSEA, so that they are similar; the pro- posed IEA is based on ORA, thus, they also have similar performances on different datasets; PLAGE and GLO- BALTEST are closely clustered together, one reason is that they both estimate a score from all pathway genes rather than individual genes (i.e., PLAGE uses weights of a metagene extracted from all pathway genes by SVD, and GLOBALTEST uses generalized linear model to give one P-value for a group of genes); in addition, MRGSE and GSVA are much different, and also different form other methods, which is possibly because they have spe- cific design principles on the measurement of pathway dysfunctions, i.e., MRGSE combines the t-statistics of individual pathway genes meanwhile GSVA uses a score as a function of the expressions of genes inside and out- side a pathway. Then, we directly grouped the datasets according to the performance of a given method, e.g., some datasets are included as K-order IEA-specific datasets, only when the rank of IEA performance compared to all methods are in the Top-K on these datasets, where K is set 3 in this study. To quantify the performance, sensitivity (i.e., P-value) and prioritization (i.e., rank) are adopted as pre- viously [29]. In previous evaluation on these datasets, PADOG displays consistently comparable performance with other methods, meanwhile, PLAGE, GLOBALTEST and MRGSE have the best performances on some Fig. 3 Category of representative gene set analysis approaches based on clustering of sensitivity performance. The evaluation of biological meaning of IEA by method comparison a Method clustering based on Euclidean distance of P-values of all pathways. b Method clustering based on Correlation distance of P-values of all pathways Fig. 3 Category of representative gene set analysis approaches based on clustering of sensitivity performance. a Method clustering based on Euclidean distance of P-values of all pathways. b Method clustering based on Correlation distance of P-values of all pathways Yu et al. BMC Genomics (2015) 16:918 Yu et al. BMC Genomics (2015) 16:918 Page 9 of 19 And seen in Table 3 and Additional file 4: Table S4 for sensitivity performance, again, every method shows its preference on particular datasets (The list of these 3- order approach-specific datasets is in Additional file 5: Table S5). It seems that GLOBALTEST and PLAGE have generally comparable performances with other methods according to their performances on many preferred datasets of other methods. Even though, IEA shows the best performance on the IEA-specific datasets, and is better than ORA on the ORA-specific datasets. This fact strongly suggests that, IEA actually can detect dysregu- lated pathways, and displays competitive performance than conventional methods in current evaluation scheme when those dysregulated pathways are just target path- ways (e.g., disease pathways in KEGG); besides, IEA is realized based on the conventional ORA, and improves ORA on the sensitivity performance, which would just be contributed by considering the new feature genes as DEVGs in dysregulated pathways. categorise of datasets [29], which already suggest the ex- istence of approach preferences. Thus, the categories of datasets induced in this work can indicate potential pref- erences of different methods on particular datasets. Of course, on the preference-specific datasets (i.e., approach-specific datasets), the preferred method should have the best performance; meanwhile, some other methods would have comparable performances. There- fore, this new evaluation scheme can supply evidences for two hypotheses: one is that the expression features of a pathway measured by a given method actually have biological meaning, and are existing or observable in real datasets corresponding to particular phenotypes (e.g., complex diseases); the other one is that a method has significant preferences (i.e., highest performance) on a group of datasets (i.e., diseases), which is comparable or not by other methods. The evaluation of biological meaning of IEA by method comparison y As seen in Table 2 for prioritization performance, every method shows its preferences on particular data- sets (The list of these 3-order approach-specific datasets is in Additional file 1: Table S1); in addition, the method comparison on more strict specificity as 2-order approach-specific datasets (K = 2) and more weak speci- ficity as 4-order approach-specific datasets (K = 4) have also been done and reported in Additional file 2: Table S2 and Additional file 3: Table S3. Combined these re- sults together, it is easily to see that: IEA, ORA and PADOG have significant preferences, due to their high- est performances than all other methods on their corres- pondingly preferred datasets; GLOBALTEST, GSVA, PLAGE and MRGSE also have significant preferences on datasets, although sometimes PADOG could have com- parable performances with them (e.g., PADOG and MRGSE are comparable when K = 3 in Table 2, but not when K = 2 in Additional file 2: Table S2; or PADOG are comparable to GLOBALTEST, GSVA, PLAGE when K = 4 in Additional file 3: Table S3, but not when K < 4); be- sides, GSA is challenged by PADOG, because on the GSA-specific datasets, PADOG always achieves similar or better performances, but not vice versa. Finally, above dataset-driven method comparison sup- plies new insights on the performance specificities of many gene-set approaches, especially for IEA; and also supports the importance and biological meaning of dys- regulated pathways identified by IEA. We can draw fol- lowing conclusions: (i) Although a few methods have consistent perfor- mances on many datasets (e.g., GLOBALTEST and PLAGE on sensitivity performance, or PADOG on prioritization performance, as shown in both this study and previous work [29]), different method still have their preferences on the expression char- acteristics of dysregulated pathways, so that each method can achieve significantly better perfor- mances on their specific datasets rather than all datasets. Especially, on the IEA-specific datasets, IEA indeed are the best one than other methods; and even on the ORA-specific datasets, IEA is better than ORA on sensitivity performance. The evaluation of biological meaning of IEA by method comparison disease pathways are possibly just the subtype- relevant pathways, so that, IEA have competitive performance in the comparison scheme. Therefore, the expression variance focused by IEA is actually full of biological meaning, and will help IEA to detect new dysregulated pathways, e.g., subtype- relevant pathways. In addition, the complex diseases concerned in IEA-specific datasets actually already have reports about the existence of subtypes on genetic level, such as dilated cardiomyopathy [46], renal cancer [47], prostate cancer [48], colorectal cancer [49], and thyroid cancer [50]. Firstly, it is the data pre-procession. Data needed in IEA have been prepared from the public resources: The gene expression data of human islets from non-diabetic and diabetic were downloaded through GEO [51]; there are two datasets, the main dataset GSE41762 [52] con- tains samples from 57 non-diabetic and 20 diabetic with 20950 genes, and the replicate dataset GSE38642 [53] contains samples from 54 non-diabetic and 9 diabetic with 19514 genes; the gene lists of 186 KEGG pathways are obtained from GSEA package [1]; the human protein interaction network (PIN) are extracted from STRING database [38] with confidence score no less than 0.9; four clinical indices are also obtained from the supple- mentary of original study [52] as sex, age, bmi, and HbA1c; meanwhile, diabetes associated genes are searched from GeneCards database [54]. (ii) (ii) Every method, or every method category, can actually capture particular dysregulated pathways. When the target pathway of a dataset just displays the expres- sion characteristics focused by an approach, such approach would have better performance on this dataset. On some specific datasets preferred by other methods, IEA should face two conditions: one is under the condition that the target pathway is not subtype-relevant pathway, and IEA will be underesti- mated but has supplied a useful down-stream analysis (i.e., map of pathways) to assistantly link the target pathway and potential subtype-relevant pathway identified; the other one is under the condition that the target pathway is a subtype-relevant pathway, and IEA should be further enhanced by integrating expression variance with other pathway pattern (e.g., linear model in GLOBALTEST or weights of overlapping genes in PADOG), which is worthy of study in future. Secondly, it is the main step of IEA. The evaluation of biological meaning of IEA by method comparison On the IEA-specific datasets, the target pathways or (i) Although a few methods have consistent perfor- mances on many datasets (e.g., GLOBALTEST and PLAGE on sensitivity performance, or PADOG on prioritization performance, as shown in both this study and previous work [29]), different method still have their preferences on the expression char- acteristics of dysregulated pathways, so that each method can achieve significantly better perfor- mances on their specific datasets rather than all datasets. Especially, on the IEA-specific datasets, IEA indeed are the best one than other methods; and even on the ORA-specific datasets, IEA is better than ORA on sensitivity performance. On the IEA-specific datasets, the target pathways or Table 2 The prioritization performance about method comparison on approach-specific datasets (K = 3) ID GSA-specific PADOG-specific IEA-specific MRGSE-specific ORA-specific GLOBALTEST-specific GSVA-specific PLAGE-specific GSA (14.93,12.82) (25.57,25.66) (44.47,29.40) (37.43,26.53) (45.72,29.72) (32.23,31.44) (22.52,26.76) (33.26,22.45) PADOG (11.98,12.48) (10.06,9.34) (29.23,23.83) (19.32,15.79) (26.56,20.69) (16.84,21.56) (15.47,12.87) (18.80,21.06) IEA (52.05,28.86) (51.15,26.26) (18.55,11.72) (68.01,16.79) (34.14,15.48) (41.20,26.26) (53.04,29.63) (51.87,31.13) MRGSE (51.97,30.59) (51.23,29.03) (65.81,31.64) (24.67,16.48) (74.35,19.55) (50.63,28.47) (45.72,27.77) (49.39,26.17) ORA (47.22,31.35) (48.91,28.29) (24.80,15.05) (69.98,17.56) (23.56,14.62) (52.38,23.61) (38.76,28.78) (53.60,26.98) GLOBALTEST (36.52,21.70) (31.10,18.93) (30.39,15.78) (35.44,18.29) (41.53,18.15) (14.66,16.19) (45.29,21.23) (26.18,22.77) GSVA (33.56,24.84) (47.83,28.90) (59.09,26.48) (52.99,27.27) (52.52,25.58) (61.99,29.17) (13.42,10.25) (62.49,25.01) PLAGE (26.44,16.96) (29.07,20.48) (42.06,34.24) (35.48,19.88) (45.77,24.91) (30.15,27.15) (31.86,16.67) (13.61,11.04) The performance of an approach on its specific dataset is highlighted in bold. And the performance of comparable approaches on some specific dataset is highlighted in bolditalic Yu et al. BMC Genomics (2015) 16:918 Page 10 of 19 Page 10 of 19 Table 3 The sensitivity performance about method comparison on approach-specific datasets (K = 3) ID GSA- specific PADOG- specific IEA-specific MRGSE-specific ORA-specific GLOBALTEST- specific GSVA-specific PLAGE- specific GSA (0.10,0.12) (0.22,0.28) (0.35,0.29) (0.042,0.014) (0.48,0.40) (0.25,0.27) (0.19,0.22) (0.21,0.22) PADOG (0.11,0.15) (0.051,0.11) (0.24,0.20) (0.057,0.011) (0.41,0.28) (0.14,0.17) (0.13,0.12) (0.12,0.15) IEA (0.11,0.17) (0.13,0.13) (0.044,0.057) (0.12,0.14) (0.012,0.0043) (0.092,0.11) (0.12,0.13) (0.10,0.12) MRGSE (0.73,0.19) (0.46,0.31) (0.59,0.27) (0.020,0.012) (0.56,0.077) (0.47,0.32) (0.46,0.31) (0.50,0.32) ORA (0.24,0.23) (0.49,0.27) (0.24,0.21) (0.50,0.60) (0.13,0.085) (0.39,0.29) (0.37,0.28) (0.40,0.29) GLOBALTEST (0.11,0.13) (0.013,0.028) (0.052,0.084) (0.00011,0.00015) (0.083,0.10) (0.011,0.044) (0.037,0.086) (0.025,0.073) GSVA (0.11,0.11) (0.32,0.28) (0.33,0.26) (0.060,0.080) (0.21,0.23) (0.24,0.26) (0.013,0.017) (0.21,0.25) PLAGE (0.097,0.11) (0.063,0.18) (0.095,0.15994) (0.010,0.014) (0.19,0.10) (0.036,0.11) (0.034,0.076) (0.022,0.066) The performance of an approach on its specific dataset is highlighted in bold. And the performance of comparable approaches on some specific dataset is highlighted in bolditalic divergent pathway associations, such as: pathway & dis- ease genes, pathway & pathway (i.e., pathway crosstalk), and subtypes of pathway & clinic (i.e., genotype- phenotype association). The evaluation of biological meaning of IEA by method comparison Different scores of pathway enrichment are calculated: (i) the conven- tional score (ORA) as P-values of hypergeometric distri- bution of DEGs in a pathway; (ii) the conventional score (GSEA) as P-values of estimated pathway enrichment; (iii) the new score (IEA) as P-values of hypergeometric distribution of differential genes (integrating DEGs and DEVGs) in a pathway calculated by the proposed HT2 approach. For ORA or IEA, the thresholds of P-value of significance test on DEGs or DEVGs are both set as 0.05, and adopted a pervious strategy to select those fea- ture genes [29]: 1) select all genes with FDR adjusted p- values no more than 0.1; 2) if the genes selected are less than 200, re-select all genes with P-values no more than 0.05 and fold-change no less than 1.5; 3) if the genes se- lected are still less than 200, directly use the top 1 % of genes ranked by P-values from least to largest. Diabetes associated genes on pathways Diabetes associated genes on pathways First of all, we investigated the overlap between prior- known disease genes (e.g., diabetes associated genes) and pathway genes. Many pathways are full of diabetes asso- ciated genes (Additional file 6: Table S6), which means pathways could have great changes during disease devel- opment and progression. These pathways would be causes or outcomes of the disease. Although IEA pays attention to the identification of dysfunctional pathways (e.g., subtype-relevant pathways) rather than discrimin- ation of causal pathways, as introduced in follows, the map of pathways can further complementally supply some clues of the causal roles of pathways at the level of network of networks [57]. Fourthly, it is the other assistant step of IEA. The DEVGs in each pathway are used to group samples in two clusters by SLC approach. For each clinical index from sex, age, bmi, and HbA1c, its subtype-factors are identified. As comparisons, the all genes of each pathway are also used to directly group samples to evaluate the significance of detected subtype-factors. Then on the main dataset GSE41762 [52], we have ob- tained feature genes as summarized in Table 4. There are 2558 DEGs and 345 DEVGs selected by IEA, many of them are also detected on the replicate dataset GSE38642 [53]. The 523 genes of DEGs are disease genes, and 658 genes are pathway genes; meanwhile, the 63 genes of DEVGs are disease genes and 79 genes are pathway genes. Obviously, there are many disease- informative or function-informative genes disregarded in conventional analysis (i.e., DEVGs rather than DEGs), and IEA can capture these genes and estimate their ef- fects in the dysfunction of pathways. Obviously, the above analysis routine can be applied on any other dataset of samples with different kinds of complex diseases. Noted, our proof-of-concept study is to combine a group of genes with differential expression and a group of genes with differential expression variance. (i) We don’t select the genes with high variances, but the genes whose expression variances can distinguish different conditions/phenotypes. If the expression variance of a gene can classify samples with different phenotypes well, the dominant component of this gene’s variance could be biological variance. Obviously, the technique variance should have no such discrimination. (ii) We have also checked the correlation between the variance of each DEVG and each clinical index. A proof-of-concept study of IEA on transcriptional analysis of complex diseases (diabetes) IEA has been applied to detect the biological malfunc- tion of complex diseases (e.g., Type II Diabetes) on the pathway level rather than gene level. IEA, as a pathway- centred analysis approach, not only supplies the conven- tional pathway enrichment analysis but also extracts Thirdly, it is one assistant step of IEA. Pathway cross- talks are evaluated by two-way RWR approach. The Yu et al. BMC Genomics (2015) 16:918 Page 11 of 19 removal of technique variance will improve the selection of DEVG and final IEA, which is our future work. interactions selected from PIN consist of differential net- work [55], where the selected interactions have signifi- cant correlation difference between diabetic and non- diabetic groups. On this differential network, two-way RWR approach is used to find the pathway crosstalks. The most significant pathway crosstalks (the threshold of P-value of significance test is set as 0.001 strictly) con- sist of the map of pathways. Besides, the enrichments of pathway genes or disease-associated genes in the high- ranked genes of RWR are also analyzed and evaluated by AUC [56], which support the efficiency of RWR on pathway-related analysis. Diabetes associated genes on pathways Many genes’ expression variances even have significant correlation with clinics, which would not be caused by technique variance too. (iii) Our experiment on microarray is an application of IEA to support the idea of combining DEG and DEVG, and the results show our method’s efficiency. Indeed, many approaches are still proposing to improve the se- lection of conventional DEG or even new DEVG. The Furthermore, in DEGs, there are 1493 gene up- regulated in disease state and 1065 genes down-regulated. Meanwhile, there are 185 genes tight-regulated in disease condition and 160 genes relax-regulated. The examples of such four expression patterns are shown in Fig. 4. MYC, known as a cancer oncogene, is also reported to be altered in diabetes [58]. Seeing Fig. 4a, MYC is indeed up- regulated in the diabetes state. Insulin is known as a main cause of diabetes [59], and its an isoform as INS-IGF2 ac- tually has down-regulation when diabetes occurs (Fig. 4b). Diabetes associated genes on pathways BMC Genomics (2015) 16:918 Page 12 of 19 Fig. 4 The cases of differential expression patterns of DEG and DEVG. For genes MYC, INS-IGF2, HOXD8, and REXO1, (a)-(d) give their expression profiles on dataset GSE41762; (e)-(h) give their expression distribution on dataset GSE41762; (i)-(l) give their expression profiles on dataset GSE38642; (m)-(p) give their expression distribution on dataset GSE38642 Fig. 4 The cases of differential expression patterns of DEG and DEVG. For genes MYC, INS-IGF2, HOXD8, and REXO1, (a)-(d) give their expression profiles on dataset GSE41762; (e)-(h) give their expression distribution on dataset GSE41762; (i)-(l) give their expression profiles on dataset GSE38642; (m)-(p) give their expression distribution on dataset GSE38642 or activation-switch of a signalling pathway, which will be studied in our future work. important role in the morphogenesis. It has tight- regulation in diabetes condition (Fig. 4c), so that, it may participate in the accurately regulation [21] of bio- logical processes associated to diabetes. By contrast, REXO1, known as Transcription elongation factor B polypeptide 3-binding protein 1, would be a cofactor involved in gene regulation [60]. This gene shows relax-regulation in diabetes condition (Fig. 4d). Thus, REXO1 would be a cause or indicator of some sub- types of diabetes. Indeed, the original study has sup- plied four clinical indices [52], we found age is mostly related to the subtypes or sample clusters of diabetes determined by REXO1 (Seeing Additional file 11: Table S11 and Additional file 12: Table S12). Diabetes associated genes on pathways HOXD8, as a gene in the homeobox family encoding a highly conserved family of transcription factors, has an Table 4 The statistic on DEGs, DEVGs and their overlapping with pathway or disease genes (Diabetes) DEGa DEVG PG_DEG DG_DEG PG_DEVG DG_DEVG DEGup DEGdown DEVGup DEVGdown GSE41762 2558 345 658 523 79 63 1493 1065 160 185 GSE38642 2306 632 647 515 167 128 1294 1012 389 243 Overlapping 836 28 246 10 219 3 489 346 15 13 Significance 0 7.4832e-07 0 3.3539e-11 0 0.00059927 0 0 5.2401e-08 3.6083e-08 aDEG points genes with differential expression; DEVG points genes with differential expression variance; PG_DEG points the pathway genes in DEGs, i.e., the overlaps between pathway genes and DEGs; DG_DEG points the disease genes in DEGs, i.e., the overlaps between disease genes and DEGs; PG_DEVG points the pathway genes in DEVGs, i.e., the overlaps between pathway genes and DEVGs; DG_DEVG points the disease genes in DEVGs, i.e., the overlaps between disease genes and DEVGs; DEGup and DEGdown point genes with up-regulation and down-regulation respectively; DEVGup and DEVGdown point genes with relax-regulation and tight-regulation respectively GSE38642 2306 632 647 515 167 128 1294 1012 389 243 Overlapping 836 28 246 10 219 3 489 346 15 13 Significance 0 7.4832e-07 0 3.3539e-11 0 0.00059927 0 0 5.2401e-08 3.6083e-08 aDEG points genes with differential expression; DEVG points genes with differential expression variance; PG_DEG points the pathway genes in DEGs, i.e., the overlaps between pathway genes and DEGs; DG_DEG points the disease genes in DEGs, i.e., the overlaps between disease genes and DEGs; PG_DEVG points the pathway genes in DEVGs, i.e., the overlaps between pathway genes and DEVGs; DG_DEVG points the disease genes in DEVGs, i.e., the overlaps between disease genes and DEVGs; DEGup and DEGdown point genes with up-regulation and down-regulation respectively; DEVGup and DEVGdown point genes with relax-regulation and tight-regulation respectively aDEG points genes with differential expression; DEVG points genes with differential expression variance; PG_DEG points the pathway genes in DEGs, i.e., the overlaps between pathway genes and DEGs; DG_DEG points the disease genes in DEGs, i.e., the overlaps between disease genes and DEGs; PG_DEVG points the pathway genes in DEVGs, i.e., the overlaps between pathway genes and DEVGs; DG_DEVG points the disease genes in DEVGs, i.e., the overlaps between disease genes and DEVGs; DEGup and DEGdown point genes with up-regulation and down-regulation respectively; DEVGup and DEVGdown point genes with relax-regulation and tight-regulation respectively Yu et al. Dysregulated pathways identified to capture DEGs and DEVGs simultaneously Obviously, the pathways high-ranked by ORA have least scores located at the right-bottom of gene-distribution graph (Fig. 5a). It means the pathways selected by ORA are full of DEGs rather than DEVGs. By contrast, in the IEA, the detected pathways are full of DEGs and DEVGs respectively and significantly (Fig. 5b). Even more, the dysregulated pathways detected by well-known GSEA [1] on these datasets shown weak performance on the identification of pathways full of DEVGs (Fig. 5c). Thus, IEA indeed can effectively detect the pathways under-scored in conventional analysis, and these dys- functional pathways would be disease-relevant or subtype-relevant. This conclusion is also supported by the similar results from the analysis on replicate data- set (Fig. 5d-f). pathways, and found the Top-30 selected pathway (labelled in red) in graph. Obviously, the pathways high-ranked by ORA have least scores located at the right-bottom of gene-distribution graph (Fig. 5a). It means the pathways selected by ORA are full of DEGs rather than DEVGs. By contrast, in the IEA, the detected pathways are full of DEGs and DEVGs respectively and significantly (Fig. 5b). Even more, the dysregulated pathways detected by well-known GSEA [1] on these datasets shown weak performance on the identification of pathways full of DEVGs (Fig. 5c). Thus, IEA indeed can effectively detect the pathways under-scored in conventional analysis, and these dys- functional pathways would be disease-relevant or subtype-relevant. This conclusion is also supported by the similar results from the analysis on replicate data- set (Fig. 5d-f). improve the robustness of dysregulated pathway identi- fication by integrating other prior information (e.g., biological network or GO annotation) [16]. In the high-ranked pathways identified by IEA, many pathways are actually full of DEVGs. These DEVGs could be further associated to potential sub- types of samples, which are discussed in follows. Be- sides, some of these pathways under-scored by other methods indeed have been reported to be altered in the disease state (e.g., diabetes). For examples, (i) 'KEGG HEMATOPOIETIC CELL LINEAGE'. Dia- betes is known to compromise the function of the bone marrow (BM) [61], and diabetic complications mainly including macrovascular events might be from the dysfunctional BM-derived hematopoietic cells. (ii) 'KEGG CYTOKINE-CYTOKINE RECEPTOR INTERACTION'. Cytokines regulate inflammatory and immune responses, which play important roles in the pathogenesis of diabetes and its microvascular complications. Dysregulated pathways identified to capture DEGs and DEVGs simultaneously In conventional analysis, the genes with differential ex- pression are focused; now, the genes with differential ex- pression variance are also attractive. In the context of differential expression variance, the dysregulated pathways are expected to have as many DEGs & DEVGs as possible, which can be captured by over-representation approach like the proposed IEA. For evaluation, a gene-distribution graph is further introduced to show the percentages of DEGs and DEVGs respectively for each pathway. In Fig. 5, a pathway is represented by a point whose indices in axis are the percentages of DEGs and DEVGs in this pathway respectively. Obviously, the pathways full of DEGs and DEVGs tend to locate at the right-up conner of such gene-distribution graph. More importantly, DEVGs (either "tight" or "relax" ex- pression) don’t mean no-changes. As the key point of our model and method, DEVG means a gene would de- note activation of a signalling pathway (or sub-pathway) in a group of samples, meanwhile, inactivation of this signalling pathway (or sub-pathway) in another group of samples. This would be a main cause of heterogeneous samples. The biological mechanism underlying this phenomenon would be the switch of pathway activation. If based on other kinds of enrichment analysis frame- work, it is possible to discuss the activation, inactivation, To validate the pathways identified by IEA and compare to other methods ORA and GSEA, we firstly calculated the percentages of DEGs and DEVGs of each KEGG pathway and plot them on the gene- distribution graph. Then we calculated the dysfunc- tional score (i.e., enrichment) and rank all KEGG Yu et al. BMC Genomics (2015) 16:918 Page 13 of 19 Fig. 5 The gene-distribution graph of pathways identified by different methods. a-c The pathways high-ranked (labelled in red) by ORA, IEA and GSEA respectively, whose results on dataset GSE41762. d-f The pathways high-ranked by ORA, IEA and GSEA respectively, whose results on dataset GSE38642 Fig. 5 The gene-distribution graph of pathways identified by different methods a-c The pathways high-ranked (labelled in red) by ORA IEA and Fig. 5 The gene-distribution graph of pathways identified by different methods. a-c The pathways high-ranked (labelled in red) by ORA, IEA and GSEA respectively, whose results on dataset GSE41762. d-f The pathways high-ranked by ORA, IEA and GSEA respectively, whose results on dataset GSE38642 pathways, and found the Top-30 selected pathway (labelled in red) in graph. Dysregulated pathway-crosstalk identified to reveal the interactive map and module among pathways Different from conventional strategy to use the over- lapping genes as pathway crosstalks [27], two-way RWR is assistantly applied to find the interactive genes between any two pathways. RWR is previously used in the rank of disease genes [28], which holds the assumption that the candidate pathogen genes are more proximate to the known disease genes than ran- domly selected genes. This assumption is expected to be hold for pathway genes too. genes. Thus, two-way RWR is effective to mimic the pathway crosstalk and construct the associations among pathways, which is obviously consistent in multiple control experiments by using different prior-known network (e.g., STRING and HPRD), or different datasets (e.g., GSE41762 and GSE38642), or even different ranked/selected feature genes (e.g., pathway genes and disease- associated genes). p y g We first evaluated the pathway genes possibly selected by RWR,which uses the identified DEGs & DEVGs in a pathway as seeds. In the high-scored genes by RWR, there is a significant amount of pathway genes (seeing Table 5), which is the same as disease genes ranked by RWR (Table 6). In the evaluation in Table 5, we have set two kinds of control experiments. One is the prior-known network used, i.e., two sources as STRING [38] and HPRD [63] are both applied; the other one is the feature genes used in the given background network, i.e., three kinds of feature genes (as all ranked genes with P-values, the ranked DEGs with P-values and the ranked genes from RWR excluding the seeds) are respectively used to calcu- late the AUC values [28] to evaluate the efficiency of selecting/ranking pathway genes or disease-associated genes. Depending on these experiments, we can find: (ii) (ii) The protein association network (as in STRING [38]) rather than protein physical network (as in HPRD [63]) would be more efficient to lead the two-way RWR to link seed genes to pathway genes or disease-associated genes. There are at least two reasons for this result: one is that the known pro- tein physical network is still greatly incomplete, by contrast protein association network would supply additional predicted interactions with high confi- dence; the other one is that, except for direct in- teractions between pathways, protein association network would cover much more indirect interac- tions or long-term interactions, which would mimic the pathway crosstalk well. Dysregulated pathways identified to capture DEGs and DEVGs simultaneously The functional variations of cyto- kines and their receptors can benefit the prediction Noted, the pathways identified by different methods can be significantly observed in the analysis on the rep- licated dataset (P-value less than 0.05, whose details are supplied in Additional file 7: Table S7). The issue of the consensus of pathway identification is not discussed more here, although some other studies have worked to Page 14 of 19 Yu et al. BMC Genomics (2015) 16:918 Table 6 The AUC of different rank lists for disease-associated genes (Diabetes) PIN STRING-based HPRD-based Data GSE41762 GSE38642 GSE41762 GSE38642 All genes 0.46994a 0.46567 0.46994a 0.46567 DEGs 0.45695 0.47277 0.45695 0.47277 twRWR 0.73546 0.73652 0.70633 0.69904 aFor feature genes like all genes and DEGs, they don’t use network information, so that, they have the same AUC values on the same dataset although different network used Table 6 The AUC of different rank lists for disease-associated genes (Diabetes) of the susceptibility and progression to Diabetic nephropathy (DN) [62]. As the potential pathogenic mediators in DN, cytokines might provide new po- tential therapeutic agents for disease treatments. Dysregulated pathway-crosstalk identified to reveal the interactive map and module among pathways In all, protein association network is efficient to detect the associations among pathways, whose usage to accurately predict physical interaction of pathways is out of this work and would be a future study. (i) As a control, when all genes are ranked according to P-values, its AUC is low. And when the selected DEGs are ranked according to P-values, the AUC is similar to that of all genes. Meanwhile, when the genes from two-way RWR excluding seeds are ranked according to proximity values, the AUC achieves highest, which support again RWR-based approach is effective to capture interactive pheno- typic genes as pathway genes or disease-associated Thus, RWR is actually effective to detect the genes interactive within known pathway genes. Then by two- way RWR, we can find the interactive genes from two pathways, and select any pathway-pair as a crosstalk sig- nificantly (Additional file 8: Table S8). All the crosstalks connect the known pathways as a map, where each crosstalk is an edge and a pathway is a node. This map of pathways is a network of networks, rather than the original background network of separate molecules. In the map of pathways, the modules of interactive path- ways can be detected, where a module represents a group of closely inter-connected pathways. In these pathway modules, two modules are obviously related to diabetes. One module (Module 1 shown in Fig. 6) is the Table 5 The AUC of different rank lists for pathway genes (Diabetes) PIN STRING-based HPRD-based Data GSE41762 GSE38642 GSE41762 GSE38642 All genes 0.4861a 0.48611 0.4861a 0.48611 DEGs 0.46342 0.51717 0.46342 0.51717 twRWR 0.83498 0.83326 0.68449 0.68023 aFor feature genes like all genes and DEGs, they don’t use network information, so that, they have the same AUC values on the same dataset although different network used Table 5 The AUC of different rank lists for pathway genes (Diabetes) Yu et al. BMC Genomics (2015) 16:918 Page 15 of 19 Fig. 6 The main topological structure of the map of pathways reconstrcucted on diabetes datasets. Module 1 is full of signalling pathways, and Module 2 is full of disease/diabetes associated pathways Fig. 6 The main topological structure of the map of pathways reconstrcucted on diabetes datasets. Module 1 is full of signalling pathways, and Module 2 is full of disease/diabetes associated pathways group of signalling pathways. Dysregulated pathway-crosstalk identified to reveal the interactive map and module among pathways Signalling pathways are known as the up-stream functions in the cascades of sig- nals, so that, they have great possibility to be the causes of the dysfunction of down-stream functions, e.g., dia- betes pathways. The other module (Module 2 shown in Fig. 6) is just the group of pathways concerning glycoly- sis, sugar metabolism, glycosaminoglycan and diabetes, which seems to be extremely a core pathway module of diabetes. Noted, the Type II diabetes pathway would have significant crosstalk with pentose phosphate path- way as shown in Fig. 6. As reported, the pentose phos- phate pathway is widely activated in diabetes and its complications [64–67], thus this pathway would be im- portant to understand the risk of diabetes diagnosis and treatment in clinical application. Obviously, the pentose phosphate pathway have no significance on the selection by IEA or other methods, and actually, its importance is reflected from the topological structure of the map of pathways. These facts reveal: (i) conventional approaches usually focus on single pathways, so that, they can some- times capture the disease associated pathways relevant to particular phenotypes but can’t distinguish or under- estimate the potential causal relationship among path- ways; (ii) the proposed IEA supplies the map of pathways to reflect the functional organization of path- ways, and disclose the key modules of pathways, such as the upstream pathways related to signalling pathways and the downstream pathways associated to diseases; (iii) on the map of pathways, those pathways full of DEVGs and DEGs tend to interact with disease path- ways, indicating the determinant of subtypes (i.e., the subtype-factors identified in follows) are actually also the potential determinants of diseases. Totally, the map of pathways supplies us a new viewpoint of functional organization at the level of network of networks. Dysregulated pathways associated to clinical indicator as subtype-factors The pathways full of DEGs and DEVGs are specially se- lected by IEA, in which the DEVGs might be the cause of potential subtypes of samples. Compared to the mean values of genes in case, the samples in control can be di- vided into two groups: one group of samples have larger gene expressions than that mean value; and the other group of samples have fewer values, or vice versa. Thus, we have applied two strategies to cluster the samples in control or case group, which can associate one pathway to some clinical index. The first common strategy (noted Page 16 of 19 Yu et al. BMC Genomics (2015) 16:918 Page 16 of 19 determination of DEVG combination at the level of pathway. In addition, the association of each pair be- tween a gene (i.e., one DEVG) and a clinical index are listed in Additional file 11: Table S11 and Additional file 12: Table S12 for normal and diseased samples respectively. as PGC) is using the expressions of whole genes in a pathway to cluster samples into two clusters, and test the significance of these two groups of samples on one clinical indicator (The test is to see if one group of sam- ples have larger or fewer clinical values than those of the other group of samples). The second strategy (just as proposed SLC) is using the discrete value of DEVGs in the same pathway to group samples and measuring their relationships with clinical indices. Noted, subtype is a potential biological explanations of DEVG, so that, we have evaluated the possible subtype- factors rather than subtypes by the association between DEVG (or DEVG-full pathways) and known clinical index. In the statistical analysis, we have analyzed age, sex, bmi, and even Hba1c which evaluates the risk of diabetes. Thus, this clinical information can help us to find subtype of genotype-phenotype associations as stated above. Of course, in breast cancer, there are some well-known subtypes determined on genotypes. To the best of our knowledge, in diabetes, the subtypes as T1D, T2D, Gestational diabetes, Surgically induced diabetes, Chemically induced diabetes, are not defined by one or two genes/proteins. Thus, our finding of subtype-factors (DEVGs or pathways) would be the causes or indicators of disease (e.g., diabetes) subtypes on genome level, which will be further studied in future. Dysregulated pathways associated to clinical indicator as subtype-factors Additional file 9: Table S9 gives the P value of the as- sociation of each pair between a pathway and a clinic index for normal samples, and Additional file 10: Table S10 gives those for diabetes. Generally, SLC tends to dis- cover more significant potential subtypes of samples cor- responding to particular clinic index (Fig. 7). Thus, DEVGs actually have more power to identify the sub- types of genotype-phenotype associations than conven- tional approaches based on differential expression only. Obviously, one pathway can associate to multiple clin- ical indices, and one clinical index can also relate to mul- tiple pathways. ‘KEGG WNT SIGNALING PATHWAY’ is consistently related to bmi in the analysis of duplicate datasets. In fact, this pathway already has a few evidences on their associations with body weight [68–70]. ‘KEGG CELL CYCLE’ is found to possibly associate with age, sex, bmi or Hba, which is known as a common factor in dis- ease development and progression [71]. Conclusion Pathway enrichment analysis is a useful tool in the study of biology or biomedicine, due to its functional screen- ing on the well-known biological processes rather than single molecules. The measurement of dysfunctions of Although each DEVG can separately determine some potential subtype-factor, here, we only discuss the Fig. 7 The summary of dysregulated pathways identified as subtype-factors corresponding to four clinical indices. For example, there are 11 pathways identified by IEA to be correlated with Hba values (i.e., based on DEVG expressions of pathway genes), but, there are only 1 pathways recognized by conventional strategy (i.e., bsaed on raw expressions of pathway genes). Similarly, for other clinical indices, IEA can also identify many subtype-factors Fig. 7 The summary of dysregulated pathways identified as subtype-factors corresponding to four clinical indices. For example, there are 11 pathways identified by IEA to be correlated with Hba values (i.e., based on DEVG expressions of pathway genes), but, there are only 1 pathways recognized by conventional strategy (i.e., bsaed on raw expressions of pathway genes). Similarly, for other clinical indices, IEA can also identify many subtype-factors Yu et al. BMC Genomics (2015) 16:918 Page 17 of 19 pathways during a phenotype change, e.g., from normal to diseased, is the key issue when applying enrichment analysis for pathway or other functional gene set. Differ- ent from differentially expressed genes focused in previ- ous methods, the genes with great differential expression variance are also attractive and important, which indi- cate another specific characteristic of a biological system in the change of phenotypes. Additional file 2: Table S2. The prioritization performance about method comparison on approach-specific datasets (K = 2). (DOCX 17 kb) Additional file 3: Table S3. The prioritization performance about method comparison on approach-specific datasets (K = 4). (DOCX 17 kb) Additional file 4: Table S4. The sensitivity performance about method comparison on approach-specific datasets (K = 4). (DOCX 17 kb) Additional file 5: Table S5. The 3-order approach-specific datasets corresponding to different methods based on sensitivity performance. (DOCX 16 kb) g p yp In the context of differential expression and expression variance, IEA is proposed to identify the pathways full of DEGs and DEVGs simultaneously, rather than conven- tional approaches focusing on only DEGs. The biological meaning of IEA has obtained strong evidences by an evaluation scheme based on method comparison. Competing interest The authors declare that they have no competing interests. Competing interest The authors declare that they have no competing interests. Additional file 13: SI document - a case study on colorectal cancer and Table A1-A3. (DOCX 3072 kb) Additional file 14: Table A4. Pathways ranking for colorectal cancer. (XLS 97 kb) Additional file 15: Table A5. Pathway-pair as a crosstalk (colorectal cancer). (XLS 133 kb) Received: 8 August 2015 Accepted: 2 November 2015 Received: 8 August 2015 Accepted: 2 November 2015 Authors’ contributions TZ and GL conceived of the study. XY carried out the experiment and analysis. XY and TZ drafted the manuscript. All authors read and approved the final manuscript. Totally, IEA supplies a new way to carry on enrich- ment analysis in the context of differential expression and expression variance, and can easily expand to handle with the analysis in other more complicated context (e.g., the differential expression covariance). It is also ne- cessary to expand IEA to functional class scoring or pathway topology based approaches in future work. References 1. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545–50. Abbreviations DEG: Differentially expressed gene; DEVG: Gene with great differential expression variance; IEA: Integrative enrichment analysis; GSEA: Gene set enrichment analysis; RWR: Random walk with restart; SLC: Supervised-like clustering approach; PIN: Protein interaction network. Additional file 1: Table S1. The 3-order approach-specific datasets corresponding to different methods based on prioritization performance. (DOCX 16 kb) Conclusion On the real datasets of disease samples, IEA indeed specific- ally identify pathways containing DEGs and DEVGs, which are usually under-scored by other methods. The map of pathways was further reconstructed based on the selected pathway crosstalks, and the module organization among pathways was also detected. The topological structure of such network of pathways re- veals the signalling pathways as upstream functions would be causes of disease, and the disease-relevant pathways as downstream functions would link to those upstream pathways by crosstalk. In addition, some disease-relevant pathways or subtype-relevant pathways are well associated with clinical indices according to their DEVGs’ relative expression level, which are usually not observed from the raw expression profiles of path- way genes. Although many identified subtype-factors haven’t clinical evidences due to the limit in the clinical application, the IEA actually show its ability to identify the risk of subtypes of genotype-phenotype associations. Those subtype-factors could help us in accurately realizing personal prevention or personal treatment [72, 73]. Besides, the additional analysis results on colorectal cancer also support these conclusions (Additional file 13: SI document - a case study on colorectal cancer and Table A1-A3; Additional file 14: Table A4; Additional file 15: Table A5). Additional file 6: Table S6. Overlaps between pathway genes and disease associated genes on diabetes datasets. (XLS 39 kb) Additional file 7: Table S7. Pathway ranking by different methods on diabetes datasets. (XLSX 22 kb) Additional file 8: Table S8. Pathway-pair as a crosstalk identified on diabetes datasets. (XLS 107 kb) Additional file 9: Table S9. Association of each pair between a pathway and a clinic index (for Normal samples) on diabetes datasets. (XLS 56 kb) Additional file 10: Table S10. Association of each pair between a pathway and a clinic index (for Diabetes samples) on diabetes datasets. (XLS 60 kb) Additional file 11: Table S11. Association of each pair between a DEVG and a clinic index (for Normal samples) on diabetes datasets. 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https://openalex.org/W4387828026	https://bluemarkpublishers.com/index.php/IJAH/article/download/15/16	English	null	Art Education and its Impact on Creativity and Critical Thinking Skills: A Review literature	International journal of arts and humanities	2,023	cc-by	5,997	\| KEYWORDS Creativity, Critical thinking, Art Education, Cognitive skills, Curriculum \| ARTICLE INFORMATION Creativity, Critical thinking, Art Education, Cognitive skills, Curriculum \| ARTICLE INFORMATION RECEIVED: 15 July 2023 ACCEPTED: 29 August 2023 PUBLISHED: 04 September 2023 PUBLISHED: 04 September 2023 ACCEPTED: 29 August 2023 \| ABSTRACT \| This study aims to review the literature on art education and its impact on creativity and critical thinking skills. The study examines various sources to understand how art education contributes to the development of these skills and how it can be integrated into traditional educational practices. The study majorly used secondary sources to gather information. The findings suggest that art education enhances creativity and critical thinking by encouraging expressive and cognitive skills. Furthermore, the study highlights the importance of integrating art education into the curriculum to foster creativity and critical thinking skills in students. Overall, this research emphasizes the value of art education in promoting these crucial skills for students' academic and personal growth. In conclusion, this review of literature provides evidence for the positive impact of art education on the development of creativity and critical thinking skills. It underscores the importance of incorporating art education in educational curricula to foster these essential skills in individuals. The findings of this study have implications for policy-makers, educators, and researchers in understanding the potential of art education in promoting creativity and critical thinking in individuals of all ages. Art Education and its Impact on Creativity and Critical Thinking Skills: A Review literature Dickson Lukaka Department of Arts and Humanities, University of Nairobi, Kenya. Corresponding Author: Dickson Lukaka, E-mail: dlukaka@gmaiol.com Department of Arts and Humanities, University of Nairobi, Kenya. Corresponding Author: Dickson Lukaka, E-mail: dlukaka@gmaiol.com International Journal of Arts and Humanities IJAH BLUEMARK PUBLISHERS Journal Homepage: http://bluemarkpublishers.com/index.php/IJAH Journal Homepage: http://bluemarkpublishers.com/index.php/IJAH BLUEMARK PUBLISHERS by Bluemark Publishers 1. Introduction Moreover, art education provides a safe and open space for students to express their thoughts, emotions, and experiences. It encourages self-reflection and self-expression, allowing students to explore and articulate their ideas and perspectives. This process of self-expression fosters critical thinking as students learn to make connections between their personal experiences and the broader human experience, developing empathy and a deeper understanding of themselves and others (Lampert, 2006). Throughout this literature review, we will examine various studies and research articles that explore the impact of art education on creativity and critical thinking skills. We will examine studies that investigate the relationship between art education and the development of creative thinking, as well as the impact of art education on critical thinking skills. Additionally, we will explore how art education can be integrated into the broader educational curriculum to enhance creativity and critical thinking across all disciplines. By understanding the existing literature on this topic, we can gain valuable insights into the benefits and potential challenges of art education in fostering creativity and critical thinking skills. This knowledge can inform educators, policymakers, and researchers in their efforts to develop effective art education programs and strategies that maximize the potential of art education in promoting creativity and critical thinking in individuals of all ages. This literature review aims to contribute to the growing body of research on art education and its impact on creativity and critical thinking skills. It seeks to provide a comprehensive overview of the current state of research and identify gaps and areas for further exploration. By examining the existing literature, this review aims to shed light on the importance of art education in the development of creativity and critical thinking skills and its potential implications for education and society as a whole. 1. Introduction Art education plays a significant role in the development of creativity and critical thinking skills in individuals. The study of art enables students to explore their creative potential and develop their capacity for critical thinking. In recent years, there has been a growing interest in the impact of art education on these skills, as educators and policymakers recognize the importance of cultivating creativity and critical thinking in the educational system (Black, 2011). Creativity is the ability to generate original ideas, insights, and solutions to problems. It is a crucial skill that is highly valued in various fields, including the arts, sciences, and business. As the world becomes increasingly complex and interconnected, the demand for creative individuals who can think outside the box and come up with innovative solutions is growing rapidly. Art education provides a unique platform for students to practice and develop their creative skills. Through engaging in various art forms such as painting, sculpture, music, and dance, students are encouraged to explore different materials, techniques, and concepts, which stimulates their imagination and promotes creative thinking (Deasy, 2002). Copyright: © 2023 the Author(s). This article is an open access article distributed under the terms and conditions of Bluemark Publishers. Published by Bluemark Publishers Critical thinking, on the other hand, is the ability to analyze, evaluate, and synthesize information in a logical and systematic manner. It involves questioning assumptions, considering multiple perspectives, and weighing evidence Critical thinking, on the other hand, is the ability to analyze, evaluate, and synthesize information in a logical and systematic manner. It involves questioning assumptions, considering multiple perspectives, and weighing evidence Copyright: © 2023 the Author(s). This article is an open access article distributed under the terms and conditions of Bluemark Publishers. Published by Bluemark Publishers P \| 1 by Bluemark Publishers Page \| 1 Art Education and its Impact on Creativity and Critical Thinking Skills: A Review literature to make informed decisions. Art education supports the development of critical thinking skills by encouraging students to engage in thoughtful analysis and interpretation of artistic works. By studying and discussing various art forms, students are exposed to different cultural and historical contexts, diverse artistic movements, and various artistic techniques. This exposure allows them to develop the ability to critically analyze and interpret works of art, understanding the intentions of the artist and the social and cultural significance of the artwork (Ingalls, 2013). 2. Impact of art education on creativity 2.1 Definition of creativity Creativity can be defined as the ability to generate new and unique ideas, perspectives, or solutions to problems. It involves thinking outside of the box, being open to new possibilities, and making connections between different ideas or concepts. Creative individuals are often known for their originality, imagination, and ability to think critically and abstractly. They are able to see patterns and relationships that others may not see, and are not afraid to take risks or challenge conventional thinking. Creativity is not limited to the arts, but can be applied to any field or discipline, including science, technology, engineering, and mathematics (STEM) (Pavlou, 2013). 2.2 Benefits of art education on creativity By engaging in art activities, students learn to think outside the box and approach problems from multiple perspectives, which can aid in their future careers (Combs, 2008). There is a significant body of research that supports the positive impact of art education on students' creativity. Art education has been found to enhance creative thinking skills, promote emotional intelligence, improve overall well- being, and develop important skills for future success. Therefore, incorporating art education into the curriculum can be beneficial for students' cognitive, social, emotional, and professional development. 2.2 Benefits of art education on creativity y Art education has long been recognized as a valuable component of a well-rounded education, providing students with opportunities for creative expression and personal growth. A growing body of research supports the notion that art education also has a positive impact on students' creativity, helping to foster the development of innovative thinking skills that are increasingly valued in today's society. One study conducted by the University of Newcastle found that art education can enhance creative thinking by promoting the exploration of novel ideas and encouraging students to think divergently. The researchers found that students who engaged in art activities were more likely to display originality and fluency in their thinking, as well as the ability to generate multiple solutions to problems (Rong, 2022). Another study conducted by the Arts Education Partnership reviewed the existing research on the impact of arts education on students' creativity. The researchers found evidence that art education can enhance students' ability to Page \| 2 IJAH 1(1): 00-00 think creatively and problem solve, as well as improve their self-confidence and self-expression. They also noted that art education may have a positive effect on students' emotional well-being and overall academic achievement (Ulger, 2018). In addition to these cognitive benefits, art education has also been found to have social and emotional benefits for students. A study published in the Journal of Educational Psychology found that participation in visual arts activities was associated with higher levels of empathy and emotional intelligence among students. The researchers suggested that engaging in art can help students develop a deeper understanding and appreciation for the perspectives and experiences of others (Uzunöz, 2017). Furthermore, exposure to art and artistic expression can also have a positive impact on students' overall well-being and mental health. Studies have found that art education can help reduce stress and anxiety, improve mood, and enhance overall psychological well-being. The act of creating art allows students to express themselves in a non- verbal way, which can be particularly beneficial for those who struggle with verbal communication or have difficulty expressing their emotions (Baş, 2022). Art education also promotes the development of important skills that are valued in the professional world. The ability to think creatively, problem solve, and think critically are all skills that are highly sought after in a variety of industries. 3. Impact of art education on critical thinking skills 3.1 Definition of critical thinking skills g Art education has long been recognized as a valuable component of a well-rounded education. It provides students with opportunities to express themselves creatively, develop their imagination, and explore different forms of communication. Beyond these benefits, art education also plays a significant role in developing critical thinking skills in students (Freedman, 2008). Critical thinking is an essential skill that enables individuals to analyze, evaluate, and generate new ideas and perspectives. It involves logical reasoning, problem-solving skills, and the ability to think independently and critically question information and ideas. Through art education, students are encouraged to think critically and develop these skills (Leong, 2010). 3.2. Benefits of art education on critical thinking skills Art education encourages students to think creatively, reflect on their choices, and make cognitive connections. It also provides opportunities for students to discuss and analyze artwork, develop communication skills, and receive feedback. Additionally, art education often promotes interdisciplinary learning, which expands students' analytical and evaluative thinking skills. Overall, art education plays a crucial role in developing and enhancing critical thinking abilities in students. 3.2. Benefits of art education on critical thinking skills Numerous studies have explored the benefits of art education on critical thinking skills, and most have found positive correlations. One study conducted by Moga (2000) found that students who received visual arts instruction demonstrated improved critical thinking skills compared to those who did not receive any art instruction. The researchers attributed this improvement to the nature of art-making, which encourages students to think creatively, reflect on their work, and make cognitive connections. Another study conducted by Padget (2012) examined the impact of art integration on critical thinking skills in elementary school students. They found that students who participated in an art-integrated curriculum showed significant gains in critical thinking abilities compared to those in a traditional curriculum. The researchers suggested that the process of making and discussing art helped students develop analytical and evaluative thinking skills. Page \| 3 Art Education and its Impact on Creativity and Critical Thinking Skills: A Review literature Additionally, a meta-analysis conducted by Santn (2017) examined multiple studies on the impact of arts education, including visual arts, music, dance, and theater, on critical thinking skills. The analysis found that arts education had a positive and significant effect on critical thinking skills across all disciplines. Specifically, the study found that arts education improved students' cognitive flexibility, problem-solving abilities, and ability to make informed judgments. There are several reasons why art education may have a positive impact on critical thinking skills. First, the process of creating art requires students to think critically about their choices, such as selecting materials, colors, and compositions. This encourages students to analyze and evaluate their decisions and consider alternative possibilities (Ulger, 2019). Furthermore, art education often involves discussing and reflecting on students' artwork. These discussions provide opportunities for students to articulate their thoughts, explain their artistic choices, and receive feedback from peers and teachers. This fosters the development of communication and reasoning skills, as students learn to articulate their ideas, defend their choices, and consider different perspectives (Black, 2011). Moreover, art education often promotes interdisciplinary learning. Students may explore connections between art and other subjects such as history, science, or literature. This interdisciplinary approach encourages students to think critically about how different disciplines intersect and influence each other, expanding their analytical and evaluative thinking skills (Freedman, 2008). In conclusion, numerous studies have highlighted the positive impact of art education on critical thinking skills. 4. Comparison of traditional education and art education 4.1 Differences in teaching methods Traditional education, also known as mainstream education, refers to the conventional methods of teaching and learning that are used in most schools and educational institutions. It focuses on academic subjects such as mathematics, science, literature, and history, with an emphasis on cognitive development and examination-based assessments (Lampert, 2006). On the other hand, art education is a specialized form of education that focuses on the creative and expressive aspects of learning. It includes visual arts, music, dance, and drama, and aims to foster creativity, imagination, and self-expression (Padget, 2012). One major difference between traditional education and art education is the teaching methods used. In traditional education, the teacher plays a central role as the primary source of knowledge and instruction. They typically deliver lectures, assign readings and homework, and assess students based on their understanding of the subject matter. The focus is on transmitting information and knowledge from the teacher to the students (Ulger, 2018). In contrast, art education places more emphasis on collaborative and hands-on learning. The teacher acts as a facilitator and guide, allowing students to explore and experiment with various art forms and techniques. Students are encouraged to express themselves creatively and think critically about their artistic choices. Instead of relying solely on textbooks and lectures, art education often incorporates experiential learning through activities such as creating art, attending performances, and visiting art exhibitions (Baş, 2022). Page \| 4 IJAH 1(1): 00-00 Another difference in teaching methods between traditional education and art education is the assessment and evaluation process. In traditional education, assessments often involve tests, quizzes, and exams to measure students' understanding and retention of knowledge. These assessments are typically focused on rote memorization and the ability to recall information (Deasy, 2002). In art education, assessment is more subjective and focuses on the process of creation and the artistic quality of the work. Students are often evaluated based on their creativity, originality, technique, and ability to communicate through their artwork. Assessments may include portfolio reviews, performances, exhibitions, and peer evaluations. This allows for a more holistic and individualized approach to evaluating student learning (Leong, 2010). Additionally, traditional education places a strong emphasis on academic achievement and standardized testing. Students are often evaluated based on their grades and test scores, and success is often measured by how well they perform in these assessments. In contrast, art education values the process of learning and self-expression. 4. Comparison of traditional education and art education 4.1 Differences in teaching methods Success is measured by students' growth in creativity, artistic skills, and ability to communicate their ideas through art (Rong, 2022). In General, traditional education and art education have distinct differences in teaching methods. Traditional education tends to focus on transmitting information through lectures and assessments that test students' retention of knowledge. Art education, on the other hand, emphasizes hands-on, experiential learning and assessment based on creativity, originality, and artistic expression. Both forms of education have their own merits and play important roles in shaping well-rounded individuals with diverse skills and abilities (Uzunöz, 2017). In General, traditional education and art education have distinct differences in teaching methods. Traditional education tends to focus on transmitting information through lectures and assessments that test students' retention of knowledge. Art education, on the other hand, emphasizes hands-on, experiential learning and assessment based on creativity, originality, and artistic expression. Both forms of education have their own merits and play important roles in shaping well-rounded individuals with diverse skills and abilities (Uzunöz, 2017). 4.2 Differences in outcomes The outcomes of traditional education mainly revolve around academic success and preparation for the workforce. Students in traditional education typically develop strong academic skills, analytical thinking, and problem-solving abilities. They are often well-prepared for standardized tests and for careers that require a high level of technical knowledge and specialized skills. Traditional education also places a strong emphasis on following rules and protocols, as well as on conformity and uniformity (Combs, 2009). In contrast, the outcomes of art education focus more on personal growth, creativity, and self-expression. Students in art education develop artistic skills, imagination, and the ability to think outside the box. They learn to express themselves through various artistic mediums, building confidence and self-esteem along the way. Art education also fosters skills like collaboration, communication, and innovation, which are valuable in both artistic and non- artistic fields. Additionally, art education promotes cultural awareness and appreciation, as students are exposed to different art forms and traditions (Ingalls, 2013). While traditional education primarily values academic achievement and career readiness, art education recognizes the importance of emotional intelligence, empathy, and holistic development. Research has shown that art education can positively impact students' social-emotional skills, mental health, and overall well-being. It can enhance critical thinking abilities, increase cultural understanding, and improve problem-solving skills (Moga, 2000). In conclusion, the outcomes of traditional education and art education differ significantly. Traditional education focuses on academic success and preparing students for the workforce, while art education emphasizes personal growth, creativity, and self-expression. Both approaches have their merits and contribute to a well-rounded education. The integration of both traditional and art education can provide a balanced and comprehensive learning experience, allowing students to develop cognitive, emotional, and creative skills that will serve them well in their personal and professional lives. Page \| 5 Art Education and its Impact on Creativity and Critical Thinking Skills: A Review literature 5.2 Lack of trained teachers Art education requires specialized knowledge and skills in areas such as drawing, painting, sculpture, and art history. Without trained teachers, students may not receive the necessary instruction and guidance to develop their artistic abilities. Additionally, the lack of trained art teachers can also result in a limited curriculum and teaching resources. Without access to professional development and training opportunities, teachers may struggle to provide engaging and meaningful art lessons (Baş, 2012). Furthermore, the lack of trained art teachers may lead to a lack of consistency and continuity in art education across different grade levels. Each teacher may have their own approach and teaching style, which can make it difficult for students to build upon their skills and knowledge from year to year (Freedman, 2008). 5. Potential barriers to implementing art education 5.1 Lack of funding Art education plays a crucial role in the overall development of students, enhancing their creativity, critical thinking skills, and cultural awareness. However, the implementation of art education programs in schools often faces various barriers, one of which is the lack of funding. Lack of funding is a significant obstacle to implementing art education programs in schools. School districts may not allocate sufficient funds for art programs due to budget constraints or prioritization of other subjects, such as math and science. As a result, schools may have limited resources for hiring qualified art teachers, purchasing art supplies, and maintaining art facilities (Rong, 2012). The lack of funding also affects the scope and quality of art education programs. Without adequate resources, schools may have to hire underqualified art teachers or reduce the number of art classes offered. This can result in limited exposure to different art forms and techniques, limiting students' artistic growth and potential (Ulger, 2018). Moreover, the lack of funding can lead to unequal access to art education. Schools in low-income communities often suffer the most from funding disparities, resulting in a lack of art programs and resources. This inequality in access to art education can exacerbate existing social and economic inequalities, as students from disadvantaged backgrounds may not have the same opportunities to develop their artistic skills and pursue creative careers (Uzunöz, 2017). Finally, the lack of funding for art education can also impact the professional development of art teachers. Without adequate resources and funding for professional development programs, art teachers may struggle to stay updated with the latest instructional strategies and techniques. This can hinder their ability to effectively teach and engage students in the artistic process (Baş, 2012). 5.3 Resistance from administrators or policy makers Art education is often seen as a non-essential or low-priority part of the education system, especially when compared to subjects like math or science. As a result, there are several potential barriers to implementing art education, including resistance from administrators or policy makers. One potential barrier is the perception that art education is not as academically rigorous as other subjects. This perception can lead administrators and policy makers to prioritize other subjects and allocate resources accordingly. Additionally, policy makers may be more inclined to invest in subjects that are seen as directly related to job skills and economic competitiveness, rather than supporting the development of artistic skills (Lampert, 2006). Resistance to art education can also arise from a lack of understanding or appreciation for the value of the arts in education. Some administrators and policy makers may view art as a hobby or a form of entertainment, rather than IJAH 1(1): 00-00 as a vital component of a well-rounded education. This perception can lead to a devaluation of art education and a reluctance to prioritize it within the curriculum (Padget, 2012). as a vital component of a well-rounded education. This perception can lead to a devaluation of art education and a reluctance to prioritize it within the curriculum (Padget, 2012). 6.2 Collaborative projects between art teachers and other subject teachers Incorporating art education into a traditional curriculum can greatly enhance students' learning experiences and provide them with a well-rounded education. One strategy for doing this is through collaborative projects between art teachers and other subject teachers. By working together, art teachers and subject teachers can design projects that integrate art concepts and skills with academic content (Padget, 2012). For example, a history teacher and an art teacher could collaborate to create a project where students design and create visual representations of key historical events or figures. This not only allows students to explore history in a creative and hands-on way, but also gives them the opportunity to develop their artistic skills and techniques. Similarly, a science teacher and an art teacher could work together to design a project where students create sculptures or models to represent scientific concepts. This allows students to apply their knowledge of science in a visual and tangible way, while also fostering their creativity and artistic expression. By incorporating art into traditional curriculum through collaborative projects, students are able to deepen their understanding of subject matter, develop their artistic abilities, and make connections between different disciplines. These projects can also help foster a sense of teamwork and collaboration among students and teachers (Ingalls Vanada, 2013). 6. Strategies for incorporating art education into traditional curriculum 6.1 Integration of art into other subjects One strategy for incorporating art education into the traditional curriculum is to integrate art into other subjects. This can be done by incorporating art activities and projects into lessons and assignments in various subject areas. For example, in a science class, students can create visual representations of scientific concepts or conduct experiments that involve artistic processes, such as painting or sculpture. In a math class, students can use art techniques to reinforce mathematical concepts, such as creating geometric patterns using different colors or shapes. In a language arts class, students can use art forms, such as drama or storytelling, to visualize and interpret literature or historical events. By infusing art into these subjects, students are able to make connections between different disciplines and engage in hands-on and creative learning experiences (Ulger, 2019). 6.3 Use of technology to enhance art education Technology has revolutionized the way we teach and learn, and it has the potential to greatly enhance art education. One way technology can be incorporated into art education is through digital art tools and software. These tools allow students to experiment with different mediums, techniques, and styles in a digital format, providing them with a new and exciting way to create art. Additionally, digital tools can also help students overcome some of the challenges associated with traditional art mediums, such as the cost and availability of materials (Rong, 2022). Another way technology can enhance art education is through virtual art galleries and online exhibitions. These platforms provide students with the opportunity to showcase their work to a wider audience and receive feedback from experts in the field. They also allow students to explore and engage with art from different cultures and time periods, expanding their knowledge and appreciation for the arts (Ulger, 2018). Incorporating technology into art education also opens up new avenues for collaboration and networking. Students can connect with artists and art professionals from around the world, participate in online art communities, and collaborate on projects with their peers. This not only enhances their artistic skills but also helps develop important communication and teamwork skills (Ulger, 2018). Page \| 7 Education and its Impact on Creativity and Critical Thinking Skills: A Review literature Art Education and its Impact on Creativity and Critical Thinking Skills: A Review literature Overall, incorporating technology into art education provides students with new and exciting opportunities to explore their creativity and engage with the arts. It helps overcome some of the challenges associated with traditional mediums and provides students with a more inclusive and global perspective on art. By embracing technology in art education, educators can create a dynamic and enriching learning environment that prepares students for success in the 21st century. 7. Conclusion This review of literature has highlighted the importance of art education in developing creativity and critical thinking skills. The findings suggest that art education provides unique opportunities for students to express themselves, think critically, and problem solve. It also enhances their ability to analyze, interpret, and evaluate information. Art education goes beyond the traditional academic subjects and allows students to think outside the box, take risks, and explore new ideas. The research reviewed in this study demonstrates that art education has a positive impact on cognition, imagination, and overall academic performance. It can also foster a sense of cultural awareness and appreciation, leading to a more inclusive and diverse society. Additionally, studies have shown that students who participate in art education are more likely to excel in other academic areas, such as math and science. While the benefits of art education are clear, there are still challenges to implementing effective programs. Limited resources, lack of access, and the prioritization of standardized testing often hinder the integration of art education into the curriculum. However, the findings of this review suggest that it is essential for educators, policymakers, and parents to recognize and support the inclusion of art education in schools. In conclusion, art education plays a crucial role in developing creativity and critical thinking skills in students. It not only enhances their cognitive abilities , but also contributes to their overall well-being and personal growth. By nurturing creativity and encouraging originality, art education prepares students for success in the 21st century workforce, where innovation and problem-solving skills are highly valued. Therefore, it is imperative that educators and policymakers prioritize and invest in art education to cultivate a future generation of individuals who are equipped with the necessary skills to navigate an ever-changing and complex world. [12] Rong, Q., Lian, Q., & Tang, T. (2022). Research on the Influence of AI and VR Technology for Students’ Concentration and Creativity. Frontiers in Psychology, 13, 767689. [1] Baş, M. T., Özpulat, F., Molu, B., & Dönmez, H. (2022). The effect of decorative arts course on nursing students' creativity and critical thinking dispositions. Nurse Education Today, 119, 105584. [10] Pavlou, V. (2013). Investigating interrelations in visual arts education: Aesthetic enquiry, possibility thinking and creativity. International Journal of Education through Art, 9(1), 71-88. [8] Lampert, N. (2006). Enhancing critical thinking with aesthetic, critical, and creative inquiry. Art Education, 59(5), 46-50. [9] Moga, E., Burger, K., Hetland, L., & Winner, E. (2000). Does studying the arts engender creative thinking? Evidence for near but not far transfer. Journal of Aesthetic Education, 34(3/4), 91-104. [7] Leong, S. (2010). Creativity and assessment in Chinese arts education: Perspectives of Hong Kong students. Research Studies in Music Education, 32(1), 75-92. [11] Padget, S. (Ed.). (2012). Creativity and critical thinking. Routledge. P \| 8 References [1] Baş, M. T., Özpulat, F., Molu, B., & Dönmez, H. (2022). The effect of decorative arts course on nursing students' creativity and critical thinking dispositions. Nurse Education Today, 119, 105584. [2] Black, J., & Browning, K. (2011). Creativity in digital art education teaching practices. Art Education, 64(5), 19-34. [3] Combs, L. B., Cennamo, K. S., & Newbill, P. L. (2009). Developing critical and creative thinkers: Toward a conceptual model of creative and critical thinking processes. Educational Technology, 3-14. [4] Deasy, R. J. (2002). Critical links: Learning in the arts and student academic and social development. Arts Education Partnership, One Massachusetts Ave., NW, Suite 700, Washington, DC 20001-1431. Web site: http://www. aep-arts. org/.. [5] Freedman, K. (2008). Leading creativity: Responding to policy in art education. International dialogues about visual culture, education and art, 39-47. [6] Ingalls Vanada, D. (2013). Practically creative: The role of design thinking as an improved paradigm for 21st century art education. [7] Leong, S. (2010). Creativity and assessment in Chinese arts education: Perspectives of Hong Kong students. Research Studies in Music Education, 32(1), 75-92. [8] Lampert, N. (2006). Enhancing critical thinking with aesthetic, critical, and creative inquiry. Art Education, 59(5), 46-50. [9] Moga, E., Burger, K., Hetland, L., & Winner, E. (2000). Does studying the arts engender creative thinking? Evidence for near but not far transfer. Journal of Aesthetic Education, 34(3/4), 91-104. [10] Pavlou, V. (2013). Investigating interrelations in visual arts education: Aesthetic enquiry, possibility thinking and creativity. International Journal of Education through Art, 9(1), 71-88. [11] Padget, S. (Ed.). (2012). Creativity and critical thinking. Routledge. [12] Rong, Q., Lian, Q., & Tang, T. (2022). Research on the Influence of AI and VR Technology for Students’ Concentration and Creativity. Frontiers in Psychology, 13, 767689. [2] Black, J., & Browning, K. (2011). Creativity in digital art education teaching practices. Art Education, 64(5), 19-34. [3] Combs, L. B., Cennamo, K. S., & Newbill, P. L. (2009). Developing critical and creative thinkers: Toward a conceptual model of creative and critical thinking processes. Educational Technology, 3-14. [4] Deasy, R. J. (2002). Critical links: Learning in the arts and student academic and social development. Arts Education Partnership, One Massachusetts Ave., NW, Suite 700, Washington, DC 20001-1431. Web site: http://www. aep-arts. org/.. [5] Freedman, K. (2008). Leading creativity: Responding to policy in art education. International dialogues about visual culture, education and art, 39-47. [6] Ingalls Vanada, D. (2013). Practically creative: The role of design thinking as an improved paradigm for 21st century art education. [7] Leong, S. (2010). Creativity and assessment in Chinese arts education: Perspectives of Hong Kong students. Research Studies in Music Education, 32(1), 75-92. [8] Lampert, N. (2006). Enhancing critical thinking with aesthetic, critical, and creative inquiry. Art Education, 59(5), 46-50. [13] Santn, M. F., & Torruella, M. F. (2017). Reggio Emilia: An essential tool to develop critical thinking in early childhood. Journal of New Approaches in Educational Research (NAER Journal), 6(1), 50-56. f pp ( ) ( ) [14] Ulger, K. (2018). The effect of problem-based learning on the creative thinking and critical thinking disposition of students in visual arts education. Interdisciplinary Journal of Problem-Based Learning, 12(1). p y f g [15] Ulger, K. (2019). Comparing the effects of art education and science education on creative thinking in high school students. Arts Education Policy Review, 120(2), 57-79. y [16] Uzunöz, F. S., & Demirhan, G. (2017). The effect of creative drama on critical thinking in preservice physical education teachers. Thinking Skills and Creativity, 24, 164-174. [13] Santn, M. F., & Torruella, M. F. (2017). Reggio Emilia: An essential tool to develop critical thinking in early childhood. Journal of New Approaches in Educational Research (NAER Journal), 6(1), 50-56. [14] Ulger, K. (2018). The effect of problem-based learning on the creative thinking and critical thinking disposition of students in visual arts education. Interdisciplinary Journal of Problem-Based Learning, 12(1). [15] Ulger, K. (2019). Comparing the effects of art education and science education on creative thinking in high school students. Arts Education Policy Review, 120(2), 57-79. [16] Uzunöz, F. S., & Demirhan, G. (2017). The effect of creative drama on critical thinking in preservice physical education teachers. Thinking Skills and Creativity, 24, 164-174. of e pp oac es i ducatio al esea c ( Jou al), 6( ), 50 56. [14] Ulger, K. (2018). The effect of problem-based learning on the creative thinking and critical thinking disposition of students in visual arts education. Interdisciplinary Journal of Problem-Based Learning, 12(1). [15] Ulger, K. (2019). Comparing the effects of art education and science education on creative thinking in high school References [2] Black, J., & Browning, K. (2011). Creativity in digital art education teaching practices. Art Education, 64(5), 19 34. [3] Combs, L. B., Cennamo, K. S., & Newbill, P. L. (2009). Developing critical and creative thinkers: Toward a conceptual model of creative and critical thinking processes. Educational Technology, 3-14. [2] Black, J., & Browning, K. (2011). Creativity in digital art education teaching practices. Art Education, 64(5), 19 34. [3] Combs, L. B., Cennamo, K. S., & Newbill, P. L. (2009). Developing critical and creative thinkers: Toward a conceptual model of creative and critical thinking processes. Educational Technology, 3-14. g p gy [4] Deasy, R. J. (2002). Critical links: Learning in the arts and student academic and social development. Arts Education Partnership, One Massachusetts Ave., NW, Suite 700, Washington, DC 20001-1431. Web site: http://www. aep-arts. org/.. g p gy [4] Deasy, R. J. (2002). Critical links: Learning in the arts and student academic and social development. Arts Education Partnership, One Massachusetts Ave., NW, Suite 700, Washington, DC 20001-1431. Web site: http://www. aep-arts. org/.. [5] Freedman, K. (2008). Leading creativity: Responding to policy in art education. International dialogues about visual culture, education and art, 39-47. [5] Freedman, K. (2008). Leading creativity: Responding to policy in art education. International dialogues about visual culture, education and art, 39-47. [6] Ingalls Vanada, D. (2013). Practically creative: The role of design thinking as an improved paradigm for 21st century art education. [7] Leong, S. (2010). Creativity and assessment in Chinese arts education: Perspectives of Hong Kong students. Research Studies in Music Education, 32(1), 75-92. [9] Moga, E., Burger, K., Hetland, L., & Winner, E. (2000). Does studying the arts engender creative thinking? Evidence for near but not far transfer. Journal of Aesthetic Education, 34(3/4), 91-104. [10] Pavlou, V. (2013). Investigating interrelations in visual arts education: Aesthetic enquiry, possibility thinking and creativity. International Journal of Education through Art, 9(1), 71-88. [11] Padget, S. (Ed.). (2012). Creativity and critical thinking. Routledge. [11] Padget, S. (Ed.). (2012). Creativity and critical thinking. Routledge. [12] Rong, Q., Lian, Q., & Tang, T. (2022). Research on the Influence of AI and VR Technology for Students’ Concentration and Creativity. Frontiers in Psychology, 13, 767689. Page \| 8 Page \| 8 Page \| 8 IJAH 1(1): 00-00 Page \| 9
https://openalex.org/W2888195290	https://www.bio-conferences.org/10.1051/bioconf/20181100022/pdf	English	null	Anatomical structure of leaves, stems and roots of hemiparasitic plants Thesium ebracteatum Hayne (Santalacea R. Br.) from the Urals	Bio web of conferences/BIO web of conferences	2,018	cc-by	1,825	1 Introduction Detailed anatomical studies of Thesium ebracteatum Hayne plants from the Urals were not carried out. This species is known from sites in central Europe, European part of Russia, Western Siberia and in the Urals. It is a native herb of forest lawns and thickets of bushes. It is perennial hemiparasitic herb with a subterranean rhizome and numerous storage lateral root with haustorial connections. The aerial shoots annual, erect or ascending at the top, 10- 30 cm tall, usually unbranched, glabrous, rarely leafy. At the top of the shoots presents bracts devoid of flowers; leaves linear, glabrous. Inflorescence is a simple, unbranched brush; flower on short stalk with one bract, bracts like the leaves but decreasing in size, perianth tubular, upward strongly wrapped. Capsule on the stalk is elliptical, with longitudinal branching veins, three times larger than the remaining part of the perianth. The paper presents the studies of anatomical structure of the leaf, stem and roots of Thesium ebracteatum plants from the Urals populations. 2 Materials and Methods The plants were collected from the mountainous areas of Middle Ural (Sverdlovsk region) in two populations nearby Nizhnyaya Irga, Ust'-Bayak. Specimens for morphological study were dried according to standard herbarium techniques and stored in the Herbarium of Institute of Plant and Animal Ecology, Ural Branch of the Russian Academy of Sciences (SVER). The materials for anatomical study were preserved in 70% alcohol in the field. Anatomical observations were performed on transverse sections of root and stem (in its top, Anatomical structure of leaves, stems and roots of hemiparasitic plants Thesium ebracteatum Hayne (Santalacea R. Br.) from the Urals Olga Kiseleva1,, and Ludmila Trofimova2 1Russian Academy of Sciences, Ural Branch: Institute Botanic Garden, 620144, Ekaterinburg, Russia 2 Ural Federal University, Ekaterinburg, 620000, Russia Abstract. The paper presents the results of structure researches of Thesium ebracteatum Hayne. plants from the Ural populations. The anatomic description includes the characteristic of leaves and roots, the stems structure in its top, middle and lower part. Some stomatografic parameters of leaves are calculated. This is the fist anatomical report on the important diagnostics signs of Thesium ebracteatum Hayne plants. BIO Web of Conferences 11, 00022 (2018) Prospects of Development and Challenges of Modern Botany BIO Web of Conferences 11, 00022 (2018) Prospects of Development and Challenges of Modern Botany https://doi.org/10.1051/bioconf/20181100022 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 3 Results and Discussion Leaf features of the species are shown in Figure 1. In surface preparations, the epidermal cells are covered by a thick cuticular layer. The leaf is amphystomatic with oval paracytic stomata. Epidermal cell walls are not undulate. The cells of the epidermis are isodiametric and polygonal with straight walls except in the coastal regions where they are narrow, elongated parallel to the veins. Upper epidermal cells have the same size as the lower ones; trichomes are absent, leaf margin with sharp papillae. Stomata orientation was random. Guard cells are elliptical, pores are elongated. Average size of guard cells in microns is 31 in lengths (varies from 16 to 38), 23 in widths (varies from 14, to 35). SI adaxial epidermis 12,8 and abaxial epidermis 11,3. The presens of stomata in adaxial and abaxial epidermis in equal parts may be the adaptive advantages to hemiparasitic nutrition. Lamina is dorsiventral. Mesophyll consists of 1 layer of elongated palisade cells and 2 or 3 layers of isodiametric, spongy parenchymatic cells have small intercellular cavities. Fig. 1. Anatomical features of Thesium ebracteatum leaves. А Lower epidermis Х400, Б Surface section of leaf Х80. 1 – epidermis cell; 2 – stoma; 3 – Midrib; 4 – vascular bundle; 5 – leaf margin with sharp papillae. Fig. 1. Anatomical features of Thesium ebracteatum leaves. А Lower epidermis Х400, Б Surface section of leaf Х80. 1 – epidermis cell; 2 – stoma; 3 – Midrib; 4 – vascular bundle; 5 – leaf margin with sharp papillae. A transverse section taken from the top, middle and lower part of the stem was observed (Fig. 2, 3А). The epidermal cells are covered by cuticular layer, trichomes are absent. Stem cortex consists of 2-5 layers of usually oval cells. Some cortex cells contain secretes at the top and middle part of the stem. The sclerenchimatic sheath is upper the vascular bundles. Cambium is distinguishable. Secondary thickening develops from a conventional cambial ring. In the lower part of the stem secondary thickening tissues predominate. Vascular bundls consists of secondary phloem and xylem. Xylem has vessels. Vessel end-walls are simple. Vessels have not vestured pits. Pith cells are large and spherical. Cavity arises in the middle and lower part of the stem. The subterranean rhizome structure is similar to those of the stem. A transverse section taken from the lateral roots was observed (Fig. 3Б). Corresponding author: kiselevaolga@inbox.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/bioconf/20181100022 BIO Web of Conferences 11, 00022 (2018) Prospects of Development and Challenges of Modern Botany average and lower part) cut by rotary microtome Microm HM (Carl Zeiss, Germany), and surface sections of leaves cut by hand. All slides were observed by light microscopes Micros (Austria) and Zeiss Axioscopus (Carl Zeiss, Germany). Well-staining sections were photographed. Stomatal index (SI) has been calculated according to Salisbury (1927) [1]. All measurements and observations were made ten times on different slides. All microscopic measurements were made with the aid of an occular micrometer. 3 Results and Discussion The features of the primary structure are retained. The typical primary root is bounded by an exodermis. The centre of the root made up entirely of many-layered cortex, what have cavities and cells with starch. The 2 https://doi.org/10.1051/bioconf/20181100022 BIO Web of Conferences 11, 00022 (2018) Prospects of Development and Challenges of Modern Botany inner side is an endodermis. Next follows the pericycle, and then the vascular system is in the middle. Vessel wall pittings and thickenings are similar to those of the stem. inner side is an endodermis. Next follows the pericycle, and then the vascular syste the middle. Vessel wall pittings and thickenings are similar to those of the stem. inner side is an endodermis. Next follows the pericycle, and then the vascular system is in the middle. Vessel wall pittings and thickenings are similar to those of the stem. Fig. 2 Thesium ebracteatum transverse section of stem A Top part Х200 1 – epidermis; 2 – cortex; 3 – vascular bundle; 4 – sclerenchyma; 5 – phloem; 6 – xylem; 7 – cambium; 8 – pith; Б Middle part Х100. 1 – epidermis; 2 – cortex; 3 – vascular bundle; 3а – phloem; 3b – xylem; 4 – sclerenchyma; 5 – pith; 6 - cavity. Fig. 2 Thesium ebracteatum transverse section of stem A Top part Х200 1 – epidermis; 2 – cortex; 3 – vascular bundle; 4 – sclerenchyma; 5 – phloem; 6 – xylem; 7 – cambium; 8 – pith; Б Middle part Х100. 1 – epidermis; 2 – cortex; 3 – vascular bundle; 3а – phloem; 3b – xylem; 4 – sclerenchyma; 5 – pith; 6 - cavity Fig. 3. Thesium ebracteatum transverse section of A Stem in the lower part Х400. 1 – sclerenchyma; 2 – phloem; 3 – cambium; 4а – secondary xylem; 4б – primary xylem; 5 – starch grains. Б Lateral root Х400. 1 – exodermis; 2 – cortex with starch grains; 3 – phloem and pericycle; 4 – xylem. Fig. 3. Thesium ebracteatum transverse section of A Stem in the lower part Х400. 1 – sclerenchyma; 2 – phloem; 3 – cambium; 4а – secondary xylem; 4б – primary xylem; 5 – starch grains. Б Lateral root Х400. 1 – exodermis; 2 – cortex with starch grains; 3 – phloem and pericycle; 4 – xylem. 4 Discussion Thesium L. with ca. 330 species counts the highest number of species of any genus in Santalales [2]. The Santalaceae family (Santalaceae R.Br.) is represented in Russia by 12 species, 3 species are found in the Urals. All of them are hemiparasitic herbs from the genus Thesium L. Several studies demonstrate geographical distribution of Thesium species [3-4], biochemical features [5], physiology of host plant-parasitic plant interaction in comparison with others parasitic plants [6], host range and selectivity [7], active ingredients of medicinal material of Thesium [8]. This reflects the general trends in the study of the biology of hemiparasitic plants. The analysis of functional features of adaptation to parasitism in plants is considered as importance [9, 11, 14]. The anatomical studies of Thesium species in the Urals have never been done before. We performed analysis of available data on the structure of root-hemiparasitic Scrophs such as Rhinanthus, Melampyrum, Odontites species and others [10-14] in comparison with Thesium ebracteatum. Root-hemiparasitic Scrophs have leaves with hydathodes and trichomes [11- 13], stems and roots with ring cambium [11, 14] that produce additional xylem tissue 3 BIO Web of Conferences 11, 00022 (2018) https://doi.org/10.1051/bioconf/20181100022 Prospects of Development and Challenges of Modern Botany embedded lignified xylem parenchyma and deficiently secondary phloem [10]. All roots hemiparasitic plants had haustorial connections, but we noticed that hemiparasitic Scrophs had significant differences from Thesium in leaves, roots and stems structure. It is confirmed that not only the parasitic way of life affects the nature of the vegetative organs adaptations. An anatomical structure of leaves, roots and stems reflects primarily belonging to the taxonomic group of hemiparasitic plants. This study was supported by the Russian Foundation for Basic Research (project no. АААА-А17- 117072810011-1). This study was supported by the Russian Foundation for Basic Research (project no. АААА-А17- 117072810011-1). References 1. E.J. Salisbury, Philos. Trans. R. Soc. London B, 1-65 (1927) 2. D.L. Nickrent, V. Malécot, R. Vidal-Russell, J. Der, Taxon 59, 538-558 (2010) 3. A. Romo, Y. Didukh, A. Boratyński, Ann. Bot. Fennici 41, 273-281 (2004) 4. K.S. Tojibaev, Czech J Genet. and Plant Breeding 46, 45-46 (2010) 5. X. Zhang, B. Liu, Q. Guo, L. Song, L. Chen, C. Wang, Biochem. Syst. and Ecol. 64, 46-52 (2016) 6. A. Fer, N. Russo, P. Simier, M.-C. Arnaud, P. Thalouarn, J Plant Phys. 143, 704-710 (1994) 7. K. Suetsugu, A. Kawakita, M. Kato, Ann. Bot. 102, 49-55 (2008) 8. F. Luo, Q. Guo, J Zhongguo Zhong Yao Za Zhi. 36, 2042-6.(2011) 9. J. Těšitel, Plant Ecol. Evol. 149, 5-20 (2016) 10. O.A. Kiseleva, Bull. Perm Univ. Biol. 3, 18-26 (2013) [In Russian] 11. J.M. Hibberd, W.D. Jeschke, J Exp. Bot. 52, 2043-2049 (2001) 12. J.M. Canne–Hilliker, C.M. Kampny, Can. J Bot. 69, 1935-1950 (1991) 12. J.M. Canne–Hilliker, C.M. Kampny, Can. J Bot. 69, 1935-1950 (1991) 13. R.N. Govier, M.D. Nelson, J.S. Pate, New Phytol. 67, 963-972 (1968) 13. R.N. Govier, M.D. Nelson, J.S. Pate, New Phytol. 67, 963-972 (1968) 14. D.D. Cameron, W.E. Seel, New Phytol. 174, 412-419 (2007) 14. D.D. Cameron, W.E. Seel, New Phytol. 174, 412-419 (2007) 4
https://openalex.org/W2743471817	https://enggheritage.com/download/13720/	English	null	Comparison Method Of Odour Impact Evaluation Using Calpuff Dispersion Modelling And On-Site Odour Monitoring	Engineering heritage journal	2,017	cc-by	6,780	1. Introduction assessing the odour concentration and set the regulation. Despite that, there are already fundamental method in sampling method and measuring the odour concentration at the source and in Malaysia MS 1963:2007 is the standard referred when running the odour sampling. Before assess the odour nuisance at the receptor, it is important in knowing the odour concentration released at the source. Odorous gases are particular kind of air pollutants. It is one of the major environmental problems that several industrial categories have to face. Odour pollution is widely recognised as the offensive air pollution due to human activity usually in populated areas. In the last 30 years, odour nuisances from different industrial sources have become a serious environmental concern [2]. By definition, odour is the aggregate of a mixture of gases on the sense of smell. Its strength is determined by using odour detection threshold (ODT). The ODT is the number of dilutions with odour free air required for an odour to be just detected by 50% of the odour panel or until the least definitely perceptible odour is achieved [3]. Nowadays, there are various methods have been developed in assessing the odour concentration. The right method need to be chosen according to the suitability of the odour assessment situation since the odour assessment depends on the measurements and usually are based on human sensitivity. According to Capelli et al., the odour characterization techniques can be categories into three types which are analytical method, sensorial method and senso-instrumental method [4]. Analytical techniques are the method where the sample will be separate, identified and quantified by using specific instrument. Method that usually being used is gas chromatography-mass spectrometry (GCMS). This method will give the concentration of the substance rather than give the odour concentration unit and it is suitable in measuring the substance that become surrogate to the odour such as hydrogen sulphide, H2S and ammonia, NH3. The results are different if sensorial and senso-instrumental method is use. Sensorial method is the method that uses the human nose as a sensor to characterize the odour by using the panel’s sensitivity on the odour sample. Olfactometer is the equipment that usually being used in running the odour analysis and the results will be in odour concentration unit [9]. Last but not least is the senso- instrumental method. 1. Introduction This third method is the method that used artificial noses that perform similar just like the function of the human olfaction system [10]. Those techniques are the method that usually used in odour sampling and all the methods have its own ways or calculation in measuring the odour concentration and estimating the odour emission rate. Offensive odours came in various kinds of sources such as fish mills, sewage treatment, petrochemical and solvent plants are common sources of odorous emissions[4]. The unpleasant smell that came from those facilities may create a poor perception from local community towards the facility, will reduce the worker efficiency and can lead to various kinds of disease. Odours that result directly or indirectly from human activities and that cause an adverse effect are often classified as contaminants and are subject to regulation[5] and to do so, proper monitoring and regulatory tools are required to minimize the impacts of odorous emissions on human populations [6]. Usually, odour concentration was expresses in OU/m3, and particularly, 1OU/m3 means the 1OU of odour in 1m3 of volume.OU/m3 represent the number of dilutions with air that needed to the odour can be detected at its detection threshold [4]. Threshold is a concentration of odour that can be detected by human nose. Olfactometer is the equipment used in measuring odour concentration and it used human odour detection threshold in knowing the value of the amount of odorants present in 1m3 of odorous gas under the standard condition [7]. Mostly the odour can be detected at 2 to 5 OU/m3[8]. However, the GCMS does not offer any practical information about the perception of the odorants, which is subjective and not generally related to their concentrations in a clear way [11]. For this reason, odors are commonly measured with a second method and third method but sensorial method is preferable at the source measurement since that method can give the emission value instead of concentration only and the absent of a variable odor background that may strongly According to Ranzato et al., assessing the odour pollution is a complex task because of first, it is hard to detect the odourants since there are a lot of numbers of odorant in the ambient air. Second, time and meteorological conditions usually the factors that change the odour intensity and third, odour perception is largely subjective [6]. Engineering Heritage Journal / Galeri Warisan Kejuruteraan 1(1) (2017) 01-05 Contents List available at RAZI Publishing Engineering Heritage Journal /Galeri Warisan Kejuruteraan Journal Homepage: http://www.razipublishing.com/journals/galeri-warisan-kejuruteraan-gwk/ Comparison Method Of Odour Impact Evaluation Using Calpuff Dispersion Modelling And On-Site Odour Monitoring Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup 1,2School of Civil Engineering, Universiti Sains Malaysia, 14300 Nibong Tebal, Pulau Pinang, Malaysia 2Solid Waste Management Cluster, Engineering Campus, Universiti Sains Malaysia 3Environmental Technology, School of Industrial Technology, Universiti Sains Malaysia, 11800Pulau Pinang, Malaysia ashikinyaacof88@gmail.com, bcenastaein@usm.my, cyusriyp@gmail.com ISSN: 2521-0904 (Print) ISSN: 2521-0440 (Online) https://doi.org/10.26480/gwk.01.2017.01.05 This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Engineering Heritage Journal / Galeri Warisan Kejuruteraan 1(1) (2017) 01-05 Contents List available at RAZI Publishing Engineering Heritage Journal /Galeri Warisan Kejuruteraan Journal Homepage: http://www.razipublishing.com/journals/galeri-warisan-kejuruteraan-gwk/ Comparison Method Of Odour Impact Evaluation Using Calpuff Dispersion Modelling And On-Site Odour Monitoring Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup 1,2School of Civil Engineering, Universiti Sains Malaysia, 14300 Nibong Tebal, Pulau Pinang, Malaysia 2Solid Waste Management Cluster, Engineering Campus, Universiti Sains Malaysia 3Environmental Technology, School of Industrial Technology, Universiti Sains Malaysia, 11800Pulau Pinang, Malaysia ashikinyaacof88@gmail.com, bcenastaein@usm.my, cyusriyp@gmail.com ISSN: 2521-0904 (Print) ISSN: 2521-0440 (Online) https://doi.org/10.26480/gwk.01.2017.01.05 This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited i M Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup 1,2School of Civil Engineering, Universiti Sains Malaysia, 14300 Nibong Tebal, Pulau Pinang, Malaysia 2Solid Waste Management Cluster, Engineering Campus, Universiti Sains Malaysia 3Environmental Technology, School of Industrial Technology, Universiti Sains Malaysia, 11800Pulau Pinang, Malaysia ashikinyaacof88@gmail.com, bcenastaein@usm.my, cyusriyp@gmail.com 2Solid Waste Management Cluster, Engineering Campus, Universiti Sains Malaysia 3Environmental Technology, School of Industrial Technology, Universiti Sains Malaysia, 11800Pulau Pinang, Malaysia ashikinyaacof88@gmail.com, bcenastaein@usm.my, cyusriyp@gmail.com This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Environmental Technology, School of Industrial Technology, Universiti Sains Malaysia, 11800Pulau Pinang, Malaysia ashikinyaacof88@gmail.com, bcenastaein@usm.my, cyusriyp@gmail.com This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited shikinyaacof88@gmail.com, cenastaein@usm.my, yusriyp@gmail.com This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Keywords: 1. Introduction acof, Nastaein Qamaruzzaman, Yusri Yusup. Comparison method of Odour Impact Evaluation Using CALPUFF Dispersion Modeling and on-site Odour Monitoring. Galeri War. Kej. 1(1) (2017) : 01–05 Cite this article as: Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup. Comparison method of Odour Impact Evaluation Using CALPUFF Dispersion Modeling and on-site Odour Monitori Cite this article as: Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup. Comparison method of Odour Impact Evaluation Using CALPUFF Dispersion Modeling and on-site Odour Monitoring. Galeri War. Kej. 1(1) (2017) : 01–05 A R T I C L E D E T A I L S Article history: Received 12 September 2016 Accepted 20 December 2016 Available online 10 January 2017 Keywords: Odour concentration, palm oil mill, olfactometry, calpuff, dispersion modeling Until now, the suitable types of odour assessment technology in Malaysia still cannot be found and at the same time, there is no odour regulation concerning the specific odour parameter stated in the Environmental Quality Act 1974. The assessment of odour pollution is still regarded as a difficult task but the development of odour pollution assessment and proper regulatory tools are urgently needed. This paper aim to compare two methods for determining odour concentration which are field inspection and atmospheric dispersion model (CALPUFF). This research goal was to compare both method estimation of odour in quantitatively and qualitatively. The comparison will highlight the strength and weakness of both methods. CALPUFF is an easy method in knowing the whole picture of the dispersion but it is still new in Malaysia, while field inspection takes into account the role of human perception but it need a lot of work to obtain the whole picture of the dispersion. Palm oil mill was chosen as the study site since palm oil mill effluent (POME) release high contribution in emitted unpleasant odour [1]. 18 possible receptors points were chosen which include residential area, institution and also public area. It was found that, both methods show that the odour concentration is higher at the receptor near the source but different in odour concentration values. Both methods have their own advantage and disadvantages and it can be conclude that, both of it provide reasonable estimation of odour nuisance but it also depends on the odour case situation such as the availability of data and the number of odour source where sometimes one of the method is the proper method to be use at that time. 2. Material and Method There are 18 receptors that already being identified affected by the offensive smell from the mill. Those receptors were identified by running the survey at the residential areas around the source. Fig. 2 shows the image of the 18 external point’s location around 4 km from the mill. For the field inspection was run in three session, morning (9 a.m to 12 p.m), evening (3 p.m to 6 p.m) and night time (12 a.m to 3 a.m). The data was taken in different hour at each receptors point to take different meteorological condition into account. 2.1 Case Study Description and Identification of the Odour Source 2.1 Case Study Description and Identification of the Odour Source A palm oil mill that located in Nibong Tebal, Pulau Pinang was chosen as the study site in this research. The study was focused on the POME treatment pond. Since the treatment pond used anaerobic process, that area is the highest area in releasing the odour gases such as NH3 and H2S [14]. Fig. 1 shows the location of the mill at the coordinate of 5° 9'13.63"N and 100°30'27.90"E. The maximum capacity of the mill is 720 metric tons/day and the average quantity of fruit to be processed is 540 metric tons/day. This POME treatment uses anaerobic digestion pond as their wastewater treatment method. CALPUFF dispersion modeling and field inspection are the method used to quantify the odor impact due to the operation of the mill. These methods were compared by run the process to the same area for three alternate days in assessing the dispersion of odour from the mill. Fig. 2 External points map layout by 1 km at each radius circle (Google Earth) Fig. 1 Palm oil mill at Nibong Tebal and its location of the effluent treatment area (Google Earth) Fig. 2 External points map layout by 1 km at each radius circle (Google Earth) In-field olfactometer was used in measuring the ambient odour concentration especially at the receptors. It is easy way in obtain the odour concentration immediately without combine with the other equipments and without being analyse in the laboratory. In-field olfactometers are portable devices with a source of clean filtered air and a dilution system based on several calibrated orifices. Scentroid SM100 field olfactometer was used in this research. Field Inspection The field inspection procedure was carried out by using portable olfactometer, SM100, by five qualified odour assessors that selected by using dynamic olfactometry procedure (MS 1963:2007, EN 13725:2003). The same panel that past in n-butanol odour sensitivity was used in identified the odour at the receptors and odour at the source. Since palm oil mill had its own special smell that can be characterize, the odour panels was trained to recognize its odour at the source and then taken to the outdoor to recognize the odour from the mill at the receptors. They need to remember the smell from the mill and had to be successful in recognizing the smell to be appointed as the odour panel in this study. All the odour characters need to be recorded during the monitoring including the background odour such as grass and dirt. The field inspection in this study is based on a number of visits at the receptors by trained odour assessors to record and identify the perceived odours. In field olfactometer was used to analyse the odour emission at the possible receptors.The goal of this work is to compare the odour impact assessed by these two new methods in Malaysia, the odour frequency by using each method and to discuss strengths and weaknesses of the two techniques emerging from the comparison. To carry out a full comparison, the odor impact predicted by the model was compared to the field inspection both qualitatively and quantitatively. 2.3 Atmospheric Dispersion Modeling 2.3 1. Introduction Because of these reasons, it is hard to identify the right and suitable method in 2 Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup/ Galeri Warisan Kejuruteraan 1(1) (2017) 01–05 taein Qamaruzzaman, Yusri Yusup/ Galeri Warisan Kejuruteraan 1(1) (2017) 01–0 Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup/ Galeri Warisan Kejuruter affect the panel response (EN 13725, 2003). Senso-instrumental or field inspection method is perfect in measuring odour at the receptors. Not only that, odour at the receptors can also be determined by using mathematical dispersion model. equation was used. It is important as the input to the air dispersion modeling: Where E is the specific odour emission rate (OUE/sm2), C is the concentration of odour (OUE/m3) that derive from olfactometry assessment, f is the volume of carrier gas per unit time (m3/s) which is 1.167E-4 m3/s and A is the cross sectional area of flux chamber (m2), 0.155 m2. According to Hudson et al., this equation is assumed that there is no background odour from the sweep air and there is complete mixing between the air flow and odorous air [15]. CALPUFF is the modeling software used in this study. CALPUFF is one of the long rang transport (LRT) dispersion modeling and it is an advanced non steady state meteorological and air quality modeling system that using the Gaussian puff model as the basic [12]. CALPUFF is being developed by Earth Tech, Inc. [15] and been recommended by the US Environmental Protection Agency (EPA) as a guideline model for LRT and on a case by case basis for near field application in 2003 [13]CALPUFF was usually being selected because it could be run easily for single sources under multiple parameters to evaluate model sensitivity [12]. 2. Material and Method The SM100 olfactometer (Scentroid, Canada) draws a sample of ambient air via venture pump and dilutes it using fresh odourless air from a compressed air tank. Fig. 1 Palm oil mill at Nibong Tebal and its location of the effluent treatment area (Google Earth) The first step for assessing the odor impact was to identify the odour concentration at the sources. There are four different treatment ponds were chosen to do the odour sampling, those are acid pond, anaerobic pond, facultative pond and also aeration pond. The odour sampling was doneduring morning (9 a.m. to 12 p.m.) and evening (3 p.m. to 6 p.m.) in three alternate days and the average was taken from it to get only one odour concentration value. Odour sampling was done by using flux hood method with vacuum chamber as the media to collect the gases. Then, the gas sample was keep in the nalophan bag and brought to the laboratory to be analyse by using olfactometer within 24 hours. Odour measurement by using olfactometer and the sampling method was based on the dynamic olfactometry procedure MS 1963:2007. Unlike other infield odour measuring devices (e.g. Nasal Ranger, St. Croix Sensory) SM100 does not rely on filters to clean ambient air for dilution. Rather, fresh odourless air is supplied by a portable light weight high pressure air tank. During the measurement, as in the laboratory dynamic olfactometry, user increase the value of detection threshold (D/T) until an individual threshold of the odour is achieved. The measurement principle was according to international standard, ASTM E679-04 (2011) and EN 13725 (2003). 3.1 Field Inspection Odour Concentration at the Receptors Field Inspection Odour Concentration at the Receptors Field inspection was run around 4 km radius from the mill at the possible affected receptors. The odour frequency from the assessment by using in-field olfactometry can be seen in Fig. 3 and Fig. 4. Each receptor recorded nine odour observations and from the results, not all the odour data was came from the mill. Only 13% of the data that really came from the mill and the others were came from background odour such as grass, smoke from open burning or from the factory stack, exhaust from vehicle and also drainage. The highest odour frequency from the mill can be detected at receptors EXT1 and EXT3 with 44% and with the maximum distance less than 1.5 km from the source. The field inspection indicated zero odour frequencies in some locations such as at EXT9 and EXT10. This situation happen because of the time during the inspection did not match the time when the odours pass through the observation point. Odour spread with the wind and wind is one of the factors that affect the gas concentration and its dispersion. The gases that disperse will expend in volume and it will incorporate dilution air from around it, thus, the gas concentration will reduce due to its dispersion. These phenomena will reduce the concentration of the gases once it is arrives at the receptor. Besides that, the gases puffs are experience random movements due to the turbulence and will deflect away from the mean direction. When it happen, even though the receptor is just near the source, but if the wind direction change, the receptor will not affected by the smell. From the contour, it is clearly shows that the higher odour emission can be detected almost 5 km radius in all direction, compare during day time where 5 km distance at only North-East direction. Wind speed and wind direction are the parameters that affected the gas concentration and its dispersion [17]. The gases that disperse far from the source will expend in volume and it will incorporate dilution air from around it, thus, the gas concentration will reduce due to its dispersion [18]. These phenomena will reduce the concentration of the gases once it is arrives at the receptor. 2.3 To calculate the odour emission, the specific odour emission rate 𝐸= 𝐶𝑓 𝐴 In this study, CALPUFF dispersion model was used to estimate odor emissions and predict odor concentrations from industrial complex area sources.Regulatory models such as CALPUFF are commonly employed to simulate the atmospheric dispersion of odorant compounds. There are three main components in CALPUFF which are CALMET, To calculate the astaein Qamaruzzaman, Yusri Yusup. Comparison method of Odour Impact Evaluation Using CALPUFF Dispersion Modeling and on-site Odour Monitoring. Galeri War. Kej. 1(1) (2017) : 01–05 3 Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup/ Galeri Warisan Kejuruteraan 1(1) (2017) 01–05 taein Qamaruzzaman, Yusri Yusup/ Galeri Warisan Kejuruteraan 1(1) (2017) 01–0 CALPUFF, and CALPOST. CALMET is a meteorological model that develops hourly wind and temperature fields on a 3D gridded modeling domain. CALPUFF is a transport and dispersion model that describes “puff” of material emitted from modeled sources, simulating dispersion and transformation processes along the way. CALPOST is used to process the files from CALPUFF, processing a summary of the simulation results in tabulated forms. CALPUFF was chosen to be use in this study because the final results it give is more accurate compare to the other dispersion modeling such as AERMOD and AUSPLUME [16]. Fig. 4 Odour frequencies from the POME at the 18 possible receptors around the mill 0 50EXT1EXT3EXT5EXT7EXT9EXT11EXT13EXT15EXT17 Frequecy of odour from mill (%) Receptors Frequencies of the odour from mill by receptors Frequencies of the odour from mill by receptors In this research, CALPUFF was configures for an analysis of terrain and land use data is at 50 km by 50 km study domain with the centered point is at the palm oil mill. The study was run for 24 hours at each three sampling days. Meteorological input prepared for CALPUFF is by using MM5 data with domain size 100 km by 100 km and 4 km resolution from the Lakes Environmental Software. Predictions of odour concentrations were generated from the contributions of odour from the four main treatment ponds. The model was run in no-obs mode since the MM5 data was fully used without backup meteorological data. The number of grid spacing is 200 km by 200 km with the grid spacing is 0.25 km. Fig. 4 Odour frequencies from the POME at the 18 possible receptors around the mill CALPUFF Atmospheric Odour Prediction 3.2 Fig. 5 shows the results of the odour dispersion for day one and three at each monitoring time. The model calculates hourly mean odour concentration. Most of the odour concentrations during day time were starts from scale 1 OU/m3 to 1E-7 OU/m3 and from the prediction, only night time the contour start with 10 OU/m3. One important consideration concerns are the shape of the odour dispersion during three monitoring time which turned out to have different orientations with same MM5 data used. This is due mainly to wind factors and some of it from geographical data. Same MM5 data does not mean same wind speed and direction being applied but all of it is depends on the date and time.Another interesting consideration derives from the comparison of the dispersion modeling results at each monitoring time is the results of the olfactometric analyses. A small different of the results from the olfactometry analyses can change drastically the odour emission at the impact area, but, it also still depends on the wind direction and its speed. 2.4 To compare these two odour assessment method, the odour level evaluation was performed by compare graphically the trend of the odour concentration at morning, evening and night time for those three days inspection. Then the comparison of the odour concentration values was examined in the field inspection versus the values estimated by CALPUFF. Besides that, this research will also computed the CALPUFF model performance and field inspection, which one is suitable to be use in Malaysia, how well CALPUFF produce the results and the relationship between the odour at the receptors with these two methods. Both method was relies on the human odour threshold acceptable level between 2 OU/m3 to 5 OU/m3 [8]. The influence of wind speed and its direction can obviously be seen in Fig 5. There are huge different during day and night time prediction. During morning and evening session, the movements of the odour particles are stretched mostly to North-East direction when most of the wind blowing on that direction at that time. Besides that, results also shows that the odorous plume was less smooth in the evening compare to morning session. This situation happens because of the wind stability factor and its speed. From the contours, the wind during morning session is more stable than evening which makes morning contour are smooth compare to the evening contour. The clear different can be seen during day and night prediction. Fig. 6 The predicted and observed odour concentration values by time for point EXT2 and EXT5 b) Fig. 6 The predicted and observed odour concentration values by time for point EXT2 and EXT5 b) b) b) b) The odour concentration values assessed by CALPUFF and field inspection in the single measurement points were showing no similarity in odour concentration values but same in the trend of the increasing and decreasing of the concentration values. From the qualitative point of view, Fig 6 shows that the trend of both predicted and observed odour concentrations mostly are the same. Both techniques were in good agreement based on the shape of the graph, where when the odour concentration for observed is increase, the prediction value is also increase. c) c) c) c) But there are also some values that did not match. On day 2 at time 56 the observed value was over predicted. Different in pattern may be the results of the limitation of field inspection since samples from the study area were analyse by human assessor as human judgment are sometimes different and not consistent. Besides that, the field inspection is limited in number of assessment for continuous period of time where the exact pattern of the odour emission cannot be determined. As Ranzato et al. said, the model simulated odours during all period of time of the domain area which make the simulation of the odour can be seen more clearly [6]. a= morning, b= evening, c= night Fig. 5 Day 1 and 3 of the odour dispersion during morning, evening and night time by using CALPUFF Atmospheric dispersion is much more advance and being suggested nowadays because it can read the odour level even at the lowest odour episode compare with the field inspection where when odour levels are lower and odor episodes are rarer it cannot easily distinguished by human assessors. Comparison between Field Inspection and CALPUFF Prediction Comparison between Field Inspection and CALPUFF Predictio 3.3 In this work, two techniques for estimating the odour nuisance was compared in terms of frequency of odor hours, caused by POME wastewater treatment plant. Those methods are field inspection, which is the analysis of reports from on-site human assessors, and the atmospheric dispersion model, CALPUFF. Their estimates of odor nuisance were generally similar but displayed some key differences. Indeed the scope of this paper was not to highlight a perfect match between the techniques, but rather to discuss differences, strengths and weaknesses emerging from the comparison. Fig. 6 The predicted and observed odour concentration values by time for point EXT2 and EXT5 From the qualitative point of view, the two techniques were in good agreement but not in quantitative point where it is hard to get the same values from both of the methods. Differences in shape could be described the limits of the field inspection methodology, since sampling in the field involved a limited number of points for discontinuous periods of time. Instead, the model simulated odors during all time steps in all the points of the spatial domain, thus it should bring out more clearly the pattern and characteristics of the plume such as its stretched and relatively-irregular shape. CALPUFF provided lower nuisance estimates than field inspection in the points where odour frequencies estimated by the field inspection were highest, this is because, in general, the model does not underestimate high frequencies, but has a reduced accuracy close to emission sources [6]. Moreover, in general it would expected that field inspection to overestimate odours rather than underestimating them, because an assessor can fail to recognize an odor episode, while it seems less likely that an assessor smells an odor which is not present. y p However, to get a good view and results from CALPUFF, it requires a great number of inputs such as detailed meteorological and geophysical data to avoid uncertainty in the simulations. Besides that, it also needs realistic emission rates value, which are sometimes difficult to estimate due to the complexity of odour substances and of emission sources such as the changing of the odour concentration with temperature. On the other hand, field measurements recorded on site should be well representative of the existing conditions. However, for the prediction, field inspection is not recommended to be used since it will be more expensive than the implementation of a model. This is because, it will need a greater number of people involved, with sufficient number of equipments and the long monitoring time required. Besides that, its performance can be influenced by sampling discontinuity in time and space, human error and subjectivity, and the simultaneous presence of more odours in the same place. For both methodologies, the core of the nuisance was centered around the odorous sources, but with different intensity peak values. The field inspection indicated zero odour frequencies in some locations, unlike CALPUFF, and estimated a high frequency of odour episodes for other points. Conclusion From the results, it is suggesting that there is room to improve the precision of the two methods and both of it can be use together to achieve a strong results and strengthen the odour assessment in Malaysia. Despite the differences between CALPUFF and field inspection estimates, their general agreement suggests that both methods provide reasonable estimates of the real odour nuisance, so that their applied use is justified. This work also shows how it is possible to assess odour impact in presence of multiple similar sources by illustrating a case study. Even though field inspection method cannot be used in the prediction purpose, it still can allowed to be use to identify the receptors or area that affected by the odour source in real situation. By using the model application, the field inspection result can be proved right in the means of the presence of the odour. 0 10 20 30 40 50 60 70 -20 0 20 40 60 80 100 120 140 Point 5: Observed and predicted odour concentration Predicted (OU/m3) Observed (OU/m3) Time Observed Predicted -0.5 0.0 0.5 1.0 1.5 2.0 2.5 Fig. 6 The predicted and observed odour concentration values by time for point EXT2 and EXT5 CALPUFF estimated a high frequency of odour episodes for other points as CALPUFF can give the whole picture of odour concentration compare to the field observation. 0 10 20 30 40 50 60 70 0 1 2 3 4 5 6 7 8 9 Time Observed Predicted Observed (OU/m3) Predicted (OU/m3) Point 2: Observed and predicted odour concentration -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 10 20 30 40 50 60 70 -20 0 20 40 60 80 100 120 140 Point 5: Observed and predicted odour concentration Predicted (OU/m3) Observed (OU/m3) Time Observed Predicted -0.5 0.0 0.5 1.0 1.5 2.0 2.5 0 10 20 30 40 50 60 70 0 1 2 3 4 5 6 7 8 9 Time Observed Predicted Observed (OU/m3) Predicted (OU/m3) Point 2: Observed and predicted odour concentration -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 4.0 3.1 Field Inspection Odour Concentration at the Receptors But when the odour did not disperse, it will not go through the dilution process resulting in high odour concentrations at the ground level [18]. Day 1 Day 3 a) a) Cite this article as: Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup. Comparison method of Odour Impact Evaluation Using CALPUFF Dispersion Modeling and on-site Odour Monitoring. Galeri War. Kej. 1(1) (2017) : 01–05 y mean direction. When it happen, even though the receptor is just near the source, but if the wind direction change, the receptor will not affected by the smell. Fig. 3 Overallpercentage (%) of odour frequencies at the receptors in three alternate days during morning, evening and night observation. Day 1 Day 3 a) a) 13 18 19 8 9 32 1 Percentage (%)of odour frequencies at the receptors around the mill mill exhaust Day 3 a) Day 3 a) a) a) a) Fig. 3 Overallpercentage (%) of odour frequencies at the receptors in three alternate days during morning, evening and night observation. 13 18 19 8 9 32 1 Percentage (%)of odour frequencies at the receptors around the mill mill exhaust exhaust Fig. 3 Overallpercentage (%) of odour frequencies at the receptors in three alternate days during morning, evening and night observation. kin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup. Comparison method of Odour Impact Evaluation Using CALPUFF Dispersion Modeling and on-site Odour Monitoring. Galeri War. Kej. 1(1) (2017 4 Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup/ Galeri Warisan Kejuruteraan 1(1) (2017) 01–05 b) b) c) c) a= morning, b= evening, c= night Fig. 5 Day 1 and 3 of the odour dispersion during morning, evening and night time by using CALPUFF 3.3 Comparison between Field Inspection and CALPUFF Prediction b) Acknowledgement Palmeri, “A comparison of methods for the assessment of odor impacts on air quality: Field inspection (VDI 3940) and the air dispersion model CALPUFF,” Atmos. Environ., vol. 61, pp. 570–579, 2012. [7] G. Schauberger, M. Piringer, and E. Petz, “Separation distance to avoid odour nuisance due to livestock calculated by the Austrian odour dispersion model (AODM),” Agric. Ecosyst. Environ., vol. 87, no. 1, pp. 13–28, 2001. [8] E. Sommer-Quabach, M. Piringer, E. Petz, and G. Schauberger, “Comparability of separation distances between odour sources d id i l d i d b i i l d [13] L. Wang, D. Parker, C. Parnell, R. Lacey, And B. 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Acknowledgement This work is supported by Universiti Sains Malaysia under the RUI grant no. 1001 / PAWAM / 814236. The authors also like to acknowledge United Oil Palm (UOP) Mill for their assistance during the sampling period. Cite this article as: Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup. Comparison method of Odour Impact Evaluation Using CALPUFF Dispersion Modeling and on-site Odour Monitoring. Galeri War. Kej. 1(1) (2017) : 01–05 astaein Qamaruzzaman, Yusri Yusup. Comparison method of Odour Impact Evaluation Using CALPUFF Dispersion Modeling and on-site Odour Monitoring. Galeri War. Kej. 1(1) (2017) : 01–05 Cite this article as: Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup. Comparison method of Odour Impact Evaluation Using CALPUFF Dispersion Modeling and on-site Odour Monitoring. Gale 5 Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup/ Galeri Warisan Kejuruteraan 1(1) (2017) 01–05 Nurashikin Yaacof, Nastaein Qamaruzzaman, Yusri Yusup/ Galeri Warisan Kejuruteraan 1(1) (2017) 01–05 References “Electronic noses for the continuous monitoring of odours from a wastewater treatment plant at specific receptors: Focus on training methods,” Sensors Actuators B Chem., vol. 131, no. 1, pp. 53–62, 2008. “Electronic noses for the continuous monitoring of odours from a wastewater treatment plant at specific receptors: Focus on training methods,” Sensors Actuators B Chem., vol. 131, no. 1, pp. 53–62, 2008. [1] [1] N. S. Azmi and K. F. M. Yunos, “Wastewater Treatment of Palm Oil Mill Effluent (POME) by Ultrafiltration Membrane Separation Technique Coupled with Adsorption Treatment as Pre-treatment,” Agric. Agric. Sci. Procedia, vol. 2, pp. 257–264, 2014. [1] N. S. Azmi and K. F. M. 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https://openalex.org/W4246524971	https://ensciencias.uab.es/article/download/v10-n2-editorial-3/2556	es		Tesis didácticas	Enseñanza de las ciencias/Enseñanza de las ciencias	2,007	cc-by	4,849	LAS PRÁCTICAS DE LABORATORIO EN LA FORMACION DEL PROFESORADO DE FISICA sistemática. Desde estas posiciones se propicia una tarea creativa de construcción de los conocimientos científicos de una manera rnás acorde con lo que es el trabajo de la ciencia (Gil 1983). nivel superior no asumimos una postura innovadora ni desarrollamos una crítica a nuestra propia tarea. Esta característica facilita la repetición, sin mayor cuestionamiento, de actividades con muchas y evidentes limitaciones didácticas. Tesis de Maestrado Autor: Eduardo M. González 1. Objetivos e hipótesis del trabajo Director: Dr. D. Gil Pérez Lugar: Departamento de Didáctica de Esta investigación ha estado dirigida a las Ciencias Experimentales. Universi- estudiar la formación que reciben los dad de Valencia futuros profesores de Física en relación Pecha: septiembre de 1991. con los trabízjos prácticos de laboratorio. Con ese fin se ha realizado un estuEste trabajo parte de reconocer una pre- dio en los cursos del ciclo básico donde ocupación creciente entre los docentes se forman dichos profesores, tomando en de Física del nivel superior, por los resul- consideración a España y a la Argentina. tados de la enseñanza que imparten. Más Las hipótesis de este trabajo han sido las en particular, esta preocupación se refie- siguientes: re a los trabajos prácticos de laboratorio que habitualmente se realizan en los cur- 1. La realización de los cursos de Física sos de Física. A pesar de que los estu- del ciclo básico de la universidad o de los diantes dedican un esfuerzo considerable centros de formación del profesorado no a esta actividad y de que, en algunos proporcionan a los estudiantes una visión casos, las instituciones invierten unacuota amplia y conectadel trabajo de laciencia. importante de recursos, los resultados son poco satisfactorios. Por el contrario, 2. Es posible transformar los trabajos parece que después de una etapa de en- prácticos de laboratorio de Física que se tusiasmo, dichos estudiantes se sienten realizan actualmente en el ciclo básico decepcionados por una tarea cuyo sentido de la universidad y de los centros de no alcanzan a comprender y que viven formación del profesorado, de tal modo como una pérdida de tiempo. que contemplen los aspectos fundamentales de una investigación científica, que Frente a este panorama se han planteado generen un mayor interés, tanto de estudiversos tipos de alternativas; algunas diantes como de docentes, y que produzsostenidas en apreciaciones didácticas can unamej0.rcomprensión de lanaturaleza fundamentadas teóricamente, otras ba- del trabajo científico. sadas más bien en ideas intuitivas en las que se depositaba grandes esperanzas. Sin embargo los resultados alcanzados a través de todas estas propuestas no han 2. Fundamentación de la primera hisatisfecho las expectativas que las mis- pótesis mas habían despertado. Muy recientemente y refiriéndose al nivel universitario a ) Se enseñíz de acuerdo con el modelo se ha hechonotar que «muchos esfuerzos de trasrnisibn de conocimientos. La made reforma e innovación, año tras año, y yoría de los docentes se ajustan a este aun década tras década han producido patrón, tanto en el ciclo medio, como en relativamente poco cambio significati- el superior. Se caracteriza por concebir vo» (Schwartz 1990). la enseñanza como un proceso de trasmisión de los conocimientos en su versión Todo este proceso se relaciona con la final. En esta orientación los trabajos emergencia de nuevas ideas y criterios prácticos son planteados generalmente didácticos -que son los que guiaron este como experiencias de ilustración o de estudio- sobre las que pueden apoyarse «verificación», es decir, como un conlas propuestas innovadoras. Estos nue- junto de experimentos-tipo donde los esvos desarrollos didácticos nos han mos- tudiantes puedan corroborar las teorías trado que no se puede plantearla enseñanza establecidasl previamente. como si el alumno partiera de cero, sin tener en cuenta sus representaciones, de b) La práct.'ca docente se realiza como una gran tenacidad, y que no se dejan una actividad empírica. Es un hecho modificar fácilmente por una enseñanza bastante conocido que los docentes del !32 C )La falta de reflexión sobre las concepciones de la ciencia. Esta desconexión o despreocupación de los docentes e investigadores por explicitar sus concepciones sobre el trabajo de la ciencia puede estar favoreciendo indudablemente que se sigan utilizando unos trabajos prácticos alejados del mismo. d ) La parcelación de la enseñanza. Las distintas actividades -teóricos, problemas y prácticos- se presentan generalmente como aspectos separados e inconexos. Esto apoyaríaelmantenimiento de unos trabajos prácticos rutinarios, que no pretenden&frktarun problema, donde se excluvan asuectos destinados a facilitar el aprendizaje conceptual y que están reducidos a sus elementos de técnicas y habilidades manipulativas. 3. Las características del trabajo científico y sus implicacisnes en la enseñanza de la ciencia El paso siguiente fue explicitar la idea de trabajo científico a la cual consideramos que tendrían que ajustarse los trabajos prácticos de laboratorio si se pretende que los estudiantes adquieran una visión correcta de la disciplina. a ) La importancia de las teorías eri el quehacer de la ciencia. El conocimiento científico está estructurado a través de las teorías científicas, que sintetizan a su vez un conjunto de experiencias, leyes y conceptos científicos. Ya no se acepta la idea de una observación edescontaminada» del fenómeno; toda observación está orientada por un concepto o una teoría, que son las que permiten dar sentido a las mediciones. b ) El papel del pensamiento divergente. Dado que no existen reglas fijas de procedimiento, la actividad de las ciencias es inmensamente rica y creativa. Si bien toma como base el conocimiento adquirido para enfrentar un problema, tiene a partir de allí un amplio campo para la diversidad, la emisión de hipótesis y la exploración de alternativas. ENSEÑANZA DE LAS CIENCIAS, 1992, 10 (2) l BIBLIOGRAFÍA Y NOTICIAS 5. Resultados alcanzados C )Carácter social de la actividad de la ciencia. La ciencia es un producto social, fruto del esfuerzo de generaciones, y por otro lado es también una tarea colectiva, de equipo. No tiene sentido plantearse la investigación como una actividad individual. Por el contrario, ésta responde cada vez más a estructuras institucionalizadas, las que expresan a su vez, los intereses, ideologías y conflictos de la comunidad. Hasta el presente se han analizado 91 guías de trabajos prácticos correspondientes a siete cursos de Física, 54 informes correspondientes a tres cursos de Física y 8 entrevistas orales a profesores cuya actividad está centrada en la realización de trabajos prácticos. Estos datos fueron obtenidos en la Universidad de Valencia, en la Universidad Politécnica de Valencia y en la Universidad Nacional de Córdoba (Argentina). 4. Diseño del experimento para la primera hipótesis Los valores obtenidos por los diferentes medios son coherentes entre sí y nos muestran que, efectivamente, en los actuales trabajos prácticos de Física que se realizan en el ciclo básico del nivel superior, no se propone a los estudiantes que se planteen un problema, que emitan hipótesis, que realicen una búsqueda bibliográfica, que diseñen el experimento, etc. También se ha mostrado que esta actividad rio conecta con nuevos problemas y que :iene un peso muy escaso en la evaluación. Se trata de un diseño de aborde múltiple, que requiere intentar validar la mayor cantidad posible de consecuencias o hipótesis operativizadas. La contrastación de la primera hipótesis se orienta en tres líneas diferentes: a ) Estudiar en qué medida los actuales trabajos prácticos tienen un carácter de mera ilustración, sin plantear a los estudiantes la realización de actividades fundamentales de la tarea de la ciencia, como es el planteamiento del problema, la consulta bibliográfica, la emisión de hipótesis, el diseño del experimento, ladiscusión colectiva de los resultados, etc. Ello implicaría, entre otras cosas, que dichos aspectos estarán ausentes en los prácticos recogidos en los libros de texto, manuales de prácticos, etc. Por otro lado, se ha mostrado, de manera tendencial, que la orientación didáctica de los docentes es tal que no incluyen entre sus objetivos los aspectos concretos del trabajo de la ciencia citados en el párrafo anterior, que no critican esas carencias en un trabajo práctico tradicional y que suSr propuestasde mejoras tampoco incluyen esos aspectos. LAS INTERACCIONES CIENCIAS FISICO-QUIMICAS / TÉCNICA / SOCIEDAD: IMPLICACIONES EN EL PROCESO DE ENSENANZAAPRENDIZAJE Tesis de Master Autora: Amparo Vilches Peña Director: Jordi Solbes Matarredona Lugar: Facultat de Físiques. Universitat de Valt?ncia. Fecha: 1990. El trabajo desarrollado en esta tesis de master, se enmarca dentro de una línea de investigación en la didáctica de las ciencias que pone en cuestión los modelos didácticos de enseñanza-aprendizaje habituales (atención casi exclusiva de los contenidos, transmisión de conoeimientos ya elaborados, aprendizaje por descubrimiento autónomo e inductivo) v se orienta en la construcción de un nue% modelo, de un nuevo paradigma basado en el aprendizaje como cambio conceptual, metodológico y actitudinal. La investigación, dentro del modelo didáctico citado, trata de contribuir a señalar la importancia de los aspectos de relaciones Ciencia / Técnica / Sociedad (C/T/S) en el proceso de enseñanza aprendizaje de las ciencias, no sólo por sus característicasmotivadoras,sino porque se piensa que su estudio, el tratamiento de dichas interacciones, contribuye a mostrar una imagen más real de la ciencia, del trabajo científico. b ) Ver en qué medida los docentes tienen una visión de los trabajos prácticos de acuerdo con esa perspectiva de ilustración, es decir, que, al plantear sus objetivos, al evaluarla tareade los estudiantes, al realizar sus críticas a los trabajos prácticos tradicionales o al plantear sus propuestas de mejoramiento, los docentes no tendrán en cuenta los aspectos del trabajo de la ciencia que se han señalado. En general, las respuestas que son desfavorables a nuestras hipótesis en algún aspecto presentan valores que oscilan alrededor del 10 96. Estas cifras son mucho menores si se consideran varios aspectos a la vez. Estos resultado son coherentes con los que: se han alcanzado en el nivel medio (Payá 1991) y son estimulantes para intentar continuar con el estudio. c ) Ver en qué medida los estudiantes adquirieren una visión deformada del trabajo de la ciencia, lo que implicaría que no serían capaces de identificar aspectos fundamentales de dicho trabajo y que manifestarían concepciones empiristas e inductivistas sobre el mismo. Referencias bibliográficas Hipótesis Gil, D., 1983. Tres paradigmas básicos en la enseñanza de las ciencias, Enseñanza de las Ciencias, 1(1), pp. 26-33. El estudio se realizó partiendo de la siguiente hipótesis: De acuerdo con ello se elaboraron, en primer lugar, cuestionarios para el análisis de las guías de trabajos prácticos y de los informes de los estudiantes, distinguiendo en este caso entre el informe en sí y las correcciones de los docentes. En segundo lugar, se elaboró un cuestionario de análisis de los libros de texto y los manuales de trabajos prácticos. Los restantes instrumentos desarollados -cinco esquemas de encuestas o entrevistas orales con sus correspondientes cuestionarios-estaban destinados a una interacción con los docentes y los estudiantes. Payá, J., 1991. Los trabajosprácticos en la enseñanza de la Física y Química: un análisis crítico y una propuesta fundamentada. Tesis doctoral. (Departamelit de Didáctica de les Ciencies Exloerimentals: Universitat de Valenci:~). ENSEÑANZA DE LAS CIENCIAS, 1992,lO (2) Schwartz, B., 1990. Guest Comment: improvirig the teaching of physics, American Journal ofphysics, 58(1 l ) , PP. 1031-1032. Se plantea, por tanto, en el trabajo, un análisis crítico de cómo se muestran estos aspectos de relaciones C/T/S en la enseñanza habitual y después las consecuencias que tiene dicha imagen en los alumnos. 1. En general la enseñanza habitual igno- ra los aspectos de interacciones C/T/S, mostrando una imagen deformada de la ciencia y los científicos, desconectada de la realidad. 11. Como consecuencia de ello, los alum- nos desconocen el papel jugado por la ciencia a lo largo de la historia de la humanidad, así como las aplicaciones técnicas, las interacciones con la saciedad y el medio en que se insertan, es decir, tienen una imagen distorsionada de la ciencia, alejada del mundo real. 233 BIBLIOGRAF~AY NOTICIAS 111. Es probable que esto sea una de las posibles causas de la actitud de rechazo y desinterés que tienen los alumnos hacia la Física y la Química y su estudio, puesta de manifiesto en los últimos años por la investigación didáctica. IV. En general, los profesores en activo piensan que la «dificultad» de la asignatura y la metodología del profesor son las causas más frecuentes de ese desinterés de los aiumnos, unido a factores externos a la propia escuela, y también, aunque en menor medida, a la desconexión de la ciencia que se estudia con la realidad, a la ausencia del estudio de las interacciones Cn/S. A continuación, se plantea la fundamentación de las hipótesis, analizando críticamente el modelo habitual de enseñanza, caracterizado por la transmisión verbal de conocimientos. el emvirismo. el crecimiento lineal y acumuiativo del desarrollo científico, poniendo de manifiesto la falta de conexión de la ciencia que se enseñaconlos problemas reales del mundo, recogiendo las aportaciones de la investigación didáctica en estos temas en los últimos años y en las posibles consecuencias de lo que todo ello plantea en los alumnos, su imagen de la ciencia y su actitud hacia la misma. Diseno experimental La operativización de la hipótesis permitió su contrastación experimental mediante un diseño que se expone detalladamente y que permitió contrastar en libros de texto, profesores y alumnos la validez de las mismas, describiendo los criterios de valoración utilizados para cada uno de los diseños particulares. Así, en primer lugar, se realizó un análisis de textos con objeto de contrastar la primera parte.de la hipótesis; después se pasaron cuestionarios a alumnos: el primero, para poner de manifiesto cuál es su actitud hacia la ciencia y su aprendizaje (tercera parte de la hipótesis), su interés o no hacia la Física y la Química, y recoger su opinión sobre la forma de aumentar el interés hacia su estudio; y el segundo, para detectar (segunda parte de la hipótesis) cuál es la imagen de la ciencia y los científicos que tienen los alumnos, su desconocimiento de las interacciones C/T/S. Por otra parte, se recogió la opinión del profesorado ( cuarta parte de la hipótesis) sobre a qué puede ser debido, en su opinión, el desinterés de los alumnos hacia la ciencia y su estudio, puesto de manifiesto por la investigación didácticaen los últimos años. Resultados A continuación se exponen los resultados obtenidos para cada uno de los dise- ños, pasando después a un análisis detallado de los mismos que conduce, como conclusión, a la confirmación y refuerzo de las hipótesis planteadas. En los resultados se señala que, generalmente, se muestra una imagen de la ciencia empirista, acumulativa y operativa, que no tiene en cuenta aspectos cualitativos de tipo histórico, tecnológico, sociológico, humanístico, etc., de relación ciencia / técnica / sociedad, y como consecuencia de ello, la mayor parte de los alumnos tienen una visión drstorsionada Y negativa de la ciencia. destacando aspectos desfavorables su caracterización; no conocen aplicaciones técnicas de la Física y la Química ni sus interacciones con la sociedad; desconocen la influencia de la ciencia en la historia cultural, económica y religiosa de la humanidad, así como su papel en la modificación del medio muestran una imagen tópica del científico y no son capaces de realizar una valoración crítica sobre el papel jugado por la Física y la Química a lo largo de la historia, y los pocos que lo hacen ponen el énfasis en sus desventajas, sus aspectosnegativos. Por otraparte, se confirm,ielpoco interés de los alumnos hacia su estudio, y gran número de los consultados creen que sería interesante para ellos y mejoraría su actitud hacia la ciencia el tratar temas de actualidad, aplicaciones técnicas de la ciencia, implicaciones sociales y medio-ambientales de la misma, salidas al exterior, etc., es decir, realizar un tratamiento de las relaciones C/T/S en las clases. - ~ Por otra parte, la mayoría del profesorado consultado (la muestra corresponde a 100 profesores, todos ellos implicados en tareas (le formación o innovación, preocupados, por tanto, de mejorar la enseñanza de la Física y la Química) piensa que una de las posibles causas de la actitud negativa de los alumnos hacia la Física y a Química, de su desinterés hacia el estudio de la ciencia, es esa imagen deformada de la misma que se presenta en general en la enseñanza, alejada del mundo real, de sus implicaciones culturales, sociales, técnicas, etc., que hace no sólo poco interesante su estudio, sino que anima al rechazo, al desinterés. se modifica dicha imagen será más fácil modificar su actitud, aumentar su interés, y se plantea por tanto la posibilidad de llevar a cabo un tratamiento detenido de las relaciones C/T/S en las clases, no sólo por su carácter motivador, porque los alumnos manifiestan un interés hacia su tratamiento, sino, sobre todo, porque se muestra así una imagen más real de lo que es la ciencia y de cómo trabajan los científicos. Las conclusiones de este trabajo, abren , por tanto, nuevos caminos, nuevas líneas de investigación prometedoras y apasionantes en las que seguimos trabajando actualmente. ~ - ~ PROPCESTA DE E V A L U A C I ~ NEN FISICA,Y ANALISIS DE LA EVALUACION HABITUAL Tesis de Master Autor: Manuel Alonso Sánchez Directores: Dr. Daniel Gil Bérez y Dr. Joaquin Martínez Torregrosa Lugar: Universitat de Val2ncia Fecha: Septiembre de 1990. Conclusiones y perspectivas En el momento actual de cambio de paradigma didáctico en la enseñanza de las ciencias, la producción de propuestas específicas de evaluación coherentes con la orientación constructivista del aprendizaje aparece como un requisito necesario para consolidar esta transformación. De hecho, la comunidad de investigadores en enseñanza de las ciencias llamó la atención recientemente sobre la necesidad de acompañar los importantes cambios que se vienen produciendo en otros apartados (introducción de conceptos, resolución de problemas, trabajos prácticos, etc.) de un cambio similar en la evaluación (Linn 1987). En las conclusiones de este trabajo que confirman en general las hipótesis planteadas, se mL.estra que si bien en la investigación se detecta la imagen negativa que tienen las alumnos de la Física y la Química, su desinterés hacia la misma, también se sacan a la luz algunas de sus posibles causas, con lo que está abierto el camino a posibles soluciones, ya que si Este trabajo ha pretendido contribuir a ese propósito, en el campo de la Física y la Química, mediante un desarrollo de la evaluación contextualizado en la orientación del aprendizaje como investigación (Gil 1986, Gil, Carrascosa, Furió y Martínez Torregrosa 199l ) , de modo que los interrogantes fundamentales que nos planteamos fueron los siguientes: I BIBLIOGRAFÍAY NOTICIAS 1. ¿Que características deberá tener un proceso de evaluación para ser coherente con los hallazgos recientes de la investigación educativa sobre el aprendizaje y la enseñanza de la Física y la Química, en particular con el modelo de aprendizaje como investigación? 2. ¿En qué medida la evaluación habitual posee las características que, según el análisis anterior, debería tener para impulsar un aprendizaje significativo de nuestra materia? 3. ¿Contribuirá el nuevo sistema de evaluación a impulsar y mejorar el proceso de aprendizaje, ayudando al cambio conceptual, metodológico y actitudinal de profesores y alumnos. La primera parte del trabajo se dedica a profundizar en tomo a la primera cuestión, es decir, en ella se desarrollan las características principales de una nueva propuesta de evaluación coherente con el aprendizaje de la Física y la Química como investigación. En este desarrollo hemos podido mostrar que una evaluación así, es decir, concebida como instrumento de aprendizaje y de mejora de la enseñanza de la Física y la Química, recoge e integra muchas de las aportaciones realizadas sobre este aspecto didáctico en los últimos años. No obstante, el esfuerzo de profundización realizado para concretar la propuesta ha fructificado también en la aparición de algunos elementos novedosos, que estaban incipientes en nuestro modelo didáctico y se concretan y desarrollan plenamente por primera vez aquí. Con la intención de mostrar, antes que nada, la plausibilidad de la nueva propuesta evaluadora y la distancia entre ella y lo que se hace habitualmente, el trabajo continúa presentando ejemplos de concreción de la misma (se presentan dos sistemas completos de evaluación para dos temas elaborados desde la óptica constructivista) y ejemplos comentados de transformación de actividades de evaluación habituales en actividades adecuadas al nuevo modelo. Finalmente, el resto del trabajo se dedica a analizar, a la luz de este desarrollo, la práctica evaluadora habitual en Física y Química, tratando de establecer con precisión sus insuficiencias y las transformaciones que sobre ella será necesario producir para hacerla coherente con el nuevo modelo. La hipótesis que ha orientado este análisis, sostiene que el tipo de evaluación que se realiza normalmente en las clases de Física y Química no se utiliza para favorecer un aprendizaje significativo y afecta negativamente a las actitudes de alumnos y profesores hacia el aprendizaje y hacia la propia evaliiación. Tras enunciar y fundamen- tar teóricamente dicha hipótesis, se procede a su operativización y al relato del diseño elal~oradopara contrastarla. Se ha optado por un diseño múltiple y coherente, abordando diversas facetas de la práctica de evaluación (profesores, exámenes, resultados y alumnos), de modo que la presentacih de los resultados de esta contrastación incluye también algunos análisis comparativos, reforzando la validez de las conclusiones obtenidas por los diferentes caminos. Resumimos ahora las aportaciones principales del trabajo, que pueden agruparse en dos grandes apartados: 1) En relación con la búsqueda del perfil y características que debería poseer un sistema de evaluación, para ser coherente con los hallazgos recientes de la investigación educativa en enseñanza de la Física y la Química, este estudio ha podido: prácticos,introduccióndeconceptos,etc.). 2) Por lo que se refiere al análisis realizado sobre la evaluación habitual, los resultados de dicho análisis han puesto de manifiesto que la práctica evaluadora habitual en Física y Química posee un perfil muy alejado del propuesto y es, en cambio, coherente con el modelo de enseñanza por transmisión de conocimientos ya elaborados, ya que: - La evaluación habitual en Física y Química se utiliza, en general, como actividad destinada a constatar el «éxito o fracaso» de los alumnos al final del desarrollo de una cierta cantidad de materia, en vez de como instrumento de aprendizaje y de mejora de la enseñanza. - Mostrar que dichas características su- ponen un cambio de una concepción de la evaluación como instrumento para constatar si los alumnos son capaces de reproducir los conocimientos «dados» en clase -al final del desarrollo de una cantidad mlís o menos extensa de materia- a una c:oncepción de la evaluación como instrumento para impulsar el aprendizaje significativo que debe incidir, por tant,>,en la mayor parte de factores que influyen en el mismo. -Mostrar que es posible la concreción de un sistema de evaluación de esa naturaleza dentro del modelo de enseñanza por investigacibn, lo que se ha puesto en evidencia mediante el desarrollo de sistemas de evaluación para dos temas y mediante la transformación de actividades habituales en actividades que fomentan un aprer dizaje basado en el cambio conceptual, metodológico y actitudinal. -Concretar 1 a idea de una concepción de la evaluación como una ocasión privilegiada para impulsar el aprendizaje significativo, mediante la invención d e actividades de autorregulación, para ser realizadas a lo largo del tema en las que los alumnos ]pueden reflexionar y aprender de sus propios avances o errores mientras realizan dicha actividad evaluadora. Éste es uno de los aspectos más novedosos del trabajo. - Los exámenes habituales incluyen so- lamente actividades que permiten -y, por tanto, fomentan- un aprendizaje de tipo repetitivo, bien sea porque dichas actividades pueden realizarse mediante una aplicación operativista de los contenidos brindados, mediante el manejo de destrezas meramente operativas o recurriendo a la simple reproducción memorística. La ausencia de actividades con énfasis en el manejo significativo de los conceptos o de cambio conceptual, en los aspectos metodológicos o en los actitudinales es prácticamente total y unporcentaje elevado de profesores no echan en falta estos aspectos cuando se les pide criticarunexamenmarcadamente «repetitivo y operativista». - Los resultados de las evaluaciones ha- bituales no son indicadores de si se ha producido o no aprendizaje significativo y, además, las calificaciones se ven afectadas por el denominado «efecto halo». Así, por ejemplo, los resultados obtenidos, a partir de las puntuaciones otorgadaspor 101profesoresaunmismoexamen, mostraron una diferencia de puntos - e n una escala de 10- entre las puntuaciones medias obtenidas en dicho examen por dos alumnos, uno supuestamente «brillante» y otro que supuestamente «no va muy bien». mas completos de evaluación para temas elaborados dt:sde la óptica de la enseñanza por investigación en los que se integranlos distiritos aspectos anteriormente señalados. -Las concepciones y expectativas de los profesores de Física y Química hacia la evaluación están muy alejadas de las orientaciones actuales sobre la enseñanza y aprendizaje. Predomina una visión de tipo determinista de la enseñanza y autoexculpatorio de los resultados de la evaluación. -Por último, enriquecer y contribuir a la validación -a añadir a los ya numerosos estudios realizados- del propio modelo - Por último, el sistema de evaluación - Aportar, a modo de concreción, siste- ENSEÑANZA DE LAS CIENCIAS, 1992, 10 (2) - de enseñanza por investigación, al estar contextualizada la nueva propuesta evaluadora en el mismo, y contribuir así a darle coherencia y a aumentar sus derivaciones en aspectos específicos (tales como resolución de problemas, trabajos habitual no contribuye a generar una ac235 BIBLIOGRAFÍA Y NOTICIAS abiertas: seguir probando que el sistema Referencias bibliogrhficas de evaluación elaborado de un modo coherente con los hallazgos de la investiga- Linn, M., 1987. Establishing a research base for science education: challención educativa, contribuye a impulsar un ges, trends and recomendations,Journal aprendizaje significativo y a mejorar la ofResearch in Science Teaching,24(3), enseñanza, generando actitudes positivas pp. 191-216. en alumrios y profesores. En relación con esta cuestión, debemos añadir a lo mostrado aquí los prometedores resultados Gil, D., 1986. La metodología científica y la enseñanza de las ciencias: unas que estarnos obteniendo al probar los sisrelaciones controvertidas, EnseRanza temas evaluadores en clase y al incorporarlosala~actividadesdeperfeccionarniento de las Ciencias, 4(2), pp. 111-121. del profesorado, los cuales muestran una alta aceptación y utilidad del modelo pro- Gil, D., Carrascosa, J., Furió, C., y Martínez Torregrosa, J., 1981. La ensepuesto para profesores y alumnos. Este trabajo, pues, continúa en la perspectiva ñanza de las ciencias en la educación secundaria. (ICE: Horsori). Todo lo expuesto, realza la importancia de una tesis doctoral que completará lo de este análisis de resultados tan rotun- desarrollado aquí y añadirá los resultados dos como preocupantes, haciendo ur- obtenidos de la puesta en funcionamiento gentelaprofundizacióndelasperspectivas del nuevo sistema evaluador. titud positiva de los alumnos hacia el aprendizaje de la Física y la Química. De un modo coherente con todo lo anterior, la evaluación fue percibida por los alumnos como una actividad no interesante, que tampoco contribuye a hacer interesante la asignatura y, lo que es peor, que genera actitudes negativas, ya que consideraron, por ejemplo, que para aprobar es mucho más importante «aprender bien las fórmulas y tener cuidado al sustituir» que «comprender en profundidad lo tratado», y no estuvieron de acuerdo en que «quienes aprueban saben lo fundamental del tema evaluado*. NOTICIAS V REUNIÓN LATINOAMERICANA SOBRE EDUCACION EN FISICA VI1 SIMPOSIO SOBRE ENSENANZA DE LA GEOLOGIA Porto Alegre (RS,Brasil), del 24 al 28 de agosto de 1992. Santiago de!Compostela, del 14 al 19 de setiembre de 1992. Con el tema básico de la Formación del Profesorado de Física en América Latina, tendrá lugar un encuentro que propone debatir cuestiones como: ¿Cuál debe ser el papel de la Historia de la Física, de la Física contemporánea o de la investigación en la Enseñanza de la Física en la formación del profesorado? ¿Cuáles son las estrategias más eficaces de formación? ¿Cuál és el papel de los resursos didácticos? ... Se pretende que los participantes aporten contribuciones en forma de vonencias o pósters. Intercambiar experiencias, opiniones y conocimientos que contribuyan a la mejora profesional de la enseñanza de la Geología, así como favorecer el conocimiento de la Geología de Galicia son los objetivos biisicos que se propone este simposio. La temática preferente señalada por el comité orgai~izadorse refiere a: 1. La Geología en los nuevos diseños curriculares. 2. Investigación didáctica en Geología. Para más información: 3. Recursos didácticos en Geología. Marco Antonio Moreira Instituto de Física - UFRGS Caixa Postal 15051 91500 Porto Alegre, RS, Brasil. 236 4. Actividades fuera del aula. En el mes de abril, ha salido a la calle una nueva revista de educación. Una revista que nace en un momento crucial, en una encrucijada del sistema educativo, y que participa del convencimiento de que la renovación es posible, puesto que existe un capital acumulado de experiencias de un gran número de profesoras y profesores. El objeto fundamental de esta nueva revista será la práctica educativa en todas sus dimensiones. Desde su inicio, la revista Aula va acompañada de un suplemento denominado Aula 2 , que plantea los objetivos, establece las actividades y contempla la evaluación de una unidad, o un aspecto de la misma, de manera que sirva como modelo para que el profesorado valore diferentes propuestas de secuenciación de contenidos, y le sirva paracontrastarla con sus propias realizaciones. 5. Nuevas tecnologías en la enseñanza / aprendizaje de la Geología. Para más información: 6. Geología y Medio Ambiente. Aula de Innovación Educativa C/ de l'Art, 81, bajos. 08026 Barcelona EXSEÑANZA DE LAS CIENCIAS, 1992, 10 (2)
https://openalex.org/W2916927402	https://europepmc.org/articles/pmc6385264?pdf=render	English	null	The role of serotonin 1B in the representation of outcomes	Scientific reports	2,019	cc-by	9,842	The role of serotonin 1B in the representation of outcomes Laura Corbit1,2, Michael Kendig Laura Corbit1,2, Michael Kendig 1,3 & Caroline Moul 1 Disrupted serotonin neurotransmission has been implicated in the etiology of psychopathic traits. Empirical research has found that people with high levels of psychopathic traits have a deficit in reinforcement learning that is thought to be linked with amygdala dysfunction. Altered serotonin neurotransmission provides a plausible explanation for amygdala dysfunction in psychopathic traits and recent research suggests that this may be associated with serotonin 1B (5-HT1B) receptor function. This research used an animal model to test the hypothesis that 5-HT1B receptors are involved in the encoding of the specific features of reinforcing outcomes. An outcome devaluation task was used to test the effect of the systemic administration of a selective 5-HT1B receptor agonist administered before encoding of “action-outcome” associations. Results showed that while administration of a 5-HT1B receptor agonist allowed rats to acquire instrumental responding for food, when the content of that learning was further probed using an outcome devaluation task, performance differed from controls. 5-HT1B agonism impaired learning about the specific sensory qualities of food rewards associated with distinct instrumental responses, required to direct choice performance when the value of one outcome changed. These findings suggest a role for 5-HT1B receptor function in the encoding of the specific features of reinforcing outcomes. Received: 21 August 2018 Accepted: 18 December 2018 Published: xx xx xxxx Received: 21 August 2018 Accepted: 18 December 2018 Published: xx xx xxxx Individuals with high levels of psychopathic personality traits (PT) are characterized by low levels of empathy, diminished feelings of shame and guilt, and limited prosocial emotions. High levels of PT are a risk factor for neg- ative outcomes that endure across the life course (e.g. criminal behaviour, relationship difficulties, and a diagnosis of antisocial personality disorder1). The negative outcomes associated with high levels of PT in adults are also relevant for children and adolescents with high levels of the developmental analogue of PT (callous-unemotional traits)2. A current focus of research is to elucidate the processes underlying the development of PT. One mecha- nism that is implicated is associative learning. Research clearly shows that individuals with high levels of PT, both with and without concurrent antisocial behaviour problems, have a deficit in reinforcement learning3–6. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1School of Psychology, University of Sydney, Sydney, Australia. 2Department of Psychology, University of Toronto, Toronto, Canada. 3School of Medical Sciences, University of New South Wales, Sydney, Australia. Correspondence and requests for materials should be addressed to C.M. (email: caroline.moul@sydney.edu.au) Received: 21 August 2018 Accepted: 18 December 2018 Published: xx xx xxxx The role of serotonin 1B in the representation of outcomes A variety of different tasks have been used to illustrate the difficulties with reinforcement learning exhibited by people with PT and, over the course of several decades, research has narrowed in on the specific nature of this problem. One of the most informative tasks has been the response-reversal learning task. These tasks typically begin with an acquisition phase in which stimuli are presented in pairs; one stimulus in each pair leads to reward when selected (e.g., win 100 points), and the other leads to a loss (e.g., lose 100 points). As such, the subject learns to respond to some stimuli and avoid others depending on whether they are rewarding or punishing. In the second phase of the task, stimulus-outcome con- tingencies are changed such that previously rewarded stimuli now lead to loss and vice versa. Thus, the subject will perform better on each trial if they stop selecting the stimulus that was previously rewarding and instead selects the one that they had previously learnt to avoid. Studies have demonstrated that while people with PT gen- erally show no deficits in acquisition (they initially learn to select the rewarding stimuli and avoid the punishing stimuli at the same rate as people without PT), they are slower to reverse their pattern of stimulus selection5,6. l l f h f b h l d h 7 8 y Potential explanations for this pattern of behaviour include an insensitivity to punishing outcomes7,8, or reward dominance (behaviour driven more by seeking reward than by avoiding punishment)9. However, further research demonstrates that the reinforcement learning deficits shown by people with PT may be independent from motivational properties. Blair, Morton et al.10 conducted a Differential Reward/Punishment Learning Task in which 2 of 10 possible pictorial stimuli were displayed together on a screen at a time. The subject was instructed to select one of the two stimuli. Half of the stimuli led to a win while the other half led to a loss. Critically, each of the 5 winning stimuli were associated with a different number of points (+1600, +800, +400, +200, +100) which 1School of Psychology, University of Sydney, Sydney, Australia. 2Department of Psychology, University of Toronto, Toronto, Canada. 3School of Medical Sciences, University of New South Wales, Sydney, Australia. Correspondence and requests for materials should be addressed to C.M. The role of serotonin 1B in the representation of outcomes (2015) found that PT in boys with antisocial behaviour problems was negatively associated with methylation at two locations in the promoter region of HTR1B: the less methylation in these locations the higher the PT. Together, the genetic and epigenetic results suggest that increased expression of HTR1B may be relevant for the development of PT. Support for this hypothesis comes from the animal literature; it has been demonstrated that increased expression of HTR1B in rats decreases fear-potentiated startle19 a response that is also reliably reduced in people with PT8.h The 5-HT1B receptor functions predominantly as a terminal autoreceptor, inhibiting serotonin release at ser- otonin terminals20,21. Thus, 5-HT1B terminal autoreceptors located on axons originating in the midbrain raphe nuclei can act as part of a negative feedback system, changing the rate of serotonin release in response to localized fluctuations in extracellular serotonin. Interestingly, a negative relationship between PT and serum serotonin lev- els was found in the HTR1B methylation research sample13 which supports a potential functional role of dimin- ished HTR1B methylation on serotonin neurotransmission.h y The overall aim of the following study was to link altered reinforcement learning to serotonergic activity and test the hypothesis that 5-HT1B receptors are involved in the encoding of the specific features of reinforcing out- comes. It was hypothesized that increased HTR1B expression, as found to be associated with PT, could be mim- icked via the use of a selective 5-HT1B agonist; the agonist would result in a downregulation of serotonin-induced activation of forebrain afferents important for detailed outcome encoding. Before conducting more in-depth studies to test the role of specific brain regions, it was first necessary to use animal models to test the basic prem- ise: that reinforcement learning processes are influenced by the activation of 5-HT1B receptors during encoding.t g pl y 1B p g g To this end, hungry animals were trained to make two instrumental responses (left and right lever presses) for distinct food outcomes (grain pellets and 20% sucrose solution). To assess the involvement of 5-HT1B in the encoding of these specific ‘action-outcome’ associations, half of the animals were given systemic administration of the 5-HT1B receptor agonist CP94253 prior to daily training sessions in which animals earned both food rewards. The remaining animals were given vehicle injections. All animals were drug-free for the subsequent test phase, which assessed animals’ sensitivity to outcome devaluation. The role of serotonin 1B in the representation of outcomes (email: caroline.moul@sydney.edu.au) Scientific Reports \| (2019) 9:2497 \| https://doi.org/10.1038/s41598-019-38938-4 1 www.nature.com/scientificreports/ was mirrored by the 5 stimuli that led to loss. During testing, all stimuli were presented with one another such that, throughout the experiment, trials could either include two stimuli that led to rewards of different values, two stimuli that led to losses of different values, or one winning stimulus and one losing stimulus. Thus, the valence (win or loss) associated with a stimulus was not sufficient for optimizing choice. Instead, participants had to recall the specific points associated with each stimulus and judge the value of those points relative to the other stimulus present on that trial (e.g., +400 is better than +200, but worse than +800). Participants with PT performed worse than controls in this task. Their ability to associate a specific value with an arbitrary stimulus and judge this value relative to alternatives to optimize performance was impaired relative to the controls. p p p Evidence from the human research literature indicates that serotonin neurotransmission plays a role in both psychopathic traits11–14 and in associative learning in humans15,16. However, serotonin has multiple classes of receptors with distinct and widespread functions17. Importantly, recent evidence specifically implicates the sero- tonin 1B (5-HT1B) receptor in the etiology of PT. Moul et al.14 found an association between a functional genetic polymorphism (rs11568817) of the serotonin 1B receptor gene (HTR1B) and callous-unemotional traits (the childhood analogue of PT) in a sample of boys with antisocial behaviour problems. This polymorphism is of specific interest as it is known to have a functional role in the serotonin system. The minor allele contributes to the creation of a transcription factor binding site that results in a 2.3-fold increase in gene expression18. Thus, this result14 would suggest that increased expression of HTR1B, linked to the presence of the minor allele of rs11568817, is associated with PT. Indeed, further research conducted by this group13 found that methylation of the promoter region of HTR1B was also associated with PT in boys with antisocial behaviour problems.l p g y p Methylation is a dynamic process that can impact gene expression by influencing the degree to which DNA is transcribed. Typically, increased methylation is associated with reduced gene expression. Moul et al. The role of serotonin 1B in the representation of outcomes In these tests animals first received free access to either pellets or sucrose solution, immediately prior to a brief test in the training chambers where responding on both levers was measured. Animals typically selectively reduce responding on the lever earning the devalued out- come. Such performance reflects intact encoding of the specific features of each outcome and association of these outcomes with the distinct responses during earlier training which permits value-based choice at test. If specific action-outcome encoding is disrupted during training, as hypothesized to occur following 5-HT1B receptor ago- nism, this should be reflected in reduced sensitivity to outcome devaluation, as measured by a failure to selectively reduce responding on the lever associated with the devalued outcome. Methods The left and right levers were assigned to earn grain pellets (45 mg, grain-based formula, Bioserv, USA) and 20% sucrose solution (w/vol; ~0.2 ml per reward) in a counterbalanced fashion. Devaluation pre-feeding was conducted in individual plastic cages with open wire tops. Rats were habituated to these cages prior to testing. Procedure. Instrumental training began with a single 40-min session of magazine training in which 15 ali- quots of 20% maltodextrin solution (0.2 ml) were delivered to the magazine. Next, all rats were trained to make both left and right instrumental responses with each lever-press reinforced with 0.1 ml of a 20% maltodextrin solution. There were two of these sessions to ensure that all rats had the opportunity to learn to respond and that any drug effects would not interfere with initial instrumental acquisition but would instead be restricted to the period of training when rats could encode the specific features of the sucrose and pellet rewards subsequently used as rewards for left and right lever press responses. Both left and right levers were trained within a single session: the left (or the right) lever was inserted into the chamber until the animal earned five outcomes. At this point the lever retracted and, 10 s later, the right (or the left) lever was inserted until five of the other outcome were earned. This process repeated until animals earned 60 total rewards, or 60 min elapsed.th h Eighteen rats successfully acquired both left and right lever press responses for 20% maltodextrin. Thirty min- utes prior to each of five subsequent training sessions, nine of these rats received CP94253 (5 mg/kg, i.p.) and the other 9 received vehicle (distilled water 1 ml/kg). Responding during these training sessions was reinforced on a random-ratio 5 schedule, meaning one outcome was delivered after 5 lever-press responses, on average. Devaluation testing. Two devaluation tests were held drug-free, ensuring that differences in responding were due to the effects of 5HT1B receptor agonism on the associations learned during prior training. Devaluation was achieved by specific satiety. Rats were tested twice on separate days with one session of retraining and drug treatment between the two tests. On each test day, rats were placed in individual feeding cages and given free access to either pellets or sucrose solution for 1-h. Methods All experimental procedures were carried out in accordance with the recommendations of the Australian code for the care and use of animals for scientific purposes 8th edition (2013) and were approved by the Animal Ethics Committee at the University of Sydney (Protocol Number 2016/1080).h y y y Twenty-four experimentally naive male adult hooded Wistar rats (University of Adelaide) were used. They were group-housed 4 per cage in a temperature- and humidity-controlled colony room maintained on a reverse dark:light cycle (lights off 0900–2100 h) and had free access to tap water throughout all experimental procedures. Chow access was initially ad-libitum during elevated plus maze testing but was then restricted to 12 g/rat/day throughout instrumental training and testing. Drug administration. The 5-HT1B agonist CP94253 (Sigma, USA) was dissolved in distilled water (5 ml/ kg) and was administered i.p. at a dosage of 5 mg/kg. This dose was based on previous studies demonstrating that this dose had no effect on milk or sucrose self-administration or locomotor activity, but was effective in reducing cocaine self-administration and reinstatement of cocaine-seeking22,23. Scientific Reports \| (2019) 9:2497 \| https://doi.org/10.1038/s41598-019-38938-4 2 www.nature.com/scientificreports/ Elevated Plus Maze test. The acute effects of 5-HT1B agonism were first assessed in the Elevated Plus Maze24, a well-validated measure of anxiety in rodents. Previous research has demonstrated an anxiogenic effect of CP9425325 and so this test was included as a positive control for behavioural effects of the selected drug dose. The apparatus was a plus-shaped maze elevated 1 m above the ground, with two closed arms surrounded by high (40 cm) Perspex walls, and two open arms. Animals are placed in the center of the maze and allowed to explore for 5-min; more time spent exploring the open arms is taken to indicate lower levels of anxiety. Instrumental training. In preparation for instrumental training a restricted feeding schedule was intro- duced wherein animals were fed a ration of 12 g chow per rat, per day. Twelve identical operant chambers (Med-Associates, USA) were used for training and test procedures. The floor consisted of steel bars with top and sidewalls made of clear Plexiglas and end walls made of aluminum. Two retractable levers were located on one side of the chamber with a recessed magazine centered between them. A 3-W, 24-V house-light provided illumi- nation. Methods They were then transferred immediately to the training cham- bers for a 5-min extinction test in which both left and right levers were presented, but presses were not reinforced. A second test was identical to the first except that rats were pre-fed the opposite food (e.g., rats that were pre-fed pellets in test 1, were pre-fed sucrose in test two, and vice versa). Sensitivity to devaluation is reflected in lower responding on the lever earning the devalued outcome relative to the lever earning the non-devalued outcome. Consumption Test. Animals were given a single consumption test to confirm the efficacy of the devaluation treatment. In this test half of the rats in each group were pre-fed pellets and the other half were pre-fed sucrose for 1-hr in the individual pre-feeding cages used for devaluation tests. These foods were then removed and all animals were given a fresh dish with 20 g of pellets. These dishes were removed 10 minutes later and consumption recorded. Lower pellet consumption following prior consumption of pellets relative to prior consumption of sucrose would confirm that specific-satiety was intact, and thus that devaluation treatment was effective. Reinforced Test. A reinforced test was conducted five days later. Half of the animals in each group were pre-fed pellets and the other half were pre-fed sucrose for 1-h. They were then transferred immediately to the training chambers where both levers remained present throughout a 20-min test. The first lever press on either lever was reinforced; thereafter, responding was reinforced using independent reinforcement schedules that ascended from random-ratio 3 (RR3) for rewards 2–5, to RR5 for rewards 6–10, and RR10 for subsequent rewards. The test lasted 20 minutes. Results l d Elevated plus maze. As shown in Fig. 1A, CP94253 administration (versus vehicle) 30-min prior to test significantly reduced time spent on the open arms (F(1, 22) = 53.21, p < 0.001) and reduced the number of entries made into the open arms of the maze (Fig. 1B; F(1, 22) = 55.30, p < 0.001). By contrast, there was no significant effect on the number of entries into closed arms (F < 1) suggesting that CP94253 did not simply reduce explora- tion or locomotor activity. Together, this pattern of results indicated that 5-HT1B agonism was anxiogenic. Elevated plus maze. Instrumental training. Results from instrumental training are shown in Fig. 2A. During the two days of instrumental pre-training when responding was reinforced with maltodextrin solution and no drug treat- ment given, response rates (lever presses/minute) for CP94253 and Vehicle groups were 1.21 ± 0.19 (SEM) and 1.15 ± 0.17 on day 1, and 2.58 ± 0.21 and 2.34 ± 0.36 on day 2. During the subsequent 5-days of instrumen- tal training with sessions conducted following CP94253 or vehicle treatment, analyses indicated that response rates increased significantly across the 5 days of instrumental training (linear trend: F(1, 16) = 43.89, p < 0.001) and that this increase did not interact with group (linear interaction trend: F(1, 16) = 1.67, p = 0.21). Averaged over the 5 days of training, CP94253-treated rats responded at a significantly lower rate than vehicle-treated Scientific Reports \| (2019) 9:2497 \| https://doi.org/10.1038/s41598-019-38938-4 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Elevated plus maze. CP94253 treatment significantly decreased time spent on open arms in the elevated plus maze, (Panel A; *p < 0.001). CP94253 treatment also decreased the number of entries into the open, but not closed arms (Panel B; p < 0.001) indicating that acute CP94253 administration increased anxiety-like behaviour and suggesting that results were unlikely to be secondary to locomotor impairment. Figure 1. Elevated plus maze. CP94253 treatment significantly decreased time spent on open arms in the elevated plus maze, (Panel A; p < 0.001). CP94253 treatment also decreased the number of entries into the open, but not closed arms (Panel B; *p < 0.001) indicating that acute CP94253 administration increased anxiety-like behaviour and suggesting that results were unlikely to be secondary to locomotor impairment. Figure 2. Instrumental training. Despite lower response rates during training (A) the CP94253 group still increased responding across days and earned a majority of the available outcomes in training sessions (B). Figure 2. Instrumental training. Despite lower response rates during training (A) the CP94253 group still increased responding across days and earned a majority of the available outcomes in training sessions (B). rats (main effect of group (F(1, 16) = 16.14, p = 0.001). Figure 2B shows that despite lower rates of respond- ing, CP94253-treated rats still earned the majority of the 60 available outcomes in each daily training session (mean = 48 on day 5 of training for CP94253 group, versus 60 for vehicle group) and thus had the opportunity to learn about specific action-outcome associations. rats (main effect of group (F(1, 16) = 16.14, p = 0.001). Figure 2B shows that despite lower rates of respond- ing, CP94253-treated rats still earned the majority of the 60 available outcomes in each daily training session (mean = 48 on day 5 of training for CP94253 group, versus 60 for vehicle group) and thus had the opportunity to learn about specific action-outcome associations. Devaluation tests. In the key test phase animals were placed in the training chambers for 5-min with both levers available and were free to respond but no rewards were delivered. Importantly, no drug was administered during devaluation tests, meaning differences in responding could only be attributed to associations formed dur- ing previous training. Figure 3A,B show bin data from the 5-min extinction test for CP94253 and vehicle groups. www.nature.com/scientificreports/ Scientific Reports \| (2019) 9:2497 \| https://doi.org/10.1038/s41598-019-38938-4 Scientific Reports \| (2019) 9:2497 \| https://doi.org/10.1038/s41598-019-38938-4 4 www.nature.com/scientificreports/ Figure 3. Devaluation test results. After 1-hr pre-feeding of one outcome, animals received a 5-min extinction test assessing responding on the levers that previously earned the now devalued and non-devalued outcomes. Analyses suggested insensitivity to devaluation treatment in CP94253-treated rats (Panel A) relative to the Control group (Panel B). (C) Responding in CP94253 and vehicle groups in the first 2 minutes of the extinction test. (D) Consumption (g) of the pre-fed outcome. Figure 3. Devaluation test results. After 1-hr pre-feeding of one outcome, animals received a 5-min extinction test assessing responding on the levers that previously earned the now devalued and non-devalued outcomes. Analyses suggested insensitivity to devaluation treatment in CP94253-treated rats (Panel A) relative to the Control group (Panel B). (C) Responding in CP94253 and vehicle groups in the first 2 minutes of the extinction test. (D) Consumption (g) of the pre-fed outcome. Data from the two tests are presented collapsed across outcome type because the pattern of responding was the same regardless of whether pellets or sucrose was the devalued outcome. Data split according to outcome are shown in Supplemental Fig. 1. A 2 × (2) × (5) ANOVA (group × [lever] × [minute]) indicated a significant 3-way interaction between group, lever, and minute (F(1, 16) = 6.51, p < 0.021) suggesting that sensitivity to devaluation varied between groups over the course of the test. In addition, this analysis found a significant overall devaluation effect (main effect of ‘lever’: F(1, 16) = 5.97, p = 0.027), that responding declined significantly over time (linear trend for ‘minute’: F(1, 16) = 21.84, p < 0.001), and was significantly lower in CP94253 animals, on average (group main effect: F(1, 16) = 12.45, p = 0.003).f Data from the two tests are presented collapsed across outcome type because the pattern of responding was the same regardless of whether pellets or sucrose was the devalued outcome. Data split according to outcome are shown in Supplemental Fig. 1. A 2 × (2) × (5) ANOVA (group × [lever] × [minute]) indicated a significant 3-way interaction between group, lever, and minute (F(1, 16) = 6.51, p < 0.021) suggesting that sensitivity to devaluation varied between groups over the course of the test. www.nature.com/scientificreports/ Thus, the insensitivity of lever-press performance to the devaluation treatment was not due to compromised sensory-specific satiety produced by CP94253, since these rats, like the vehicle group, were sensitive to the effects of pre-feeding when measured in consumption. Although consumption was similar between groups, it is possible that the resultant experience of satiety and, consequently, the efficacy of the devaluation treatment, differed between groups. Figure 4 shows the results from a follow-up consumption test held to confirm the impaired sensitivity to devaluation measured by instrumental responding was not due to ineffective satiety treatment. Thus, rats in CP94253 and vehicle groups were pre-fed either sucrose or pellets (n = 4 or 5 per group; Fig. 4A) for an hour – identical conditions to those preceding deval- uation tests – prior to a 10-min test in which all rats were given a dish of pellets to eat (Fig. 4B). A 2 × 2 (group [CP94253 or vehicle] × pre-feeding [sucrose or pellets]) ANOVA found significantly lower pellet consumption in animals pre-fed pellets than those pre-fed sucrose (pre-feeding main effect: F(1, 14) = 61.89, p < 0.001) with no main effect of group (F(1, 14) = 3.05, p = 0.102) and no group × pre-feeding interaction (F < 1). Therefore, the insensitivity to devaluation shown by CP94253 rats in Fig. 3 was not attributable to a failure in sensory-specific satiety. y To explore the possibility that the lower overall responding in the drug group could have impacted our ability to detect a devaluation effect, we next examined sensitivity to devaluation in the lower- and higher-responding half of each group. After conducting a median split based on training response rates, we found the devaluation effect to be similar between the high- and low-responding subsets within each group. Thus, both high and low responding controls appeared sensitive to devaluation and both high and low responding CP94353 animals were not. (Supplemental Fig. S2). Reinforced test. A final test confirmed that, following pre-feeding, the deficit observed in CP94253 animals was rescued when lever-pressing in test conditions was reinforced by the delivery of the outcomes, as in train- ing. These results are shown in Fig. 5, with total responding across a 20-min test shown in panel A and the same data presented in 4-min bins separated by group in panels B and C. www.nature.com/scientificreports/ In addition, this analysis found a significant overall devaluation effect (main effect of ‘lever’: F(1, 16) = 5.97, p = 0.027), that responding declined significantly over time (linear trend for ‘minute’: F(1, 16) = 21.84, p < 0.001), and was significantly lower in CP94253 animals, on average (group main effect: F(1, 16) = 12.45, p = 0.003).f f ( ) p ) Figure 3 and the 3-way interaction reported above suggested a group difference in sensitivity to devaluation in the early portion of the test. A 2 × (2) ANOVA applied to responding in the first 2-min of the test showed a signif- icant group × [lever] interaction (F(1, 16) = 5.74, p = 0.029), a significant overall effect of lever (F(1, 16) = 5.32, p = 0.035) and a significant main effect of group (F(1, 16) = 16.33, p < 0.001). These results, shown in Fig. 3C and consistent with performance in the test overall, indicated that the group treated with CP94253 showed an ina- bility to selectively direct responding toward the lever earning the nondevalued outcome. Tests of simple effects confirmed no effect of devaluation in the CP94253-treated group (F(1, 16) = 0.004; p = 0.95) in contrast to a robust effect of devaluation evident in rats treated with vehicle during training (F(1, 16) = 11.05, p < 0.004). The difference between groups cannot be explained by a difference in consumption during the 1-hr pre-feeding phase as consumption of neither pellets (t(16) = 1.08, p = 0.30) nor sucrose solution (t(16) = 0.13, p = 0.90) differed between groups (Fig. 3D). Scientific Reports \| (2019) 9:2497 \| https://doi.org/10.1038/s41598-019-38938-4 5 www.nature.com/scientificreports/ Figure 4. Devaluation consumption test. Animals were pre-fed either sucrose or pellets and then tested for further consumption of pellets. Animals in both CP94253 and vehicle groups reduced pellet consumption when pre-fed pellets compared to those pre-fed sucrose. Thus, the insensitivity of lever-press performance to the devaluation treatment was not due to compromised sensory-specific satiety produced by CP94253, since these rats, like the vehicle group, were sensitive to the effects of pre-feeding when measured in consumption. Figure 4. Devaluation consumption test. Animals were pre-fed either sucrose or pellets and then tested for further consumption of pellets. Animals in both CP94253 and vehicle groups reduced pellet consumption when pre-fed pellets compared to those pre-fed sucrose. www.nature.com/scientificreports/ Responding for pellets versus sucrose was collapsed because this factor did not alter the pattern of responding (no significant interaction or main effects when this factor was included in preliminary analyses). Data are instead organized according to whether the outcome earned by responding was currently devalued or non-devalued. Data were analysed in a 2 × [2] × [5] ANOVA (group × [lever] × [bin]), which found a significant overall devaluation effect (main effect of ‘lever’: F(1, 16) = 13.20, p = 0.002) that increased across time F(1, 16) = 4.25, p = 0.056), but critically, did not interact with group (lever x group interaction: F < 1). Overall responding during the test did not differ significantly between groups (F(1, 16) = 1.13, p = 0.30) and there were no other significant interaction effects. Discussion This suggests that they could use feedback from outcome delivery to direct responding appropriately and that their deficit was limited to choice responding based on an internal representation of the specific outcomes and/or action-outcome contingencies.hf g The hypothesis of this research was that a 5-HT1B agonist would mimic the effects of increased HTR1B expres- sion, as found to be associated with psychopathic personality traits in humans13, and would therefore be asso- ciated with diminished encoding of the specific features of outcome stimuli. This hypothesis was supported, as reflected by the apparent inability of the drug-treated animals to preferentially select the lever that lead to a non-devalued reward. As such, the serotonin 1B receptor is a plausible target for research into the cognitive sub- strates of psychopathic personality traits and the results support suggestions that the function of the serotonin system is altered in psychopathy.i y y y In addition to associative learning deficits reported to be associated with PT, the amygdala has been found to differ in size26 and function27,28 in individuals with PT compared to those without and these differences may contribute to the etiology of PT. Furthermore, individuals with amygdala lesions show some similarities to people with PT such as difficulties in correctly recognizing others’ emotions and response-reversal deficits4,29. The altered amygdala function observed in individuals with PT together with what is known about the role of the amygdala in associative learning may yield insight into the observed deficits in learning tasks including devaluation. A recently developed model, the Differential Amygdala Activation Model (DAAM)30 argues that while there is substantial evidence to implicate amygdala function in PT, rather than treating the amygdala as a unitary structure, experi- mental findings are better explained by considering the distinct contributions of two important subregions of the amygdala; the central amygdala (CeA) and the basolateral amygdala (BLA). The model then draws from the ani- mal associative learning literature to describe how understanding amygdala function could offer an explanation for the deficits in reinforcement learning shown by individuals with PT. Discussion Here we found that while peripheral administration of a selective 5-HT1B receptor agonist (CP94253) allowed rats to acquire instrumental responding for food, when the content of that learning was further probed using an outcome devaluation task, performance differed from controls. CP94253 administered prior to learning about the unique outcomes associated with each lever resulted in animals being unable to selectively adjust responding to reflect the altered value of one, but not the other reward, in the test phase. The results indicate that 5-HT1B ago- nism impairs learning about the specific sensory qualities of food rewards associated with distinct instrumental responses, required to direct choice performance when the value of one outcome has changed. The fact these deficits were detected when animals were tested drug-free is notable as it indicates that 5-HT1B agonism altered the details of what was encoded during acquisition which then impacted responding even when animals were drug-free at test. This deficit cannot be explained by inability to discriminate between the different rewards or by Scientific Reports \| (2019) 9:2497 \| https://doi.org/10.1038/s41598-019-38938-4 6 www.nature.com/scientificreports/ Figure 5. Reinforced test results. When lever pressing was reinforced by reward delivery after devaluation treatment, both groups selectively directed responding towards the lever earning the non-devalued outcome (Panel A). Analyses of data in 4-min bins showed this pattern was comparable for CP94253 (Panel B) and Control groups (Panel C). Figure 5. Reinforced test results. When lever pressing was reinforced by reward delivery after devaluation treatment, both groups selectively directed responding towards the lever earning the non-devalued outcome (Panel A). Analyses of data in 4-min bins showed this pattern was comparable for CP94253 (Panel B) and Control groups (Panel C). the efficacy of the satiety treatment itself since both drug and vehicle groups showed a robust devaluation effect measured in consumption and ate comparable amounts of the outcomes during devaluation treatment itself (i.e., during induction of devaluation by specific-satiety). Further, in animals trained under 5-HT1B agonism sensitiv- ity to devaluation emerged when the outcomes were made available in a separate reinforced test. Discussion Behaviour that is inflexible and that does not respond appropri- ately to changes in the reinforcing properties of different outcomes may appear to be: risky (in the context of an increase in the probability of a negative outcome) or unkind (in the context of a dyadic interaction in which the behaviour causes a decreasingly positive response in the dyadic partner). In these situations, it is the continued engagement of a previously reinforced behaviour that gives rise to two of the personality features associated with PT. The DAAM hypothesizes that the chronic underactivation of the BLA is due, in part, to altered serotonin neurotransmission. For example, within the amygdala, a high density of serotonergic innervation has been found in the basal and lateral, but not central amygdaloid nuclei20,36. As such, within the amygdala, altered serotonergic transmission is likely to selectively alter BLA function and behaviours that rely on these nuclei. Indeed, localized infusions of serotonin selective reuptake inhibitors (SSRIs) into the BLA have behavioural effects, such as a reduc- tion in the conditioned-fear response37. Together, there is substantial overlap between the symptoms of PT and the behavioural effects of BLA lesions; serotonin function within the BLA may link the two. f y While the current data provide a useful proof of concept demonstration of the importance of 5-HT1B receptors in outcome encoding, there are several limitations to the current study. Importantly, while 5-HT1B agonism with CP94253 produces effects that are similar to those produced by BLA lesions, since this research used systemic administration of the agonist it is not known exactly which 5-HT1B receptors were stimulated. Future studies administering CP94253 directly to the BLA would extend the current findings and more directly test the tenets of the DAAM model.h The CP94253-treated group responded less in training and consequently had less exposure to the two dis- tinct outcomes which may have impacted their ability to form outcome-specific representations. Some findings suggest that response reinforcer associations may continue to grow with extended training38 and so stronger associations in the control group may contribute to a larger devaluation effect, although others have found that extended training, even under choice conditions, can favor habitual responding39,40. Discussion i g y Lesion studies in animals have demonstrated that the CeA and BLA simultaneously and independently form associations between a stimulus or response and different aspects of the outcome; the CeA is involved with the encoding of the general motivational significance and valence of an outcome (good/bad) while the BLA is involved with encoding the specific sensory features of an outcome, linking those with the reinforc- ing properties of that outcome and updating that representation when value changes. As such, much like rats administered CP94253, while rats with lesions of the BLA acquire instrumental responding for food reward, they non-selectively reduce responding when one instrumental outcome is devalued31,32. The DAAM posits that in people with PT, the amygdala is differentially activated such that the BLA is chronically underactivated while the CeA is functioning normally; and this suggestion has recently gained empirical support from brain imaging stud- ies33,34. Since associating stimuli or responses with specific features of reinforcing events relies on the BLA, altered function of this structure would result in a style of encoding that neglects the specific features (and particular Scientific Reports \| (2019) 9:2497 \| https://doi.org/10.1038/s41598-019-38938-4 7 www.nature.com/scientificreports/ value) of an outcome; information that is required in order to facilitate flexible and appropriate responding to associated stimuli or responses (e.g., to choose a course of action based on the value of different outcomes or to modify performance when expected value changes). Basic reinforcement processes, e.g., learning to perform a response for reward, should nonetheless be intact as this can be supported by a typically functioning CeA. There is some empirical evidence to support the suggestion that the reinforcement learning deficits seen in people with PT are linked to diminished encoding of the specific features of outcomes. In a learning task that was designed to put specific feature encoding in opposition with that of general motivational valence, a positive association was found between PT and general motivational valence encoding in both a sample of healthy adult males and a clinical sample of children with conduct problems35. In other words, people with PT had a style of encoding that neglected specific-features of outcomes. Discussion However, we think this is unlikely to fully explain the current results because previous studies employing a similar design but using more limited training (1–3 days), and thus reduced outcome exposure, have nonetheless generated robust devaluation effects41–43 indicating that the exposure in the drug group ought to have been sufficient to allow learning about the distinct outcomes, at least in intact animals. In fact, Colwill and Rescorla (1998)38 found that as few as 40 response-outcome pairings was sufficient to produce considerable response-reinforcer learning detectable with devaluation testing and so even if associations were stronger in control animals, CP94253-treated animals had enough training to allow specific associations to form yet they showed no indication of any selective sensitivity to the devaluation treatment. Future studies using yoked procedures to equate the number of response-outcome pairings across groups could more fully examine this issue. p g g p y It is possible that drug-induced satiety accounts for the lower instrumental response rates and reduced number of earned rewards during training as previous research has found that CP94253 promotes satiety44,45. While the choice of drug dose was based on previous reports that CP94253 in this dose range does not affect self-administration of sucrose or sweetened milk22,23 it is possible that motivation to respond for food reward was reduced in the drug group if these rats were experiencing drug-induced satiety. Nonetheless, we do not believe this potential effect of the drug can account for the test data of this experiment as no drug was given on the test day, and any lasting effects of the drug seem unlikely since consumption during the induction of specific-satiety was similar for the two groups. It is notable that devaluation in animals trained under CP94253 produced a nonselective decrease in responding at test rather than having no effect on performance. We believe that this is largely due to the method of devaluation. When animals are satiated prior to testing, control animals with intact representation of the specific features and value of each outcome are able to direct their responding for the still-valued outcome. However, if the feature-specific outcome representation needed to choose between the two responses is compromised, as we hypothesize for the CP94253 group, responding for reward in general may be indiscriminately diminished when animals are not hungry. Scientific Reports \| (2019) 9:2497 \| https://doi.org/10.1038/s41598-019-38938-4 Discussion Notably, this is the exact pattern seen following multiple demonstrations of impaired sensitivity to outcome devaluation following manipulation of the BLA, and indeed, in addition to indiscriminate responding, the overall rates of responding (<5 lever presses/minute) are similar31,32,46–48. It has also been reported that CP94253 can have anxiogenic effects25. Indeed, this effect was utilized in this experiment as a positive control to provide evidence for sufficient drug dosage. As found previously, CP94253 appeared to have an anxiogenic effect on the animals in this experiment as drug-administered animals spent less time in the open arms of the elevated plus maze. There is no clear consensus on what the effect of an anxious state is on instrumental learning, but it should be considered as a potential confound. While any effects of anxiety on outcome encoding are largely untested, studies in both humans and animals suggest acute stress before, or dur- ing, testing can shift responding from goal-directed to habitual control49–51. For example, Pritchard et al. (2018)49 found that the induction of stress during the extinction test following specific satiety of one of two possible out- comes disrupts outcome devaluation while leaving reacquisition intact. p g q While the drug dosage used in this experiment induced anxiety in a test designed to bring out such ffects, insensitivity to devaluation is unlikely to be a result of anxiety produced by the drug. First, while drug 8 www.nature.com/scientificreports/ administration before training may have produced anxiety, since the drug was not administered prior to testing, the animals ought not to have been anxious at test. Any effects must then relate to how the drug affected what ani- mals learned during training. Given that research suggests that stress affects instrumental behaviour primarily via processes involved in performance rather than acquisition49–53 it is unlikely that our insensitivity to devaluation effect was driven by anxiety states during acquisition. Second, evidence from the Posttraumatic Stress Disorder literature suggests that anxious states are associated with an increase of perceptual coding of stimuli54. It is the perceptual information (the features of the rewarding outcome) that seems lacking in the drug-administered rats and so even if rats were somewhat anxious during training this does not explain the pattern of results. Nonetheless, this finding is problematic for a model of PT. Typically, PT is characterized by low levels of anxiety. Discussion However, because 5-HT1B receptors can act as either autoreceptors or heteroreceptors, it is possible that CP94253 may have different effects depending on what type and population of 5-HT1B receptors are affected following systemic administration. Future research may benefit from more localized manipulation of 5-HT1B receptors that may be able to isolate effects of the drug on reinforcement learning from those on anxiety. Specifically, intra-BLA infusions of CP94253 would give a more accurate estimate of the role of 5-HT1B terminal autoreceptors within the BLA on reinforcement learning. Alternatively, viral gene transfer could be used to target only the 5-HT1B recep- tors acting as autoreceptors to assess whether either the anxiogenic or the cognitive effects of CP94253 are linked with differences in receptor function. While such strategies have less translational appeal, they nonetheless could help isolate the locus of receptors essential to the clinical trait, and it is possible that in time these populations could be selectively targeted with less invasive techniques. y g q Finally, because the rats were trained under drug and tested drug free, we cannot rule out state-dependent learning effects. However, here our explicit aim was to manipulate 5-HT1B function during the acquisition of two specific action-outcome associations to assess whether this altered the nature of reward encoding. Future studies specifically targeting BLA may mitigate any potential state dependent learning effects. if In summary, the DAAM model hypothesizes that hypofunction of the BLA resulting from increased expres- sion of HTR1B leads to altered encoding of rewarding events; while basic reinforcement mechanisms are intact, individuals with PT lack detailed representations of specific outcomes and thus perform poorly when specific out- come values are changed, and representations require updating. We were able to mimic this effect with systemic administration of a 5-HT1B receptor agonist, administration of which lead to a reduced sensitivity to outcome devaluation. Future research targeting the BLA could reduce the side effect profile and provide further support for the DAAM model. Learning that is insensitive to differences in the specific features of outcomes and changes in the value of some of those outcomes, results in behaviour that is inflexible and unreceptive. In dyadic contexts where the constant monitoring of, and sensitivity to social cues is critical, this could manifest as a lack of respon- siveness to the changing behaviours and expressions of others. Discussion The individual would appear to be unempathic and emotionally indifferent; two of the hallmark features of psychopathy. Data Availabilityh y The datasets generated during and/or analysed during the current study are available from the correspond author on reasonable request. References e e e ces 1. Hare, R. D. Psychopathy as a Risk Factor for Violence. Psychiatric Quarterly 70, 181–197, https://doi.org/10.1023/a:1022094925150 (1999). 1. Hare, R. D. Psychopathy as a Risk Factor for Violence. Psychiatric Quarterly 70, 181–197, https://doi.org/10.1023/a:1022094925150 (1999). 2. Viding, E., Fontaine, N. M. G. & McCrory, E. J. Antisocial behaviour in children with and without callous-unemotional traits Journal of the Royal Society of Medicine 105, 195–200, https://doi.org/10.1258/jrsm.2011.110223 (2012). 2. Viding, E., Fontaine, N. M. G. & McCrory, E. J. Antisocial behaviour in children with and without callous-unemotional traits. Journal of the Royal Society of Medicine 105, 195–200, https://doi.org/10.1258/jrsm.2011.110223 (2012). 3. Budhani, S. & Blair, R. J. Response reversal and children with psychopathic tendencies: success is a function of salience of contingency change. Journal of child psychology and psychiatry, and allied disciplines 46, 972–981, https://doi. org/10.1111/j.1469-7610.2004.00398.x (2005). 3. Budhani, S. & Blair, R. J. Response reversal and children with psychopathic tendencies: success is a function of salience of contingency change. Journal of child psychology and psychiatry, and allied disciplines 46, 972–981, https://doi. org/10.1111/j.1469-7610.2004.00398.x (2005). g j 4. 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Incentive memory: evidence the basolateral amygdala encodes and the insular cortex retrieves outcome values to guide choice between goal-directed actions. The Journal of neuroscience: the official journal of the Society for Neuroscience 33, 8753–8763, https://doi.org/10.1523/jneurosci.5071-12.2013 (2013).h 48. Johnson, A. W., Gallagher, M. & Holland, P. C. The basolateral amygdala is critical to the expression of pavlovian and instrumental outcome-specific reinforcer devaluation effects. The Journal of neuroscience: the official journal of the Society for Neuroscience 29, 696–704, https://doi.org/10.1523/jneurosci.3758-08.2009 (2009). p g j ( ) 49. Pritchard, T. L., Weidemann, G. & Hogarth, L. Negative emotional appraisal selectively disrupts retrieval of expected outcome values required for goal-directed instrumental choice. Cognition and Emotion 32, 843–851, https://doi.org/10.1080/02699931.2017.13590 17 (2018). p g j 49. Pritchard, T. L., Weidemann, G. & Hogarth, L. Negative emotional appraisal selectively disrupts retrieval of expected outcome values required for goal-directed instrumental choice. Cognition and Emotion 32, 843–851, https://doi.org/10.1080/02699931.2017.13590 17 (2018). Scientific Reports \| (2019) 9:2497 \| https://doi.org/10.1038/s41598-019-38938-4 10 www.nature.com/scientificreports/ 0. Fournier, M., d’Arripe- Longueville, F. & Radel, R. Effects of psychosocial stress on the goal-directed and habit memory system during learning and later execution. Psychoneuroendocrinology 77, 275–283, https://doi.org/10.1016/j.psyneuen.2016.12.008 (2017) 50. Fournier, M., d’Arripe- Longueville, F. & Radel, R. Additional Information upplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-38938-4. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-38938-4. Competing Interests: The authors declare no competing interests. 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https://openalex.org/W4320037126	https://www.nature.com/articles/s41467-023-36492-2.pdf	English	null	A spectral method for assessing and combining multiple data visualizations	Nature communications	2,023	cc-by	13,969	A spectral method for assessing and com- bining multiple data visualizations Rong Ma 1, Eric D. Sun2 & James Zou 2 Rong Ma 1, Eric D. Sun2 & James Zou 2 Received: 26 July 2022 Accepted: 3 February 2023 Check for updates Dimension reduction is an indispensable part of modern data science, and many algorithms have been developed. However, different algorithms have their own strengths and weaknesses, making it important to evaluate their relative performance, and to leverage and combine their individual strengths. This paper proposes a spectral method for assessing and combining multiple visualizations of a given dataset produced by diverse algorithms. The pro- posed method provides a quantitative measure – the visualization eigenscore – of the relative performance of the visualizations for preserving the structure around each data point. It also generates a consensus visualization, having improved quality over individual visualizations in capturing the underlying structure. Our approach is ﬂexible and works as a wrapper around any visua- lizations. We analyze multiple real-world datasets to demonstrate the effec- tiveness of the method. We also provide theoretical justiﬁcations based on a general statistical framework, yielding several fundamental principles along with practical guidance. Data visualization and dimension reduction is a central topic in sta- tistics and data science, as it facilitates intuitive understanding and global views of high-dimensional datasets and their underlying struc- tural patterns through a low-dimensional embedding of the data1,2. The past decades have witnessed an explosion in machine learning algo- rithms for data visualization and dimension reduction. Many of them, such as Laplacian eigenmap3, kernel principal component analysis (kPCA)4, t-SNE5, and UMAP6, have been regarded as indispensable tools and state-of-art techniques for generating graphics in academic and professional writings7, and for exploratory data analysis and pattern discovery in many research disciplines, such as astrophysics8, com- puter vision9, genetics10, molecular biology11, especially in single-cell transcriptomics12, among others. Thus, for a given dataset, selecting the most suitable visualization method and along with its tuning parameters calls for a method that provides quantitative and objective assessment of different visualiza- tions of the dataset. On the other hand, as different methods are usually based on distinct ideas and heuristics, they would generate qualitatively diverse visualizations of a dataset, each containing important features about the data that are possibly unique to the visualization method. Article https://doi.org/10.1038/s41467-023-36492-2 1Department of Statistics, Stanford University, Stanford, CA, USA. 2Department of Biomedical Data Science, Stanford University, Stanford, CA, USA. e mail jamesz@stanford edu 1Department of Statistics, Stanford University, Stanford, CA, USA. 2Department of Biomedical Data Science, Stanford University, Stanford, CA, USA. e-mail: jamesz@stanford.edu Overview of the method Speciﬁcally, the proposed method takes as input a collection of visualizations, or low-dimensional embeddings of a dataset, here- after referred as candidate visualizations, and summarizes each visualization by a normalized pairwise-distance matrix among the samples. With respect to each sample in the dataset, we construct a comparison matrix from these normalized distance matrices, char- acterizing the local concordance between each pair of candidate visualizations. Based on eigen-decomposition of the comparison matrices, we propose a quantitative measure, referred as visualiza- tion eigenscore, that quantiﬁes the relative performance of the candidate visualizations in a sample-wise manner, reﬂecting their local concordance with the underlying low-dimensional structure contained in the data. To obtain a meta-visualization, the candidate visualizations are combined together into a meta-distance matrix, deﬁned as a row-wise weighted average of those normalized distance matrices, using the corresponding eigenscores as the weights. The meta-distance matrix is then used to produce a meta-visualization, based on an existing method such as UMAP or kPCA, which is shown to be more reliable and more informative compared to individual candidate visualizations. Our method is schematically summarized in Fig. 1 and Algorithm 1, and detailed in Method section. The thus obtained meta-visualization reﬂects a joint perspective aggregating various aspects of the data that are oftentimes captured separately by individual candidate visualizations. Here we present a spectral method for assessing and combining multiple visualizations of a given dataset produced by diverse algo- rithms, allowing for different settings of tuning parameters for indi- vidual algorithms. The proposed method provides a quantitative measure – the visualization eigenscore – of the relative performance of the visualizations for preserving the structure around each data point. Fig. 1 \| A graphical illustration of the proposed method. The algorithm takes as input the normalized pairwise distance matrices associated to a collection of can- didate visualizations (viz1 to viz4) of a dataset. For each sample of the dataset, we compute the similarity matrix between the rows of the normalized distance matrices associated to the sample (rows highlighted in the same color), and then deﬁne the corresponding eigenscores as the ﬁrst eigenvector of the similarity matrix. The size of the circles in the similarity matrices and the vectors of eigen- scores indicate the magnitude of the entries (assumed to be non-negative). The meta-distance matrix is deﬁned such that its rows are the eigenscore-weighted average of the rows in the normalized distance matrices. A spectral method for assessing and com- bining multiple data visualizations Meanwhile, due to the noisiness and high- dimensionality of many real-world datasets, their low-dimensional visualizations necessarily contain distortions from the underlying true structures, which again mayvary from one visualization to another.It is therefore of substantial practical interest to combine strengths and reach a consensus among multiple data visualizations, in order to obtain an even better meta-visualization of the data that captures the most information and is least susceptible to the distortions. Naturally, a meta-visualization would also save practitioners from painstakingly selecting a single visualization method among many. However, the wide availability and functional diversity of data visualization methods also brings forth new challenges to data analysts and practitioners13,14. On the one hand, it is critically important to determine among the extensive list which visualization method is most suitable and reliable for embedding a given dataset. In fact, even for a single visualization method, such as t-SNE or UMAP, oftentimes there are multiple tuning parameters to be determined by the users, and different tuning parameters may lead to distinct visualizations15,16. Quantitative assessment of dimension reduction and data visua- lization algorithms have been studied extensively. For example, many evaluation methods are based on distortion measures from metric geometry17–20, whereas some other methods rely on information- Nature Communications\| (2023) 14:780 1 https://doi.org/10.1038/s41467-023-36492-2 Article theoretic precision-recall measures21,22, co-ranking structure23, or graph-based criteria16,24. See also recent reviews by Bertini et al.25, Nonato and Aupetit13 and Espadoto et al.14. However, most of these existing methods evaluate data visualizations by comparing them directly with the original dataset, without accounting for its noisiness. The thus obtained assessment may suffer from intrinsic bias due to ignorance of the underlying true structures, only approximately represented by the noisy observations. It also generates a consensus visualization, having improved quality over individual visualizations in capturing the underlying structure. Our approach is ﬂexible and works as a wrapper around any visuali- zations. In particular, our approach only needs access to the low- dimensional embeddings rather than the raw data; as a result, the users can use our method even if they don’t have access to the original data, which is often the case. Results Compared to the quantitative assessment of data visualizations, there is a scarcity of meta-visualization methods that combine strengths of multiple data visualizations. Pagliosa et al.26 proposed an interactive method that assesses and combines different multi- dimensional projection methods via a convex combination techni- que. However, for supervised learning tasks such as classiﬁcation, there is a long history of research on designing and developing meta- classiﬁers that combine multiple classiﬁers27–31. Compared with meta- classiﬁcation, the main difﬁculty of meta-visualization lies in the identiﬁcation of a common space to properly align multiple visuali- zations, or low-dimensional embeddings, whose scales and coordi- nate bases may drastically differ from one to another. Moreover, unlike many meta-classiﬁers, which combines presumably indepen- dent classiﬁers trained over different datasets, a meta-visualization procedure typically relies on multiple visualizations of the same dataset, and therefore has to deal with more complicated correlation structure among the visualizations. The current study provides the ﬁrst meta-visualization method that can ﬂexibly combine any num- ber of visualizations, and has interpretable and provable perfor- mance guarantee. Article Article θ > 0, we generate fY* ig1 ≤i ≤n uniformly from each of the following three structures: θ > 0, we generate fY* ig1 ≤i ≤n uniformly from each of the following three structures: Numerically, through extensive simulations and analysis of mul- tiple real-world datasets with diverse underlying structures, we show the effectiveness of the proposed eigenscores in assessing and ranking a collection of candidate visualizations, and demonstrate the super- iority of the ﬁnal meta-visualization over all the candidate visualiza- tions in terms of identiﬁcation and characterization of these structural patterns. To achieve a deeper understanding of the proposed method, we also develop a formal statistical framework, that rigorously justiﬁes the proposed scoring and meta-visualization method, providing the- oretical insights on the fundamental principles behind the empirical success of the method, along with its proper interpretations, and guidance on practice. (i) Finite point mixture with r = 5: fY* ig1 ≤i ≤n are independently sampled from the discrete set fγ1,γ2,:::,γr + 1g Rp with equal probability, where γi’s are arbitrary orthogonal vectors in Rp with the same length, i.e., ∥γi∥2 = θ for 1 ≤i ≤r + 1. (i) Finite point mixture with r = 5: fY* ig1 ≤i ≤n are independently sampled from the discrete set fγ1,γ2,:::,γr + 1g Rp with equal probability, where γi’s are arbitrary orthogonal vectors in Rp with the same length, i.e., ∥γi∥2 = θ for 1 ≤i ≤r + 1. (ii) Smiley face with r = 2: fY* ig1 ≤i ≤n are generated independently and uniformly from a two-dimensional smiley face structure (Supplementary Fig. 1 left) of diameter θ, isometrically embedded in Rp and subject to an arbitrary rotation. (iii) Mammoth manifold with r = 3: fY* ig1 ≤i ≤n are generated inde- pendently uniformly from a three-dimensional mammoth manifold (Supplementary Fig. 1 right) of diameter θ, isome- trically embedded in Rp and subject to an arbitrary rotation. The main features of the method can be summarized as follows: • We propose a computationally efﬁcient spectral method for assessing and combining multiple data visualizations. The method is generic and easy to implement: it does not require knowledge of the original dataset, and can be applied to a large number of data visualizations generated by diverse methods. The thus generated datasets cover diverse structures including Gaussian mixture clusters (i), mixed-type nonlinear clusters (ii), and a connected smooth manifold (iii). Simulation Studies: Visualizing Noisy Low-Dimensional Structures To evaluate the proposed eigenscores, for each setting and each i ∈{1, 2, . . . , n}, we compute cos ﬀðbsi,siÞ : = ðbsiÞ > si kbsik2ksik2 , for the angle between the eigenscores bsi (see Methods) and the true local con- cordance si deﬁned as To demonstrate the wide range of applicability and the empirical advantage of the proposed method, we consider visualization of three families of noisy datasets, each containing a distinct low-dimensional structure as its underlying true signal. We assess performance of the eigenscores and the quality of the resulting meta-distance matrix based on 16 candidate visualizations produced by multiple visualiza- tion methods. si : = ððP ð1Þ i: Þ >P * i:,ðP ð2Þ i: Þ >P * i:,:::,ðP ðKÞ i: Þ >P * i:Þ 2 RK, ð1Þ ð1Þ where P * i: is the i-th row of the normalized distance matrix P * for the underlying noiseless samples fY* ig1 ≤i ≤n, deﬁned as in (9) with XðkÞ i ’s replaced by Y* i’s. Table 1 shows empirical mean and standard error (SE) of the averaged cosines 1 n Pn i = 1 cos ﬀðbsi,siÞ, over the family of datasets under the same low-dimensional structure associated with various θ as shown in Fig. 2a. Our simulations showed that cos ﬀðbsi,siÞ≈1, indicating that the eigenscores bsi essentially characterize the true concordance between the patterns contained in each candidate visualization and that of the underlying noiseless samples, evaluated locally with respect to sample i. This justiﬁes the proposed eigenscore as a precise measure of performance of the candidate visualizations in preserving the underlying true signals. For a given sample size n, we generate p-dimensional noisy observations fYig1 ≤i ≤n from the signal-plus-noise model Yi = Y* i + Zi, where fY* ig1 ≤i ≤n are the underlying noiseless samples (signals), and fZig1 ≤i ≤n are the random noises. Speciﬁcally, we generate true signals fY* ig1 ≤i ≤n from various low-dimensional structures isometrically embedded in the p-dimensional Euclidean space. Each of the low- dimensional structures lie in some r-dimensional linear subspace, and is subject to an arbitrary rotation in Rp, so that these signals are generally p-dimensional vectors with dense (nonzero) coordinates. Then we generate i. i. d. noise vector Zi from the standard multivariate normal distribution N ð0,IpÞ, and use the p-dimensional noisy vector Yi = Y* i + Zi as the ﬁnal observed data. Article • We establish rigorous theoretical justiﬁcations of the method under a general signal-plus-noise model in the large-sample limit. We prove the convergence of the eigenscores to certain underlying true concordance measures, the guaranteed perfor- mance of the meta-visualization and its advantages over alternative methods, its robustness against possible adversarial candidate visualizations, along with their conditions, interpreta- tions, and practical implications. • We establish rigorous theoretical justiﬁcations of the method under a general signal-plus-noise model in the large-sample limit. We prove the convergence of the eigenscores to certain underlying true concordance measures, the guaranteed perfor- mance of the meta-visualization and its advantages over alternative methods, its robustness against possible adversarial candidate visualizations, along with their conditions, interpreta- tions, and practical implications. Article As a result, the ﬁrst family of datasets was set to have p = 500 and n = 900, and were obtained by ﬁxing var- ious values of the SNR parameter θ, and generating Y* i 2 Rp from the above setting (i) to obtain the noisy dataset fYig1 ≤i ≤n as described above. Similarly, the second and the third families of datasets were obtained by drawing Y* i 2 Rp from the above settings (ii) and (iii), respectively, and generating datasets fYig1 ≤i ≤n with p = 300 and n = 500, for various values of θ. • For any collection of visualizations of a dataset, our method provides a quantitative measure – eigenscore – of the relative performance of the visualizations for preserving the structure around each data point. The eigenscores are useful on their own rights for assessing the local and global reliability of a visuali- zation in representing the underlying structures of the data, and in guiding selection of hyper-parameters. • The proposed method automatically combines strengths and ameliorates weakness (distortions) of the candidate visualiza- tions, leading to a meta-visualization, which is provably better than all the candidate visualizations under a wide range of set- tings. We show that the meta-visualization is able to capture diverse intrinsic structures, such as clusters, trajectories, and mixed low-dimensional structures, contained in noisy and high- dimensional datasets. For each dataset fYig1 ≤i ≤n, we consider 12 existing data visuali- zation tools including principal component analysis (PCA), multi- dimensional scaling (MDS), Kruskal’s non-metric MDS (iMDS)32, Sam- mon’s mapping (Sammon)33, locally linear embedding (LLE)34, Hessian LLE (HLLE)35, isomap36, kPCA, Laplacian eigenmap (LEIM), UMAP, t-SNE and PHATE37. For methods such as kPCA, t-SNE, UMAP and PHATE, that require tuning parameters, we consider two different settings (Sup- plementary ﬁle Section A.2) of tuning parameters for each method, denoted as kPCA1 and kPCA2, etc. Therefore, for each dataset we obtain K = 16 candidate visualizations corresponding to different combinations of visualization tools and tuning parameters. Applying our proposed method, we obtain eigenscores fbsig1 ≤i ≤n for the candi- date visualizations. We also compare two meta-distances based on the 16 visualizations, which are, the proposed spectral meta-distance matrix (meta-spec) based on the eigenscores, and the naive meta- distance matrix (meta-aver) assigning equal weights to all the candi- date visualizations, as in (4). Overview of the method The meta-distanceleads to a meta-visualization, expected to be more concordant with the underlying true structure than individual candidate visualizations. Fig. 1 \| A graphical illustration of the proposed method. The algorithm takes as input the normalized pairwise distance matrices associated to a collection of can- didate visualizations (viz1 to viz4) of a dataset. For each sample of the dataset, we compute the similarity matrix between the rows of the normalized distance matrices associated to the sample (rows highlighted in the same color), and then deﬁne the corresponding eigenscores as the ﬁrst eigenvector of the similarity matrix. The size of the circles in the similarity matrices and the vectors of eigen- scores indicate the magnitude of the entries (assumed to be non-negative). The meta-distance matrix is deﬁned such that its rows are the eigenscore-weighted average of the rows in the normalized distance matrices. The meta-distanceleads to a meta-visualization, expected to be more concordant with the underlying true structure than individual candidate visualizations. Nature Communications\| (2023) 14:780 2 2 https://doi.org/10.1038/s41467-023-36492-2 Nature Communications\| (2023) 14:780 Article Article https://doi.org/10.1038/s41467-023-36492-2 Fig. 2 \| Results from simulation studies. a Boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of the mean concordance with the underlying true pattern for 15 candidate visualizations (HLLE omittted due to very low concordance) and the two meta-distance matrices under each simula- tion setting (Left: n = 900 independent samples generated from the Gaussian mixture model; Middle: n = 500 independent samples generated from the smiley face model; Right: n = 500 independent samples generated from the mammoth model) across various values of the SNR value θ. See Supplementary Fig. 2 for complete plots. b Results for n = 900 independent samples generated under the Gaussian mixture model. Top: examples of candidate visualizations along with their sample-wise concordance fðP ðkÞ i: Þ >P * i:g1 ≤i ≤n with the structure of noiseless samples, and the proposed meta-visualization using UMAP and the concordance fðP m i: Þ >P * i:g1 ≤i ≤n for the proposed meta-distance. Bottom: boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of concordance measures as grouped by the true clusters. See Supplementary Fig. 3 for more examples. complete plots. b Results for n = 900 independent samples generated under the Gaussian mixture model. Top: examples of candidate visualizations along with their sample-wise concordance fðP ðkÞ i: Þ >P * i:g1 ≤i ≤n with the structure of noiseless samples, and the proposed meta-visualization using UMAP and the concordance fðP m i: Þ >P * i:g1 ≤i ≤n for the proposed meta-distance. Bottom: boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of concordance measures as grouped by the true clusters. See Supplementary Fig. 3 for more examples. Fig. 2 \| Results from simulation studies. a Boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of the mean concordance with the underlying true pattern for 15 candidate visualizations (HLLE omittted due to very low concordance) and the two meta-distance matrices under each simula- tion setting (Left: n = 900 independent samples generated from the Gaussian mixture model; Middle: n = 500 independent samples generated from the smiley face model; Right: n = 500 independent samples generated from the mammoth model) across various values of the SNR value θ. See Supplementary Fig. 2 for Fig. 2 \| Results from simulation studies. Simulation Studies: Visualizing Noisy Low-Dimensional Structures In this way, we simulated noisy observations fYig1 ≤i ≤n of an intrinsically r-dimensional structure. For our simulations, for some given signal-to-noise ratio (SNR) parameter To assess the quality of two meta-distance matrices, for each dataset, we compare the mean concordance 1 n Pn i = 1 ðP ðkÞ i: Þ >P * i: between Nature Communications\| (2023) 14:780 3 Article a Boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of the mean concordance with the underlying true pattern for 15 candidate visualizations (HLLE omittted due to very low concordance) and the two meta-distance matrices under each simula- tion setting (Left: n = 900 independent samples generated from the Gaussian mixture model; Middle: n = 500 independent samples generated from the smiley face model; Right: n = 500 independent samples generated from the mammoth model) across various values of the SNR value θ. See Supplementary Fig. 2 for distance is substantially more concordant with the underlying true patterns, than every candidate visualization and the naive meta-dis- tance, indicating the superiority of the proposed meta-distance. To further demonstrate the advantage of the spectral meta-distance and its beneﬁts to the ﬁnal meta-visualization, we compared our pro- posed meta-visualization using UMAP, and candidate visualizations of a dataset under setting (i) with θ = 5, and present their sample-wise concordance fðP ðkÞ i: Þ >P * i:g1 ≤i ≤n for each k, and fðP m i: Þ >P * i:g1 ≤i ≤n of the the normalized distance of each candidate visualization and that of the underlying noiseless samples, and the mean concordance 1 n Pn i = 1 ðP m i: Þ >P * i: between the obtained meta-distance and that of the underlying noiseless samples (see Methods). Figure 2a and Supple- mentary Fig. 2 show boxplots of these mean concordances for the 16 candidate visualizations and the two meta-distances under each setting of underlying structures across various values of θ. We observe that for each of the three structures, our proposed meta- Nature Communications\| (2023) 14:780 Nature Communications\| (2023) 14:780 4 https://doi.org/10.1038/s41467-023-36492-2 Article Fig. 3 \| Visualization of 590 fragments of texts from eight religious and biblical books. a Three examples of candidate visualizations. The samples are marked by eight different symbols and colors according to their associated books. More examples are included in Supplementary Fig. 5. b Boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of eigenscores for all 16 candidate visualizations, each containing n = 590 samples. c The proposed spectral meta-visualization using UMAP. d Median silhouette indi- ces over n = 590 samples for the 16 candidate and 2 meta-visualizations. Article The error bars of the meta-visualizations indicate the variability (95% conﬁdence interval over 50 rounds of repetitions) due to the visualization method (UMAP) applied to the meta-distance matrix (13). Source data are provided as a Source Data ﬁle. Fig. 3 \| Visualization of 590 fragments of texts from eight religious and biblical proposed spectral meta-visualization using UMAP. d Median silhouette indi- ces over n = 590 samples for the 16 candidate and 2 meta-visualizations. The error bars of the meta-visualizations indicate the variability (95% conﬁdence interval over 50 rounds of repetitions) due to the visualization method (UMAP) applied to the meta-distance matrix (13). Source data are provided as a Source Data ﬁle. Fig. 3 \| Visualization of 590 fragments of texts from eight religious and biblical books. a Three examples of candidate visualizations. The samples are marked by eight different symbols and colors according to their associated books. More examples are included in Supplementary Fig. 5. b Boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of eigenscores for all 16 candidate visualizations, each containing n = 590 samples. c The contains examples of candidate visualizations obtained by PHATE, t- SNE, and kPCA, whose median eigenscores were ranked top, middle and bottom among all the visualizations (Fig. 3b), respectively. More examples are included in Supplementary Fig. 5. In each visualization, the samples (text fragments) were colored by their associated books, showing how well the visualization captures the underlying clusters of the samples. The usefulness and validity of the eigenscores in Fig. 3b can be veriﬁed empirically, by visually comparing the clarity of cluster patterns demonstrated by each candidate visualizations in Fig. 3a and in Supplementary Fig. 5. Figure 3c is the proposed meta- visualization (hereafter we used meta-spec and meta-aver to refer to the ﬁnal meta-visualizations rather than the meta-distance matrices as in Section 2) of the samples by applying UMAP to the meta- distance matrix, which shows substantially better clustering of the text fragments in accordance with their sources. In addition, the meta-visualization also reﬂected deeper relationship between the eight religious books, such as the similarity between the two Hin- duism books YOG and UPA, the similarity between Buddhism (BUD) and Taoism (TTC), the similarity between the four Christian books BOE1, BOE2, BOP, and BOW, as well as the general discrepancy between Asian religions (Hinduism, Buddhism, Taoism) and non- Asian religions (Christianity). Article All of these important phenomena, while salient in our meta-visualization, only appeared vaguely in very few candidate visualizations such as those produced by PHATE (Fig. 3a) and UMAP (Supplementary Fig. 5). proposed meta-distance (Fig. 2b and Supplementary Fig. 3). We observe that, while each individual method may capture some clus- ters in the dataset but misses others, the proposed meta- visualization is able to combine strengths of all the candidate visualizations in order to capture all the underlying clusters. Finally, to demonstrate the ﬂexibility of our method with respect to higher intrinsic dimension r, under the setting (i), we further evaluated the performance of different methods for r ∈{15, 30, 50}. Supplementary Fig. 4 shows consistent and superior performance of the proposed method compared to the other approaches. Nature Communications\| (2023) 14:780 Visualizing cell cycles based on the normalized pairwise-distance matrices of the embed- dings deﬁned in (9). The Silhouette index (see Supplementary ﬁle Section A.2 for its deﬁnition), deﬁned for each individual sample in a visualization, measures the amount of discrepancy between the within- class distances and the inter-class distances with respect to a given sample. As a result, for a given visualization, its Silhouette indices altogether indicate how well the underlying cluster pattern is pre- served in a visualization, and higher Silhouette indices indicate that the underlying clusters are more separate. Empirically, we observed a notable correlation (ρ = 0.679) between the median Silhouette indices and the median eigenscores across the candidate visualizations (Sup- plementary Fig. 6). In addition, for each candidate visualization, we found that samples with higher Silhouette index tend to have higher eigenscores (Supplementary Fig. 7), demonstrating the effectiveness of eigenscores, and its beneﬁts on the ﬁnal meta-visualization. In Fig. 3d, we show that, even taking into account the stochasticity of the visualization method (UMAP) applied to the meta-distance matrix, our meta-visualization had the median Silhouette index much higher than those of the candidate visualizations, as well as that of the meta- visualization based on the naive meta-distance (meta-aver). It is of interest to note that meta-spec was the only visualization with a posi- tive median Silhouette index, showing its better separation of clusters compared with other visualizations. Importantly, the proposed meta- visualization was not sensitive to the speciﬁc visualization method applied to the meta-distance matrices – similar results were obtained when we replaced UMAP by PHATE, the method having the highest median eigenscore in Fig. 3c, or t-SNE, for meta-visualization (Sup- plementary Fig. 6). Our second real data example concerns visualization of a different low- dimensional structure, namely, a mixture of cycle and clusters, con- tained in the gene expression proﬁle of a collection of mouse embryonic stem cells, as a result of the cell cycle mechanism. The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. Identifying the cell cycle stages of individual cells analyzed during development is important for understanding its wide-ranging effects on cellular phy- siology and gene expression proﬁles. Speciﬁcally, we consider a dataset containing n = 288 mouse embryonic stem cells40, whose underlying cell cycle stages were determined using ﬂow cytometry sorting. Visualizing clusters of religious texts Cluster data are ubiquitous in scientiﬁc research and industrial appli- cations. Our ﬁrst real data example concerns n = 590 fragments of text, extracted from English translations of eight religious books or sacred scripts including Book of Proverb (BOP), Book of Ecclesiastes (BOE1), Book of Ecclesiasticus (BOE2), Book of Wisdom (BOW), Four Noble Truth of Buddhism (BUD), Tao Te Ching (TTC), Yogasutras (YOG) and Upanishads (UPA)38. All the text were pre-processed using natural language processing into a 590 × 8265 Document Term Matrix that counts frequency of 8265 atomic words, such as truth, diligent, sense, power, in each text fragment. In other words, each text fragment was treated as a bag of words, represented by a vector with 8265 features. The word counts were centred and normalized before downstream analysis. y As in our simulation studies, we still consider K = 16 candidate visualizations generated by 12 different methods with various tuning parameters (see Supplementary ﬁle Section A.2 for details). Figure 3a To quantitatively evaluate the preservation of the underlying clustering pattern, we computed for each visualization the Silhouette indices39 with respect to the underlying true cluster membership, Nature Communications\| (2023) 14:780 5 https://doi.org/10.1038/s41467-023-36492-2 Article Fig. 4 \| Visualization of the cell cycle of 288 mouse emryonic stem cells. a Three examples of candidate visualizations. The cells are marked by three different symbols and colors according to their associated cell cycle stages. More examples are included in Supplementary Fig. 16. b Boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of eigenscores for all 16 candidate visualizations, each containing n = 288 samples. c The proposed meta-visualization using kPCA. d Median Silhouette indices versus Kendall’s tau statistics for the 16 candidate and the 2 meta-visualizations (Red: proposed spectral meta-visualiza- tion; Orange: naive simple average meta-visualization). For both metrics, a higher value indicates a better visualization of the respective structure (cluster/cycle). Source data are provided as a Source Data ﬁle. Fig. 4 \| Visualization of the cell cycle of 288 mouse emryonic stem cells. a Three examples of candidate visualizations. The cells are marked by three different symbols and colors according to their associated cell cycle stages. More examples are included in Supplementary Fig. 16. b Boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of eigenscores for all 16 candidate visualizations, each containing n = 288 samples. c The proposed meta-visualization using kPCA. Visualizing clusters of religious texts d Median Silhouette indices versus Kendall’s tau statistics for the 16 candidate and the 2 meta-visualizations (Red: proposed spectral meta-visualiza- tion; Orange: naive simple average meta-visualization). For both metrics, a higher value indicates a better visualization of the respective structure (cluster/cycle). Source data are provided as a Source Data ﬁle. Fig. 4 \| Visualization of the cell cycle of 288 mouse emryonic stem cells. a Three Fig. 4 \| Visualization of the cell cycle of 288 mouse emryonic stem cells. a Three examples of candidate visualizations. The cells are marked by three different symbols and colors according to their associated cell cycle stages. More examples are included in Supplementary Fig. 16. b Boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of eigenscores for all 16 candidate visualizations, each containing n = 288 samples. c The proposed meta-visualization Visualizing cell cycles b Boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of eigenscores for all 16 count data were preprocessed and normalized (Methods), leading to a dataset consisting of standardized expression levels of 500 most variable genes for the 421 cells. visualization by applying kPCA to the meta-distance matrix. Compar- ing with Fig. 4a, the proposed meta-visualization showed better clus- tering of the cells according to their cell cycle stages, as well as a more salient cyclic structure underlying the three cell cycle stages (Supple- mentary Figure 9). To quantify the performance of each visualization in terms of these two underlying structures (cluster and cycle), we considered two distinct metrics, namely, the median Silhouette index with respect to the underlying true cell cycle stages, and the Kendall’s tau statistic41 between the inferred relative order of the cells and their true orders on the cycle. Speciﬁcally, to infer the relative order of cells, we projected the coordinates of each visualization to the two- dimensional unit circle centred at the origin (Supplementary Fig. 9), and then determined the relative orders based on the cells’ respective projected positions on the unit circle. Figure 4d shows that the pro- posed meta-visualization was signiﬁcantly better than all the candidate visualizations and the naive meta-visualization in representing both aspects of the data. Again, we obtained 16 candidate visualizations as before, and applied our proposed method. In Fig. 5a–c we show examples of candidate visualizations, boxplots of the eigenscores, and the meta- visualization using kPCA. The global (Fig. 5b) and local (Supple- mentary Fig. 10) variation of eigenscores demonstrated contribution of different visualizations to the ﬁnal meta-visualization according to their respective performance. We observed that some candidate visualizations such as kPCA, UMAP (Fig. 5a) and PHATE (Supple- mentary Fig. 11) to some extent captured the underlying trajectory structure consistent with the time course of the cells. However, the meta-visualization in Fig. 5c showed much more salient patterns in terms of both the underlying trajectory and the cluster pattern among the cells, by locally combining strengths of the individual visualizations (Supplementary Fig. 10). We quantiﬁed the perfor- mance of visualizations from these two aspects using the median Silhouette index with respect to the underlying true cluster mem- bership (i.e., batches of time course) and Kendall’s tau statistic between the inferred cell order and the true order along the pro- gression path. Visualizing cell cycles Among them, one-third (96) of the cells are in the G1 stage, one-third in the S stage, and the rest in the G2M stage. The raw count data were preprocessed and normalized, leading to a dataset consist- ing of standardized expression levels of 1147 cell-cycle-related genes for the 288 cells (Methods). We obtained 16 candidate visualizations as before, and applied our proposed method. Figure 4a contains examples of candidate visualizations obtained by t-SNE, LEIM, and kPCA, whose median eigenscores were ranked top, middle and bottom among all the visualizations, respectively, and the cells were colored according to their true cell cycle stages. Figure 4b contains the boxplots of eigen- scores for the candidate visualizations, indicating the overall quality of each visualization. The variation of eigenscores within each candidate visualization suggests that different visualizations have their own unique features and strengths to be contributed to the meta- visualization (Supplementary Fig. 8). Figure 4c is the proposed meta- Nature Communications\| (2023) 14:780 6 https://doi.org/10.1038/s41467-023-36492-2 Article Fig. 5 \| Visualization of 421 cells undergoing differentiation. a Three examples of candidate visualizations. The individual cells are marked by ﬁve different symbols and colors according to the time points they were captured and sequenced. More examples are included in Supplementary Fig. 11. b Boxplots (center line, median; box limits, upper and lower quartiles; points, outliers) of eigenscores for all 16 candidate visualizations, each containing n = 421 samples. c The proposed meta- visualization using kPCA. d Median Silhouette indices versus Kendall’s tau statistics for the 16 candidate and the 2 meta-visualizations (Red: proposed spectral meta- visualization; Orange: naive simple average meta-visualization). Source data are provided as a Source Data ﬁle. candidate visualizations, each containing n = 421 samples. c The proposed meta- visualization using kPCA. d Median Silhouette indices versus Kendall’s tau statistics for the 16 candidate and the 2 meta-visualizations (Red: proposed spectral meta- visualization; Orange: naive simple average meta-visualization). Source data are provided as a Source Data ﬁle. Fig. 5 \| Visualization of 421 cells undergoing differentiation. a Three examples of candidate visualizations. The individual cells are marked by ﬁve different symbols and colors according to the time points they were captured and sequenced. More examples are included in Supplementary Fig. 11. Visualizing cell cycles To infer the relative order of the cells from a visuali- zation, we ordered all the cells based on the two-dimensional embedding along the direction that explained the most variability of the cells. In Fig. 5d, we observed that, the proposed meta- visualization had the largest median Silhouette index as well as the largest Kendall’s tau statistic, compared with all the candidate visualizations and the naive meta-visualization, showing the super- iority of the proposed meta-visualization in both aspects. Nature Communications\| (2023) 14:780 Computational cost For datasets of moderate size as the ones analyzed in the previous sections, the proposed method had a computational cost compar- able to that of t-SNE or UMAP for generating a single candidate visualization (Supplementary Fig. 12). As for very large and high- dimensional datasets, there are a few features of the proposed algorithm that make it readily scalable. First, although our method relies on computing the leading eigenvector of generally non-sparse matrices, these matrices (i.e., Gi in Algorithm 1) are of dimension K × K, where K – the number of candidate visualizations – is usually much smaller compared to the sample size n or dimensionality p of the original data. Thus, for each sample i, the computational cost due to the eigendecomposition is mild. Second, given the candidate visualizations, our proposed algorithm is independent of the dimensionality (p) of the original dataset, as it only requires as input a set of low-dimensional embeddings produced by different visuali- zation methods. Third, since our algorithm computes the eigen- scores and the meta-distance with respect to each sample individually, the algorithm can be easily parallelized and carried out in multiple cores to further reduce time cost. Yi = Y* i + Zi, i = 1, 2,:::,n, ð2Þ ð2Þ Yi = Y* i + Zi, i = 1, 2,:::,n, ð2Þ where fY* ig1 ≤i ≤n are the underlying noiseless samples and fZig1 ≤i ≤n are the random noises. Recall that fPðkÞg1 ≤k ≤K are the distance matrices associated to the candidate visualizations (see Methods). Then, for the candidate visualizations, we consider a scaled signal-plus-noise expression p To demonstrate the computational efﬁciency of the proposed method for large and high-dimensional datasets, we evaluated the proposed method on real single-cell transcriptomic datasets43 of various sample sizes (n ∈{1000, 2000, 4000, 8000, 14000} cells of nine different cell types from the neurogenic regions of mice) and dimensions (p ∈{500, 1000, 2000} genes). For each dataset, we obtained 11 candidate visualizations and applied Algorithm 1 to generate the ﬁnal meta-visualization (Methods). Supplementary Fig. 13b contains boxplots of median Silhouette indices for each candidate visualizations and the meta-visualization (highlighted in red) with respect to the underlying true cell types, showing the stable and superior performance of the proposed method under various sample sizes and dimensions. In Supplementary Fig. Article https://doi.org/10.1038/s41467-023-36492-2 We denote these visualizations as two-dimensional embeddings fXðkÞ i g1 ≤i ≤n R2 for k ∈{1, 2, . . . , K}. As can be seen from Methods Section, there are two key ingredients of our proposed method, namely, the eigenscores fbsig1 ≤i ≤n for evaluating the candidate visuali- zations, and the meta-distance matrix P m that combines multiple candidate visualizations to obtain a meta-visualization. To formally study their properties, we introduce a generic model for the collection of K candidate visualizations produced by multiple visualization methods, with possibly different settings of tuning parameters for a single method as considered in previous sections. Speciﬁcally, we assume fYig1 ≤i ≤n are generated as Computational cost 13a, we com- pared the running time for generating the 11 candidate visualizations, and that for generating the meta-visualizations based on Algorithm 1, on a MacBook Pro with 2.2 GHz 6-Core Intel Core i7. In general, as n became large, the running time of the proposed algorithm also increased, but remained much less than that for generating the candidate visualizations. The difference in time cost became more signiﬁcant as n increased, demonstrating that for very large and high- dimensional datasets the computational cost essentially comes from generating candidate visualizations, rather than from the meta- visualization step. In particular, for dataset of sample size as large as 8000 and of dimension 2000, it took about 60 mins to generate all the 11 candidate visualizations, and took about additional 12 mins to generate the meta-visualization. Moreover, Supplementary Fig. 13a also demonstrated that, for each n, when p increased, the running time for generating the candidate visualizations was longer, but the time cost for meta-visualization remained about the same (dif- ference less than one minute). We also note that users often create multiple visualizations for data exploration, and our approach can simply reuse these visualizations with little additional computational cost. PðkÞ i: = ci,kðP* i: + hðkÞ i Þ, k = 1, 2,:::,K, ð3Þ ð3Þ induced by (2), where ci,k≥0 is a global scaling parameter, P* i: is the i-th row of the pairwise distance matrix P* = ðk Y* i Y* jk2Þ1 ≤i,j ≤n of the underlying noiseless samples, and hðkÞ i is a random vector character- izing the relative distortion of PðkÞ i: associated to the k-th candidate visualization, from the underlying true pattern P* i:. Before character- izing the distributions of fhðkÞ i g1 ≤k ≤K, we point out that, in principle, the relative distortions fhðkÞ i g1 ≤k ≤K are jointly determined by the random noises fZig1 ≤i ≤n in (2), and the features and relations between of the speciﬁc visualization methods. Importantly, in line with what is often encountered in practice, equation (3) allows for ﬂexible and possibly distinct scaling and directionality for different candidate visualiza- tions, by introducing the visualization-speciﬁc parameter ck, and by focusing on the pairwise distance matrices, rather than the low- dimensional embeddings fXðkÞ i g1 ≤i ≤n themselves. Computational cost 1 ≤i ≤n To quantitatively describe the variability of the distortions fhðkÞ i g1 ≤k ≤K across K candidate visualizations, we assume (C1a) fhðkÞ i g1 ≤k ≤K are identically distributed sub-Gaussian vectors with parameter σ2, that is, for any deterministic unit vector g 2 Rn, we have E expfðhðkÞ i Þ >gg ≤expðσ2=2Þ, and that k hðkÞ i k2 2 = cσ2nð1 + o ð1ÞÞ for some constant c > 0 with high prob- ability, that is, with probability at least 1 −n−D for some large constant D > 0 for all sufﬁciently large n. This assumption makes (3) a generative model for fPðkÞ i: g1 ≤k ≤K with ground truth P* i: and random distortions, where the variance parameter σ describes the average level of the distortions of candidate visualiza- tions from the truth after proper scaling. In relation to (2), such a condition can be satisﬁed when the signal structure fY* ig1 ≤i ≤n is ﬁnite, the noise fZig1 ≤i ≤n is sub-Gaussian, and the dimension reduction map underlying the candidate visualization is bounded and sufﬁciently smooth. See Supplementary ﬁle Section B.2 for details. In addition, we also need to characterize the correlations among these random dis- tortions, not only because the candidate visualizations are typically obtained from the same dataset fYig1 ≤i ≤n, but also because of the possible similarity between the adopted visualization methods, such as MDS and iMDS, or t-SNE under different tuning parameters. Speciﬁcally, for any j, k ∈{1, 2, . . . , K}, we deﬁne the cross-visualization covariance Σjk = EhðjÞ i ðhðkÞ i Þ >, and quantify the level of dependence between a pair of candidate visualizations by ρjk = ∥Σjk∥/σ2. By Condition (C1a), we have ρjj ≤1 for all j. For all correlation parameters fρjkg1 ≤j,k ≤K, we assume Visualizing trajectories of cell differentiation Our third real data example concerns visualization of a mixed pattern of a trajectory and clusters underlying the gene expression proﬁles of a collection of cells undergoing differentiation42. Speciﬁcally, 421 mouse embryonic stem cells were induced to differentiate into primitive endoderm cells. After the induction of differentiation, the cells were dissociated and individually captured at 12- or 24-hour intervals (0, 12, 24, 48 and 72 h), and each cell was sequenced to obtain the ﬁnal total RNA sequencing reads using the random displacement ampliﬁcation sequencing technology. As a result, at each of the ﬁve-time points, there were about 70 to 90 cells captured and sequenced. The raw 7 Nature Communications\| (2023) 14:780 (C1b) The matrix R = ðρjkÞ1 ≤j,k ≤K satisﬁes ρ ≔∥R∥= o(K). visualizations, suggesting a competitive performance of the meta- visualization. In particular, if in addition to Conditions (C1a) (C1b) and (C2) we also have kP* i:k2 σ ﬃﬃn p ≤C, that is, the magnitude of the random dis- tortions from the true structure P* i: is relatively large, then each can- didate visualization necessarily has at most mediocre performance, i.e., max1 ≤k ≤K cos ﬀðPðkÞ i: ,P* i:Þ<1 δ in probability. In such cases, the proposed meta-distances is still consistent and thus strictly better than all candidate visualizations. Theorem 2 justiﬁes the superior perfor- mance of the spectral meta-visualization demonstrated in previous sections, compared with 16 candidate visualizations. (C1b) The matrix R = ðρjkÞ1 ≤j,k ≤K satisﬁes ρ ≔∥R∥= o(K). Condition (C1b) covers a wide range of correlation structures among the candidate visualizations, allowing in particular for a subset of highly correlated visualizations possibly produced by very similar methods. The parameter ρ characterizes the overall correlation strength among the candidate visualizations, which is assumed to be not too large. As a comparison, note that a set of pairwise independent candidate visualizations implies that ρ ≈1, whereas a set of identical candidate visualizations have ρ ≈K. In particular, the requirement ρ = o(K) can be satisﬁed if, for example, among K candidate visualiza- tions, there are subsets of at most ﬃﬃﬃﬃ K p visualizations that are produced by very similar procedures, such as by the same method under dif- ferent tuning parameters, sothat ρ ≤ ﬃﬃﬃﬃ K p = oðKÞ. When Condition (C1b) fails, as all the candidate visualizations are essentially similarly dis- torted from truth, combination of them will not be substantially more informative than each individual visualization. k Among the three conditions required for the consistency of the proposed meta-distance matrix, Condition (C2) is most critical as it describes the minimal SNR requirement, that is, how much infor- mation the candidate visualizations altogether should contain about the underlying true structure of the data. In this connection, our theoretical analysis indicates that, in fact, such a signal strength condition is also necessary, not only for the proposed method, but for any possible methods. More speciﬁcally, in Supplementary ﬁle Section B.6, we proved (Theorem 4) that, it’s impossible to construct a meta-distance matrix that is consistent when Condition (C2) is violated. This result shows that the settings where our meta- visualization algorithm works well is essentially the most general setting possible. (C2) For (σ, ρ) deﬁned in (C1a) and (C1b), it holds that kP* i:k2 σ ﬃﬃn p ≫ ﬃﬃﬃﬃﬃﬃﬃﬃﬃ ρ=K p and K = o(n) as n →∞. (C2) For (σ, ρ) deﬁned in (C1a) and (C1b), it holds that kP* i:k2 σ ﬃﬃn p ≫ ﬃﬃﬃﬃﬃﬃﬃﬃﬃ ρ=K p and K = o(n) as n →∞. In our numerical studies, in addition to the proposed meta-visualiza- tion, we also considered the meta-visualization based on the naive meta-distance matrix P a, whose rows are Our algorithm is expected to perform well if ﬃﬃﬃﬃﬃﬃﬃﬃﬃ ρ=K p is small rela- tive to the overall SNR. The condition K = o(n) is easily satisﬁed for a sufﬁciently large dataset. > > > P a i: = 1 K X K k = 1 P ðkÞ i: 2 Rn, ð4Þ ð4Þ y g Recall that si = ððP ð1Þ i: Þ >P * i:,ðP ð2Þ i: Þ >P * i:,:::,ðP ðKÞ i: Þ >P * i:Þ: The following theorem concerns the convergence of eigenscores to the true con- cordance si, and is proved in Supplementary ﬁle Section B.3. which is a simple average across all the candidate visualizations. We observed in all our real-world data analyses that, such a naive meta- visualization only had mediocre performance compared to the candidate visualizations (Figs. 3, 4, and 5), much worse than the proposed spectral meta-visualization. The empirical observations suggest the advantage of informative weighting for combining candidate visualizations. Theorem 1. Under Conditions (C1a) (C1b) and (C2), for each i ∈{1, 2, . . . , n}, it holds that cos ﬀðbsi,siÞ = ðbsiÞ > si kbsik2ksik2 ! 1 in probability as n →∞. Theorem 1 implies that, as long as the candidate visualizations contain sufﬁcient amount of information about the underlying true structure, and are not terribly correlated, the proposed eigenscores fbsig1 ≤i ≤n are quantitatively reliable, as they converge to the actual quality measures fsig1 ≤i ≤n asymptotically. In other words, the eigen- scores provide a point-wise consistent estimation of the concordance between the candidate visualizations as summarized by fPðkÞg1 ≤k ≤K and the underlying true patterns P, justifying the empirical observa- tions in Table 1. Importantly, Condition (C2) suggests that our pro- posed eigenscores may beneﬁt from a larger number K of candidate visualizations, or a smaller overall correlation ρ, that is, a collection of functionally more diverse candidate visualizations. The empirically observed suboptimality of the non-informative weighting procedure can justiﬁed rigorously by theory. (C1b) The matrix R = ðρjkÞ1 ≤j,k ≤K satisﬁes ρ ≔∥R∥= o(K). Under Condition (C1a), it holds that E k hðkÞ i k2 σ ﬃﬃﬃn p . Hence, we can use the quantity kP i:k2 σ ﬃﬃn p to characterize the overall SNR in the can- didate visualizations as modelled by (3), which reﬂects the average quality of the candidate visualizations in preserving the underlying true patterns around sample i. Before stating our main theorems, we ﬁrst introduce our main assumption on the minimal SNR requirement, that is, Nature Communications\| (2023) 14:780 Robustness of spectral weighting against adversarial visualizations (C2) For (σ, ρ) deﬁned in (C1a) and (C1b), it holds that kP* i:k2 σ ﬃﬃn p ≫ ﬃﬃﬃﬃﬃﬃﬃﬃﬃ ρ=K p and K = o(n) as n →∞. Theoretical guarantees We develop a general and ﬂexible theoretical framework, to investi- gate the statistical properties of the proposed methods, as well as the fundamental principles behind its empirical success. Throughout, for a matrix A = ðaijÞ 2 Rn × n, we deﬁne its spectral norm as k A k = supkxk2 ≤1 k Axk2. For sequences {an} and {bn}, we write an = o(bn) or bn ≫an if limnan=bn = 0, and write an ≍bn if there exists constants C1, C2 > 0 such that C1bn ≤an ≤C2bn for all n. We consider visualizing a p-dimensional dataset fYig1 ≤i ≤n containing n samples. From fYig1 ≤i ≤n, suppose we obtain a collection of K (candidate) visualizations of the data, produced by various visualization methods. Nature Communications\| (2023) 14:780 8 https://doi.org/10.1038/s41467-023-36492-2 Article (C1b) The matrix R = ðρjkÞ1 ≤j,k ≤K satisﬁes ρ ≔∥R∥= o(K). Limitations of original noisy high-dimensional data The proposed eigenscores provide an efﬁcient and consistent way of evaluating the performance of the candidate visualizations. As mentioned in Introduction, a number of metrics have been proposed to quantify the distortion of a visualization by comparing the low- dimensional embedding directly with the original high-dimensional data. Such metrics essentially treat the original high-dimensional data as the ground truth, and do not take into account the noisiness of the high-dimensional data. However, for many datasets arising from real-world applications, the observed datasets, as modelled by (2), are themselves very noisy, which may not make an ideal refer- ence point for evaluating a visualization that probably has already signiﬁcantly denoised the data through dimension reduction. For example, all the three real-world datasets we have considered con- tain much more features than number of samples. In each case, there are some underlying clusters among the samples, but the original datasets showed signiﬁcantly weaker cluster structure compared to most of the 16 candidate visualizations (Supplementary Figure 14), suggesting that directly comparing a visualization with the noisy high-dimensional data may be misleading. In this respect, our theo- rems indicate that the proposed spectral method is able to precisely assess and effectively combine multiple visualizations to better grasp the underlying noiseless structure P, without referring to the original noisy datasets, making it more robust, ﬂexible, and computationally more efﬁcient. Although originally developed for data visualization, the pro- posed method can be useful for other supervised and unsupervised machine learning tasks, such as combining multiple algorithms for clustering, classiﬁcation, or prediction. For example, for a given data- set, if one has a collection of predicted cluster memberships produced by multiple clustering algorithms, one could construct cluster mem- bership matrices with (i, j)-th entry being 0 if sample i and j are not assigned to the same cluster and being 1 otherwise. Then we may deﬁne the similarity matrix as in (11), obtain the eigenscores for the candidate clusterings, and a meta-clustering using (13). It is of interest to know its empirical performance and if the fundamental principles unveiled in the current work continue to hold for such broader range of learning tasks. Discussion poor (adversarial) candidate visualizations to be combined with other relatively good visualizations, the proposed method still perform well thanks to the consistent eigenscore weighting in light of Theorem 1. On the other hand, no matter how strong the SNR is for those well- conditioned candidate visualizations, the method based on non- informative weighting is strictly sub-optimal. Indeed, when k P i:k2≫σ ﬃﬃﬃn p E k hik2, although we have cos ﬀðPðkÞ i: ,P* i:Þ ! 1 in probability for all k 2 C0, the non-informative weighting would suffer from the non-negligible negative effects from the adversarial visuali- zations in C1, causing a strict deviation from P; see, for example, Figs. 3–5 (b)(d) for empirical evidences from real-world data. poor (adversarial) candidate visualizations to be combined with other relatively good visualizations, the proposed method still perform well thanks to the consistent eigenscore weighting in light of Theorem 1. On the other hand, no matter how strong the SNR is for those well- conditioned candidate visualizations, the method based on non- informative weighting is strictly sub-optimal. Indeed, when k P i:k2≫σ ﬃﬃﬃn p E k hik2, although we have cos ﬀðPðkÞ i: ,P* i:Þ ! 1 in probability for all k 2 C0, the non-informative weighting would suffer from the non-negligible negative effects from the adversarial visuali- zations in C1, causing a strict deviation from P; see, for example, Figs. 3–5 (b)(d) for empirical evidences from real-world data. We developed a spectral method in the current study to assess and combine multiple data visualizations. The proposed meta-visualization combines candidate visualizations through an arithmetic weighted average of their normalized distance matrices, by their corresponding eigenscores. Although the proposed method was shown both in theory and numerically to outperform the individual candidate visualizations and their naive combination, it is still unclear whether there exists any other forms of combinations that lead to even better meta- visualizations. For example, one could consider constructing a meta- distance matrix using the geometric or harmonic (weighted) average, or an average based on barycentric coordinates44. We plan to investi- gate such problems concerning how to optimally combining multiple visualizations in a subsequent work. (C2) For (σ, ρ) deﬁned in (C1a) and (C1b), it holds that kP i:k2 σ ﬃﬃn p ≫ ﬃﬃﬃﬃﬃﬃﬃﬃﬃ ρ=K p and K = o(n) as n →∞. Our next theorem concerns the behavior of the proposed meta-distance matrix P m and the naive meta-distance matrix P a when combining a mixture of well-conditioned candidate visualizations, as character- ized by our assumptions (C1a) (C1b) and (C2), and some adversarial candidate visualizations whose pairwise-distance matrices does not contain any information about the true structure. Speciﬁcally, we suppose among all the K candidate visualizations, there is a collec- tion C0 of (1 −η)K well-conditioned candidate visualizations for some small η ∈(0, 1), and a collection C1 of ηK adversarial candidate visualizations. Our second theorem concerns the guaranteed performance of our proposed meta-distance matrix and its improvement upon the individual candidate visualizations in the large-sample limit. Theorem 3. For any i ∈{1, 2, . . . , n}, suppose among all the K candidate visualizations, there is a collection C0 of (1 −η)K candidate visualiza- tions for some small η ∈(0, 1) satisfying Conditions (C1a) (C1b) and (C2), and a collection C1 of ηK adversarial candidate visualizations such that ðPðkÞ i: Þ >P* i: = 0 for all k 2 C1. Then, for the proposed meta-distance P m, we still have cos ﬀðP m i: ,P* i:Þ ! 1 in probability as n →∞. However, for the naive meta-distance P a, even if k P* i:k2≫σ ﬃﬃﬃn p , we have cos ﬀðP a i:,P* i:Þ<1 η in probability as n →∞. Theorem 2. Under Conditions (C1a) (C1b) and (C2), for each i ∈{1, 2, . . . , n}, it holds that cos ﬀðP m i: ,P* i:Þ ! 1 in probability as n →∞. Moreover, for any constant δ ∈(0, 1), there exist a constant C > 0 such that, whenever k P* i:k2 ≤Cσ ﬃﬃﬃn p , we have max1 ≤k ≤K cos ﬀðPðkÞ i: ,P* i:Þ<1 δ in probability as n →∞. Theorem 2 is proved in Supplementary ﬁle Section B.4. In addition to the point-wise consistency of P m as described by cos ﬀðP m i: ,P* i:Þ ! 1 in probability, Theorem 2 also ensures that the proposed meta- distance is in general no worse than the individual candidate Theorem 3 is proved in Supplementary ﬁle Section B.5. By Theo- rem 3, on the one hand, even when there are a small portion of really Nature Communications\| (2023) 14:780 9 9 Article https://doi.org/10.1038/s41467-023-36492-2 https://doi.org/10.1038/s41467-023-36492-2 Beneﬁts of including more functionally diverse visualizations Beneﬁts of including more functionally diverse visualizations Our theoretical analysis implies that the proposed meta-visualization may beneﬁt from a large number (larger K) of functionally diverse (small ρ) candidate visualizations. To empirically verify this theoretical observation, we focused on the religious and biblical text data and the mouse embryonic stem cells data, and obtained spectral meta- visualizations based on a smaller but relatively diverse collection of 5 candidate visualizations, produced by arguably the most popular methods, namely, t-SNE, PHATE, UMAP, PCA and MDS, respectively. Compared with the 16 candidate visualizations considered earlier, here we have presumably similar ρ but much smaller K. As a result, for the religious and biblical texts data, the meta-visualization had a median Silhouette index 0.187 (Supplementary Figure 15), which was smaller than the median Silhouette index 0.275 based on the 16 candidate visualizations as in Fig. 3d; for the cell cycle data, the meta- visualization had a median Silhouette index -0.062 and a Kendall’s tau statistic 0.313, both smaller than the respective values based on the 16 candidate visualizations as in Fig. 4d. On the other hand, we also evaluated the effect when ρ is increased but K remains ﬁxed. Speciﬁ- cally, we obtained 16 candidate visualizations, all produced by PHATE with varying nearest neighbor parameters, the ﬁnal spectral meta- visualization had a median Silhouette index 0.094, which was even lower than the above meta-visualization based on ﬁve distinct meth- ods, although being still slightly better than the 16 PHATE-based can- didate visualizations (Supplementary Figure 15). These empirical evidences were in line with our theoretical predictions, suggesting beneﬁts of including more diverse visualizations. P ðkÞ = ½DðkÞ 1PðkÞ, ð5Þ ð5Þ where PðkÞ = ðk XðkÞ i XðkÞ j k2Þ1 ≤i,j ≤n and DðkÞ = diag ðk PðkÞ 1: k2,:::, k PðkÞ n: k2Þ. 2. Obtain eigenscores: for each i ∈{1, 2, . . . , n}, j 1 ≤i,j ≤n 2. Obtain eigenscores: for each i ∈{1, 2, . (i) calculate the similarity matrix Gi = ððP ðk1Þ i: Þ >P ðk2Þ i: Þ1 ≤k1,k2 ≤K: ð6Þ ð6Þ (ii) perform eigen-decomposition of Gi and deﬁne the eigenscores bsi = ð^si,1,^si,2,:::,^si,KÞ : = ∣bui∣, ð7Þ ð7Þ where bui is the eigenvector of Gi associated to its largest eigenvalue. i 3. Construct meta-distance matrix: for each i ∈{1, 2, . . . , n}, calcu- late the eigenscore-weighted average 3. Eigenscore and meta-visualization methodology Throughout, without loss of generality, we assume that for visualiza- tion purpose the target embedding is two-dimensional, although our discussion applies to any ﬁnite-dimensional embedding. Algorithm 1. Spectral assessment and combination of multiple data visualizations Algorithm 1. Spectral assessment and combination of multiple data visualizations ðkÞ Input: candidate visualizations fXðkÞ i g1 ≤i ≤n for k ∈{1, 2, . . . , K}. Input: candidate visualizations fXðkÞ i g1 ≤i ≤n for k ∈{1, 2, . . . , K}. Input: candidate visualizations fXðkÞ i g1 ≤i ≤n for k ∈{1, 2, . . . , K}. 1. Construct normalized pairwise-distance matrices: for each k { } l l Input: candidate visualizations fXð Þ i g1 ≤i ≤n for k ∈{1, 2, . . . , K}. 1. Construct normalized pairwise-distance matrices: for each k ∈{1, 2, . . . , K}, calculate 1 ≤i ≤n 1. Construct normalized pairwise-distance matrices: for each k ∈{1, 2, . . . , K}, calculate where PðkÞ = ðk XðkÞ i XðkÞ j k2Þ1 ≤i,j ≤n 2 Rn × n, ð10Þ ð10Þ P m i: = X K k = 1 ^si,k P ðkÞ i: 2 Rn: ð13Þ ð13Þ is the un-normalized Euclidean distance matrix, and DðkÞ = diag ðk PðkÞ 1: k2, k PðkÞ 2: k2,:::, k PðkÞ n: k2Þ is a diagonal matrix with its diagonal entries being the ℓ2-norms of the rows fPðkÞ 1: ,:::,PðkÞ n: g of P(k). As a result, the normalized distance matrix P ðkÞ has its rows being unit vectors, and is invariant to any scaling and rotation of the visualization fXðkÞ i g1 ≤i ≤n. ðkÞ Then, the meta-distance matrix is deﬁned as P m 2 Rn × n whose i-th row is P m i: . To obtain a meta-visualization, we take the meta-distance matrix P m and apply an existing visualization method that allows for the meta- distance P m (or its symmetrized version P m + ðP mÞ >) as its input. l f h ll l l Then, the meta-distance matrix is deﬁned as P m 2 Rn × n whose i-th row is P m i: . To obtain a meta-visualization, we take the meta-distance matrix P m and apply an existing visualization method that allows for the meta- distance P m (or its symmetrized version P m + ðP mÞ >) as its input. i i l f h i 1 2 i ll l i i l i 1 ≤i ≤n The normalized distance matrices fP ðkÞg1 ≤k ≤K summarize the candidate visualizations in a compact and efﬁcient way. Their scale- and rotation-invariance properties are particularly useful for compar- ing visualizations produced by distinct methods. Intuitively, for each i = 1, 2, . . . , n, we essentially apply a principal component (PC) analysis to the normalized distance matrix Pi = P ð1Þ i: P ð2Þ i: ::: P ðKÞ i: h i 2 Rn × K: Speciﬁcally, by deﬁnition45 the leading eigenvector bui of Gi = P > i Pi 2 RK × K is the ﬁrst PC loadings of Pi, whereas the ﬁrst PC is deﬁned as the linear combination Pibui = PK k = 1 ^ui,k P ðkÞ i: . Article Article https://doi.org/10.1038/s41467-023-36492-2 Importantly, the eigenscores essentially take the underlying true signals rather than the noisy observations fYig1 ≤i ≤n as its referential target for performance assessment, making the method easier to implement and less susceptible to the effect of noise in the original data (Supplementary Figure 14). In order that the proposed method is invariant to the respective scale and coordinate basis (i.e., directionality) of the low-dimensional embeddings generated from different visualization method, we start by considering the normalized pairwise-distance matrix for each visualization. where Under the condition that the ﬁrst PC loadings are all nonnegative (which is ensured with high probability under condi- tion (C2) below), the ﬁrst PC Pibui is exactly the meta-distance P m i: deﬁned in (13) above. When interpreted as PC loadings, the leading eigenvector bui of Gi contains weights for different vectors fP ðkÞ i: g1 ≤k ≤K so thatthe ﬁnal linear combination Pibui has the largest variance, that is, summarizes the most information contained in Pi. It is in this sense that the meta-distance P m i: is a consensus across fP ðkÞ i: g1 ≤k ≤K. Sample-wise eigenscores for assessing visualizations Our spectral method for assessing multiple visualizations is based on the normalized distance matrices fP ðkÞg1 ≤k ≤K. For each i ∈{1, 2, . . . , n}, we deﬁne the similarity matrix Gi = ððP ðk1Þ i: Þ >P ðk2Þ i: Þ1 ≤k1,k2 ≤K 2 RK × K, ð11Þ ð11Þ which summarizes the pairwise similarity between the candidate visualizations with respect to sample i. By construction, the entries of Gi are inner-products between unit vectors, each representing the normalized distances associated with sample i in a candidate visualization. Naturally, a larger entry ðP ðk1Þ i: Þ >P ðk2Þ i: indicates higher concordance between the two candidate visualizations. Then, for each i ∈{1, 2, . . . , n}, we deﬁne the vector of eigenscores bsi = ð^si,1,:::,^si,KÞ for the candidate visualizations with respect to sample i as the absolute value of the eigenvector bui 2 RK of Gi associated to its largest eigenvalue, that is, For our own numerical studies, we used UMAP for meta-visualizing datasets with cluster structures, and used kPCA for meta-visualizing all the other datasets with smoother manifold structures, such as trajec- tory, cycle, or mixed structures. The choice of UMAP in the former case was due to its advantage in treating large numbers of clusters without requiring prior knowledge about the number of clusters6,16; whereas the choice of kPCA in the latter case was rooted in its advantage in cap- turing nonlinear smooth manifold structures46. In each case, the hyper- parameters used for generating the meta-visualization were deter- mined without further tuning – for example, when using UMAP for meta-visualization, we set the hyper-parameters the same as those associated to the UMAP visualization which achieved higher median eigenscore than other UMAP visualizations. Moreover, while in general UMAP/kPCA works well as a default method for meta-visualization, our proposed algorithm is robust with respect to the choice of this ﬁnal visualization method. In our numerical analysis, we observed empiri- cally that other methods such as t-SNE and PHATE could also lead to meta-visualizations with comparably substantial improvement over individual candidate visualizations in terms of the concordance with the underlying true low-dimensional structure of the data (see Sup- plementary Figure 6). In addition, the meta-visualization shows robustness to potential outliers in the data (Figs. 4 and 5). bsi : = ∣bui∣, ð12Þ ð12Þ where the absolute value function ∣⋅∣is applied entrywise. Beneﬁts of including more functionally diverse visualizations Construct meta-distance matrix: for each i ∈{1, 2, . . . , n}, calcu- late the eigenscore-weighted average P m i: = X K k = 1 ^si,k P ðkÞ i: , ð8Þ ð8Þ and deﬁne P m 2 Rn × n whose i-th row is P m i: . i: 4. Obtain meta-visualization: apply an existing visualization method (e.g., UMAP or kPCA) to P m to obtain a meta- visualization. 4. Obtain meta-visualization: apply an existing visualization method (e.g., UMAP or kPCA) to P m to obtain a meta- visualization. Output: the eigenscores fbsig1 ≤i ≤n, and the meta-visualization. Output: the eigenscores fbsig1 ≤i ≤n, and the meta-visualization. Nature Communications\| (2023) 14:780 10 Meta-visualization using eigenscores Speciﬁcally, for each k ∈{1, 2, . . . , K}, we deﬁne the normalized pairwise-distance matrix Using the above eigenscores, one can constructa meta-distance matrix properly combining the information contained in each candidate visualization. Speciﬁcally, for each i ∈{1, 2, . . . , n}, we deﬁne the vector of meta-distances with respect to sample i as the eigenscore-weighted average of all the normalized distances respect to sample i, that is, P ðkÞ = ½DðkÞ 1PðkÞ 2 Rn × n, ð9Þ P ðkÞ = ½DðkÞ 1PðkÞ 2 Rn × n, ð9Þ ð9Þ where where References 1. Donoho, D. 50 years of data science. J. Comput. Graph. Stat. 26, 745–766 (2017). 1. Donoho, D. 50 years of data science. J. Comput. Graph. Stat. 26, 745–766 (2017). Reporting summary y g (C2’) The candidate visualizations altogether contains sufﬁcient amount of information about the underlying true structures. (C2’) The candidate visualizations altogether contains sufﬁcient amount of information about the underlying true structures. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Intuitively, Condition (C1’) concerns diversity of methods in pro- ducing candidate visualizations, whereas Condition (C2’) is related to the quality of the candidate visualizations. In practice, Condition (C2’) is satisﬁed when the signal-to-noise ratio in the data, as described by (2), is sufﬁciently large, so that the adopted visualization methods perform reasonably well on average. On the other hand, a sufﬁcient condition for (C1’) is that, at most ﬃﬃﬃﬃ K p out of K candidate visualizations are very similarly distorted from the true patterns in terms of the normalized distances P ðkÞ. This would allow, for example, groups of up to 3 to 4 candidate visualizations out of 10 to 15 visualizations being produced by very similar procedures such as the same method under different hyper-parameters. Data availability The religious and biblical text data38 are downloaded from UCI Machine Learning Repository [https://archive.ics.uci.edu/ml/machine- learning-databases/00512/]. The cell cycle analysis is based on the mouse embryonic stem cell data40 available in EMBL-EBI with accession code [E-MTAB-2805]. The cell trajectory analysis is based on the mouse embryonic stem single cell data42, available in Gene Expression Omnibus with accession code [GSE98664]. The single-cell tran- scriptomic dataset43 used for evaluating computational cost is acces- sible at BioProject with accession code [PRJNA795276]. Source data are provided with this paper. Cell cycle data The raw count data were preprocessed, normalized, and scaled by following the standard procedure (R functions CreateSeuratObject, NormalizeData and ScaleData under default settings) as incorpo- rated in the R package Seurat. We also applied the R function FindVariableFeatures in Seurat to identify 2000 most variable genes for subsequent analysis. The ﬁnal p = 1147 cell-cycle related genes were selected based on two-sample t-tests. The pre- processed data are provided in the Source Data ﬁle. The 16 can- didate visualizations were generated the same way as in the pre- vious example. 5. van der Maaten, L. & Hinton, G. Visualizing data using t-SNE. J. Mach. Learn. Res. 9, 2579–2605 (2008). 6. McInnes, L., Healy, J. & Melville, J. Umap: Uniform manifold approximation and projection for dimension reduction. arXiv pre- print arXiv:1802.03426 (2018), http://arxiv.org/abs/1802.03426. 7. Chen, C.-h., Härdle, W. K. & Unwin, A.Handbook of Data Visualization (Springer Science & Business Media, 2007), https://link.springer. com/book/10.1007/978-3-540-33037-0. https://doi.org/10.1007/ 978-3-540-33037-0. Simulations For each simulation setting, we let the diameter of the underlying structure vary within a certain range so that the ﬁnal results are comparable across different structures. The ﬁnal boxplots in Fig. 2a, and Supplementary Figures 2 and 4 summarize the simulation results across 20 equispaced diameter values for each underlying structure. The R codes of the method, and for reproducing our simulations and data analyses are available at our GitHub repository meta- visualization47https://github.com/rongstat/meta-visualization. Religious and biblical texts data 2. Chen, M., Hauser, H., Rheingans, P. & Scheuermann, G.Foundations of Data Visualization (Springer, 2020), https://link.springer.com/ book/10.1007/978-3-030-34444-3. https://doi.org/10.1007/978-3- 030-34444-3. 2. Chen, M., Hauser, H., Rheingans, P. & Scheuermann, G.Foundations of Data Visualization (Springer, 2020), https://link.springer.com/ book/10.1007/978-3-030-34444-3. https://doi.org/10.1007/978-3- 030-34444-3. Each visualization method was applied to the Document Term Matrix, with 8265 centred and normalized features and 590 samples (text fragments). The raw data are provided in the Source Data ﬁle. For the 16 PHATE-based candidate visualizations obtained in Supplementary Figure 15, we consider 16 values of nearest neighbor parameter knn ranging from 2 to 150 with equal space. 3. Belkin, M. & Niyogi, P. Laplacian eigenmaps for dimensionality reduction and data representation. Neural Comput. 15, 1373–1396 (2003). 4. Schölkopf, B., Smola, A. & Müller, K.-R. Kernel principal component analysis. In International Conference on Artiﬁcial Neural Networks, 583–588 (Springer, 1997), https://link.springer.com/chapter/10. 1007/BFb0020217. https://doi.org/10.1007/BFb0020217. Sample-wise eigenscores for assessing visualizations As explained in our main text, the nonnegative components of bsi quantify the relative performance of K candidate visualizations with respect to sample i, with higher eigenscores indicating better per- formance. Consequently, for each candidate visualization fXðkÞ i g1 ≤i ≤n, one obtains a set of eigenscores f^si,kg1 ≤i ≤n summarizing its perfor- mance relative to other candidate visualizations in a sample-wise manner. Ranking and selection among candidate visualizations can be achieved based on various summary statistics of the eigenscores, such as mean, median, or coefﬁcient of variation, depending on the speciﬁc applications. In particular, when some candidate visualiza- tions are produced by the same method but under different tuning parameters, the eigenscores can be used to select the most suitable tuning parameters for visualizing the dataset. However, a more substantial application of the eigenscores is to combine multiple data visualizations into a meta-visualization, which has improved signal-to-noise ratio and higher resolution of the structural informa- tion contained in the data. where the absolute value function ∣⋅∣is applied entrywise. As explained in our main text, the nonnegative components of bsi quantify the relative performance of K candidate visualizations with respect to sample i, with higher eigenscores indicating better per- formance. Consequently, for each candidate visualization fXðkÞ i g1 ≤i ≤n, one obtains a set of eigenscores f^si,kg1 ≤i ≤n summarizing its perfor- mance relative to other candidate visualizations in a sample-wise manner. Ranking and selection among candidate visualizations can be achieved based on various summary statistics of the eigenscores, such as mean, median, or coefﬁcient of variation, depending on the speciﬁc applications. In particular, when some candidate visualiza- tions are produced by the same method but under different tuning parameters, the eigenscores can be used to select the most suitable tuning parameters for visualizing the dataset. However, a more substantial application of the eigenscores is to combine multiple data visualizations into a meta-visualization, which has improved signal-to-noise ratio and higher resolution of the structural informa- tion contained in the data. Under a generic signal-plus-noise model, we obtain explicit the- oretical conditions under which the performance of the proposed spectral method is guaranteed. These conditions provide proper interpretations and guidance on the application of the method, such as how to more effectively prepare the candidate visualizations. Cell differentiation data 8. 8. Traven, G. et al. The galah survey: classiﬁcation and diagnostics with t-SNE reduction of spectral information. Astrophys. J. Suppl. Ser. 228, 24 (2017). 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Funding support was provided by Professor David Donoho at Stanford University (R.M.), Knight-Hennessy Scholars program (E.D.S.), Paul and Daisy Soros Fellowship for New Americans (E.D.S.), the National Science Foundation Graduate Research Fellowship Program (E.D.S.), NSF CAREER 1942926 (J.Z.), NIH P30AG059307 (J.Z.), 5RM1HG010023 (J.Z.) and grants from the Silicon Valley Foundation (J.Z.) and the Chan- Zuckerberg Initiative (J.Z.). R.M. would like to thank David Donoho and Rui Duan for helpful dis- cussions. Funding support was provided by Professor David Donoho at Stanford University (R.M.), Knight-Hennessy Scholars program (E.D.S.), Paul and Daisy Soros Fellowship for New Americans (E.D.S.), the National Science Foundation Graduate Research Fellowship Program (E.D.S.), NSF CAREER 1942926 (J.Z.), NIH P30AG059307 (J.Z.), 5RM1HG010023 (J.Z.) and grants from the Silicon Valley Foundation (J.Z.) and the Chan- Zuckerberg Initiative (J.Z.). 27. Woods, K., Kegelmeyer, W. P. & Bowyer, K. 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E.D.S. contributed to the numerical analysis and software implementation. R.M. designed and developed the theoretical results for the study. R.M. prepared a draft of the manuscript. J.Z. and E.D.S. edited the manuscript. 30. Liu, Z.-G., Pan, Q., Dezert, J. & Martin, A. Combination of classiﬁers with optimal weight based on evidential reasoning. IEEE Trans. Fuzzy Syst. 26, 1217–1230 (2017). 31. Mohandes, M., Deriche, M. & Aliyu, S. O. Classiﬁers combination techniques: A comprehensive review. IEEE Access 6, 19626–19639 (2018). Competing interests p g The Authors declare no competing interests. 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Correspondence and requests for materials should be addressed to James Zou. Peer review information Nature Communications thanks the anon- ymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Reprints and permissions information is available at http://www.nature.com/reprints © The Author(s) 2023 © The Author(s) 2023 Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional afﬁliations. Nature Communications\| (2023) 14:780 14
https://openalex.org/W3014959192	https://ieeexplore.ieee.org/ielx7/6287639/8948470/09051727.pdf	English	null	Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Function by Using Artificial Bee Colony Optimization	IEEE access	2,020	cc-by	11,938	HABIB UR RAHMAN HABIB 1,2, UMASHANKAR SUBRAMANIAM3, (Senior Member, IEEE), ASAD WAQAR 4, BASHAR SAKEEN FARHAN 5, KOTB M. KOTB 6,7, AND SHAORONG WANG 1 1School of Electrical and Electronics Engineering, Huazhong University of Science and Technology, Wuhan 430074, China 2Department of Electrical Engineering, Faculty of Electrical and Electronics Engineering, University of Engineering and Technology Taxila, Taxila 47050, Pakistan Taxila 47050, Pakistan 3Renewable Energy Laboratory, College of Engineering, Prince Sultan University, Riyadh 11586, Saudi Arabia 4Department of Electrical Engineering, Bahria University, Islamabad 44000, Pakistan 5Electrical Engineering Department, Engineering College, Al-Iraqia University, Baghdad 10053, Iraq 6Electrical Power and Machines Engineering Department, Faculty of Engineering, Tanta University, Tanta 31512, Egypt 7Electric Power Department, Faculty of Engineering and Informatics, Budapest University of Technology and Economics, 1111 Budapest, Hungary Corresponding author: Habib Ur Rahman Habib (hr_habib@hust.edu.cn) ABSTRACT The worldwide demand for reduction of CO2 pollution, with more penetration of renewable energy sources and an increased number of electric vehicles (EVs), demonstrates the importance of economic dispatch (ED) with taking into account the reduction of CO2 emission. ED is a classical problem in which EVs impose more penetration as a dynamic load, and its impact as vehicle-to-grid (V2G) is the possible future trend with cost minimization. Based on the integration of EVs and hybrid renewable sources concerning both economic dispatch and pollution minimization, the multi-objective function is converted into a single comprehensive objective by using the judgment matrix methodology. In this paper, the investigation involves the minimization of the cost of all three objectives viz. operation cost, pollution cost, and carbon emissions with ED by incorporating V2G technology. The algorithms which include particle swarm optimization, as well as artiﬁcial bee colony, are applied under various operation and control strategies. The proposed models are veriﬁed and analyzed with different case studies. In terms of operation economics, the simulation results validate the superior performance of EVs based microgrid (MG) model in the coordinated charging and discharging mode. Further, the comparison of both algorithms shows better results with the ABC algorithm in terms of cost minimization of all objectives. ABC is better in V2G based microgrid with coordinated charging and discharging mode while its performance is signiﬁcant during a large number of EVs (i.e., 700 EVs). Moreover, the load shedding scenarios are integrated which enables the MG system to operate in dual mode (i.e., seamless transition). Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Function by Using Artificial Bee Colony Optimization HABIB UR RAHMAN HABIB 1,2, UMASHANKAR SUBRAMANIAM3, (Senior Member, IEEE), ASAD WAQAR 4, BASHAR SAKEEN FARHAN 5, KOTB M. KOTB 6,7, AND SHAORONG WANG 1 Received March 14, 2020, accepted March 27, 2020, date of publication March 31, 2020, date of current version April 14, 2020. Received March 14, 2020, accepted March 27, 2020, date of publication March 31, 2020, date of current version April 14, 2020. Received March 14, 2020, accepted March 27, 2020, date of publication March 31, 2020, date of current version April 14, 2020. Digital Object Identifier 10.1109/ACCESS.2020.2984537 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 INDEX TERMS Vehicle-to-grid (V2G), electrical vehicles (EVs), particle swarm optimization (PSO), artiﬁcial bee colony (ABC), dynamic ED, operating cost (OC), pollutant treatment cost (PTC), carbon dioxide emission (CE). I. INTRODUCTION For the time being, the integration of renewable energy sources (RESs) has been signiﬁcantly increased due to the global energy decline as well as the increase of environmental concerns represented in greenhouse gases (GHG emission). However, with distributed energy resources (DERs), the con- cept of microgrid (MG) has been exaggerated [1]. It has reshaped the power grid to fully utilize both the renewable energy sources (RESs) and the distributed energy generation (DEG). Along with the revolution of MGs, the later penetration of electric vehicles (EVs) of clean energy sources that have almost zero pollutant emissions as compared to conventional combustion engines is considered the beginning of a new era [2]. Although the combustion engines are considered more economical, EVs cost is expected to be decreased when the improvement of the energy storage technology with declining fossil fuels will happen soon. The charging of EVs affects the main grid performance in terms of increasing peak hours, and ﬂuctuations of the voltage and frequency [3], [4]. With the intervention of vehicle-to-grid (V2G) technol- ogy [5], the above mentioned issues will become obsolete. With the utilization of bidirectional converters, bidirectional power ﬂow is, therefore, possible to have access to power from the utility grid and even to supply it as DERs with the use of power converters while EVs are plugged-in. However, by controlling the charging/discharging process of EVs that installed in an MG, peak load could be shifted, and therefore, a reliable and economical grid operation can be achieved [6], [7]. The OC was taken as an objective function by authors in [16] and was not considered in [17], even though the V2G operation was taken in both cases. In [18], a single cost function was assumed, and the performance of differ- ent algorithms was analyzed. Carbon dioxide emission (CE) with other objective functions was considered in [19] as multi-objective functions without considering OC, CE and pollutant treatment cost (PTC). Authors considered the only grid-connected case in [20] without considering renewables penetration with V2G technology, while no optimization with cost function was carried out. A single objective function was examined in [21] and the performance of multi-optimization algorithms was investigated without considering the V2G scenario. Authors in [22], minimization of OC was rec- ognized as a single objective function without considering OC, CE, and PTC. HABIB UR RAHMAN HABIB 1,2, UMASHANKAR SUBRAMANIAM3, (Senior Member, IEEE), ASAD WAQAR 4, BASHAR SAKEEN FARHAN 5, KOTB M. KOTB 6,7, AND SHAORONG WANG 1 In this paper, the main contribution involves penetration of EVs as dynamic load and its V2G impact in a coordinated or uncoordinated way, application of ABC algorithm for this particular load problem with improved results, and inclusion of short-term load shedding scenarios. INDEX TERMS Vehicle-to-grid (V2G), electrical vehicles (EVs), particle swarm optimization (PSO), artiﬁcial bee colony (ABC), dynamic ED, operating cost (OC), pollutant treatment cost (PTC), carbon dioxide emission (CE). INDEX TERMS Vehicle-to-grid (V2G), electrical vehicles (EVs), particle swarm optimization (PSO), artiﬁcial bee colony (ABC), dynamic ED, operating cost (OC), pollutant treatment cost (PTC), carbon dioxide emission (CE). NOMENCLATURE ABBREVIATIONS PG Power Grid WT Wind Turbine MT Micro Gas Turbine The associate editor coordinating the review of this manuscript and approving it for publication was Yanbo Chen . PV Photovoltaic DE Diesel Engine DG Diesel Generator FC Fuel Cell BSS Battery Storage System PSO Particle Swarm Optimization TP Thermal Plants DERs Distributed Energy Resources NOMENCLATURE ABBREVIATIONS PG Power Grid WT Wind Turbine MT Micro Gas Turbine MT Micro Gas Turbine The associate editor coordinating the review of this manuscript and approving it for publication was Yanbo Chen . is work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi RES Renewable Energies Sources MOED Multi-Objective ED ABC Artiﬁcial Bee Colony OC Operation Cost SOED Single Objective Economic Dispatch PTC Pollutant Treatment Cost LSS Load Shedding Scenarios CE Carbon Dioxide Emissions CHP Combined Cooling Heating and Power GA Genetic Algorithm IP Interior Point PS Pattern Search EP Evolutionary Programming EBCO Enhanced Bee Colony Optimization HIC Hybrid Imperialist Competitive GWA Grey Wolf Algorithm FLC Fuzzy Logic Controller DP Dynamic Programming MPC Model Predictive Control COM Classical Optimization Method ARO Adjustable Robust Optimization V2G Vehicle-To-Grid SOC State of charge Economic dispatch (ED) is considered the fundamen- tal concern for operation engineers working with any MG system. Dynamic ED transforms the MG model into discrete-time equations with small steps (i.e., in minutes which can be solved with 1 or 5 minutes steps) followed by static ED for solving each minute’s intervals [8]. In this way, this strategy will work as the consecutive scheduling according to the real MG operation. HABIB UR RAHMAN HABIB 1,2, UMASHANKAR SUBRAMANIAM3, (Senior Member, IEEE), ASAD WAQAR 4, BASHAR SAKEEN FARHAN 5, KOTB M. KOTB 6,7, AND SHAORONG WANG 1 g p Many researchers have chosen multi-objective function with consideration of different objectives, which include operating costs along with the emission and environmen- tal considerations with the help of various dispatch algo- rithms mentioned in [9], [10]. Many kinds of researches have been carried out about the ED as mentioned above problems which include minimum fuel cost, low SO2 emis- sions, low NOx emissions in [11]. At the same time, uni- ﬁed consideration of SO2 and NOx altogether was not assumed in terms of environmental pollution. In [12], GHG emission was analyzed, except CO2 emissions, as an indi- vidual objective function entity rather than combining it with pollutant emissions. NSGA-II was implemented in [11] and [13]; however, it was only possible for solving one dura- tion of schedule, but the dynamic ED which was designed to handle the consecutive durations, was observed in [12]. The charging/discharging and demand-side phenomena for photovoltaic based grid-connected MG with EVs, transfer- able loads and distributed resources were examined in [14]. The operating cost (OC) is considered as objective function with four cases. While multi-objective seeker optimization was applied with fuzzy logic. The authors in [15] proposed a power routing strategy for EVs based unbalanced hybrid MG system. The objective function involves minimization of power loss and power imbalance factor along with improved system load ability as well as voltage proﬁle. For DC MG system, fast EVs charging scheme is proposed in [2] with virtual-battery based droop control with improved bus voltage, ED and battery State of Charge (SOC). II. THE LOAD MODELS OF EVS A. SPATIAL AND TEMPORAL ATTRIBUTES In this paper, the main contribution involves penetration of EVs as dynamic load and its V2G impact in a coordinated or uncoordinated way, application of ABC where µs = 8.920; σs = 3.240; µt = 17.470; σt = 3.410. where µs = 8.920; σs = 3.240; µt = 17.470; σt = 3.410. EVs data follows a normal distribution, and our data which is taken from [29], follows a normal distribution, and there- fore, it is selected for this research study. I. INTRODUCTION Model predictive control was applied in [23], [24] with consideration of OC and PTC, while the V2G scenario was out of scope. Only the V2G case without optimization cost functions was studied in [25]. A single 62077 62077 VOLUME 8, 2020 VOLUME 8, 2020 H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi TABLE 1. Summary of related research on EVs. TABLE 1. Summary of related research on EVs. algorithm for this particular load problem with improved results, and inclusion of short-term load shedding scenarios. objective function was solved with the V2G case in [26] without keeping in view the integration of renewable energy resources as well as multi-objective cost functions (OC, PTC, CE). V2G with OC and CE in [27] was elaborated without PTC. In [28], V2G with OC and PTC was expressed without CE cost function and PV generation. V2G with OC and PTC was taken in [28] and [29] while the additional cost factor of CE was also considered in [29]. Table 1 shows a summary of relevant research that was performed on EVs in recent times. It is observed from Table 1 that almost all of the literature considered particular objectives out of all three proposed objectives mentioned in this study. II. THE LOAD MODELS OF EVS A. SPATIAL AND TEMPORAL ATTRIBUTES A. SPATIAL AND TEMPORAL ATTRIBUTES The proposed study presented the EVs system for domestic hybrid PEVs. The owner’s driving preferences are considered as a spatial characteristic of EVs. The probability function with consideration of daily mileage of EVs is expressed as [30]: fs(x) = 1 x . 1 σs √ 2π exp −(ln x −µs)2 2σ 2s ! (1) (1) The contribution of this paper can be highlighted as fol- lows: ED optimization considering OC, PTC, and CE using ABC algorithm, the effect of EV on ED with different charg- ing modes and scheduling strategies, The DES consisting of PV, WT, DE, FC and Battery Storage System (BSS), the results of ABC algorithms are compared with the results of PSO algorithms. Also, the similar relationship for ending time is [30]: Also, the similar relationship for ending time is [30]: ft(x) =    1 σt √ 2π exp −(x −µt)2 2σ 2t ! , µt −12 < x < 24 1 σt √ 2π exp −(x −(µt −24))2 2σ 2t ! , 0 < x < µt −12 (2) ft(x) =   ! µt −12 < x < 24 1 σt √ 2π exp −(x −(µt −24))2 2σ 2t ! , 0 < x < µt −12 (2) (2) g After a comprehensive review of literature studies, it is concluded that Economic dispatch (ED) is a classical problem in which the existing approaches rarely used detailed analy- sis of EVs as dynamic load in both coordinated as well as un-coordinated way. At the same time, ABC is not applied to this particular type of ED problem. Furthermore, short-term load shedding scenarios are also not discussed in the literature studies during ED. Due to energy shortage and greenhouse gas (GHG) emissions globally, the new future trend involves high penetration of EVs and its application as a vehicle to grid (V2G). Therefore, more advanced algorithms like ABC are required to solve more complicated ED problems. As com- pared to other existing approaches, the ABC algorithm does not trap in local minima during the search of the best opti- mal solution. The speciﬁc attribute of the ABC algorithm is the limit cycle which is identiﬁed by a scout bee to avoid local minima. B. AUTONOMOUS MODE The preference is the owners who can conveniently initiate EV charging, keeping in view any of the policies imposed by the government while the EV scheduling process is not active. The unidirectional power ﬂow with the charging period is expressed as: TC = SW100 100PCηC_EV (3) (3) where W100 shows the power utilization (kWh/100km), PC is charging power (kW), ηC_EV is charging efﬁciency. 62078 VOLUME 8, 2020 U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Function FIGURE 1. Flow diagram for computational load (a) autonomous mode. FIGURE 1. Flow diagram for computational load (a) autonomous mode. where N is the sum of vehicles; Pi(t) is the charging power; i is duration interval t (kW). Fig. 1 (a) shows the autonomous mode ﬂowchart. The sum of the charging load PEVload (t) can be calcu- lated by adding the values of every duration interval. As for charging periods of EVs are not dependent on one another; therefore, the daily load curve can be found with the following relationship as: 3) CARBON EMISSIONS COST min F = {f1(x), f2(x), . . . , fk(x)} min C3 = K X i=1 CCO2γiCO2Pi + CCO2γGridCO2PGrid (13) (13) s.t. ( gi(x) ≤0; i = 1, 2, . . . , q hj(x) = 0; j = 1, 2, . . . , p (9) (9) where CCO2γiCO2 and γGridCO2 are the carbon treatment cost, coefﬁcient emissions, and coefﬁcient of grid emissions, respectively. where x = (x1, x2, . . . , xn) are decision variables based on n-dimension; fk(x) is the kth objective, gi(x) ≤ 0 and hj(x) = 0 are inequality and equality constraints respectively. ED objectives of MG which are analyzed in this paper are as follows: Then expression for the charge duration is as follows: Then expression for the charge duration is as follows: ) The pollutant treatment cost (C2) of the MG is: TC = WEV PCηC_EV (8) (8) min C2 = K X i=1 X k (Ckγik)Pi + X k (CkγGridk)PGrid (12) (12) Equation (4) will provide a total EV charging load. A coordi- nated mode ﬂow chart is shown in Fig. 1 (b). where K, k, Ck, γik, γGridk, and PGrid are the sum of total DGs; pollutants emissions type; treatment cost; pollutant emissions coefﬁcient; coefﬁcient of grid pollutant emissions; and grid output power respectively. B. CONVERSION TO SINGLE OBJECTIVE FUNCTION j( ) q y q y p y ED objectives of MG which are analyzed in this paper are as follows: The judgment matrix methodology (JMM) expressed in [31] ﬁnds the weight of the objective, which is also a reﬂection of the situation and the owner’s priority. The proposed study applied JMM to ﬁnd the weight coefﬁcients for every objec- tive and ﬁnally combine in a single objective, as follows [29]: A. COST FUNCTIONS The function based on multiple objectives problem with n- dimension decision variables is mathematically expressed in the following way [29]: 3) CARBON EMISSIONS COST The carbon emission cost (C3) can be expressed as: 3) CARBON EMISSIONS COST The carbon emission cost (C3) can be expressed as: C. COORDINATED MODE The coordinated mode (V2G) is meant to control EVs prop- erly and centrally by keeping in view the electricity pricing policy and the behavior of the owners. Grid-connected EVs PEVload (t) = N X i=1 Pi(t) (4) (4) 62079 VOLUME 8, 2020 62079 H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi FIGURE 1. (Continued.) Flow diagram for computational load (b) coordinated mode. FIGURE 1. (Continued.) Flow diagram for computational load (b) coordinated mode. The actual discharging time TdisC when EVs are discharg- ing can be calculated as follows: which are scheduled are analyzed. The assumption is made that these EVs can be completely scheduled. EVs will be charged during off-peak load durations, while EVs will be discharged during peak load hours. TdisC = Tend_disC −Tstart_disC (6) (6) The maximum discharging duration can be calculated from the battery SOC, daily mileage, and discharging power as follows: The EV charging demand is the sum of total utilization in the everyday period, which includes daily transport utilization and discharge capacity as follows: Tmax _disC = (SOCmax −SOCmin) CEV PdisC − SW100 100PdisC (5) (5) WEV = PdisCTmax _disC −PdisCTdisC (7) (7) VOLUME 8, 2020 62080 VOLUME 8, 2020 ergy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Function H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi 3) CARBON EMISSIONS COST The carbon emission cost (C3) can be expressed as: 3) CARBON EMISSIONS COST The carbon emission cost (C3) can be expressed as: 1) OPERATING COST The OC (C1) of the MG is expressed: C = ω1C1 + ω2C2 + ω3C3 (14) (14) min C1 = CFuel + COM + CDC + MCGRID + (1 −M)CLS (10) (10) where ω1, ω2, ω3 are the weight coefﬁcients for each objec- tive. The main idea of the judgment matrix method is to ﬁnd a judgment matrix that which objective has more priority. Based on the analytic hierarchy process expressed in [32], the criteria are shown in Table 2: where CFuel, COM, CGRID, CLS, and M are fuel cost; O&M cost; interaction cost between MG and power grid (PG); compensation cost for interruptible load; grid connection/ disconnection indication respectively. M = 1 indicates MG is connected with PG, while M = 0 shows a standalone mode of the MG. TABLE 2. The criteria for the judgment matrix. In the cost functions and formulas (10), the energy loss cost is not considered. Economic dispatch (ED) problems typically handle generation sources while distribution and transmission losses are generally out of the scope of the ED problem. We picked reference paper problems [29] and applied the ABC algorithm while no change in the problem is made. Every objective (OC, PTC, CE) is graded in three cate- gories. Table 2 shows the judgment numbers with priorities, and the matrix can be expressed: The depreciation cost (CDC) is determined [29]: CDC = InCost ∗ h d(1+d)l (1+d)l−1 i Pmax ∗8760 ∗cf ∗Pi (11) J =   1 3 5 1 3 1 3 1 5 1 3 1   (15) (11) (15) where Pi, i, cf , InCost, d, l and Pmax are output power; capacity factor; installation cost; interest rate (8%); DG life- time; and DG maximum power respectively. After calculation, weight coefﬁcients are found as ω1 = 0.6370, ω2 = 0.2583, ω3 = 0.1047. According to the judgment number in table 2, there are many matrices which can be expressed, but the authors choose the formulas (15) due to the following reasons: About the pollutant emissions types and the corresponding cost, we only considered three pollutants, namely CO2, SO2, and NOx. The emission parameters along with the corre- sponding weight as well as processing costs, are mentioned in Table 5. • Because the multi-objective problem can be represented in two ways, namely single objective, and Pareto-front. 62081 VOLUME 8, 2020 H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi • As the single objective representation of the multi- objective problem is straightforward. Therefore, a judg- ment number matrix is used. where PG(t)PG(t −1)rmax and t are outputs, maximum ramp rate, and time interval, respectively. Regarding the ramp rates of different generators, the data is limited in research papers understandably, due to the con- ﬁdential bidding of various market players, often data in research papers may not be a real picture. Formula 18 shows a ramp rate for the DEs, which is taken from the base paper. Future work will further include the different ramp rates of various generators. All factors will be considered comprehen- sively in the theoretical part. • In multi-objective optimization, the objectives to be improved are conﬂict. 5) LINE TRANSMISSION CAPACITY Power ﬂow between MG and PG is expressed as: • The use of weights can be adapted as a single objective function for a multi-objective problem. −PL max ≤PGrid ≤PL max (20) where PL max is the maximum capacity. −PL max ≤PGrid ≤PL max (20) where PL max is the maximum capacity. −PL max ≤PGrid ≤PL max (20) is the maximum capacity −PL max ≤PGrid ≤PL max (20) (20) where PL max is the maximum capacity. • In the multi-objective problem, the solution is not unique, and there is no unique, natural, and canonical solution concept. where PL max is the maximum capacity. where PL max is the maximum capacity. IV. ECONOMIC DISPATCH STRATEGIES Due to clean and renewable energy from PV and wind, the maximum utilization of PV-wind is ensured in this study. Different scheduling strategies for both operating modes of the MG are as follows: C. CONSTRAINTS 1) POWER BALANCE OF MG 1) POWER BALANCE OF MG SOCmin ≤SOC(t) ≤SOCmax (21) −PBS max ≤PBS(t) ≤PBS max (22) SOCend = SOCstart + N−1 X t=0 PBS(t)ηC1t = SOCstart SOCend = SOCstart + N−1 X t=0 PBS(t) ηD 1t = SOCstart (23) N X i=1 Pi + PGrid + PBS = PLoad + PEVload (16) (21) (22) (21) (22) (16) where PLoad, PEVload, PEVload, and PBS are load; EV power; and BSS output respectively. 2) POWER LIMITS (23) DGs power limits are expressed as: Pi min ≤Pi ≤Pi max (17) (17) where SOCmin and SOCmax are the minimum and maximum SOC limits of the battery storage.PBS max, PBS(t) ηC, ηD are minimum and maximum value; maximum BSS power; charging and discharging efﬁciencies. SOCstart And SOCend are the SOCs in the beginning and end of a cycle, respectively. Taking into account that the dynamic ED strategy for the MG is performed in cycles, an assumption may be applied that the battery SOC is equal between the beginning and the end of a cycle, as shown in formula (23). where Pi min and Pi max are the minimum and maximum limit [29]. In the constraints of formulas (17), the limit of reactive power and voltage is not considered. As the EV load is active, we only considered active power, P. In ED problems, only limits of active power (P) are considered. Further, we do not measure voltage because it is not the load ﬂow problem. Also, the DE ramp rate is considered as 1 kW/min, which is taken from the base paper. Because the voltage limit is directly related to power quality and power system security, further discussion and analysis about the voltage limit will be considered in the subsequent paper involving power ﬂow analysis. 4) BATTERY SOC BSS of EVs in terms of SOC is expressed as: SOCEV min ≤SOCEV (t) ≤SOCEV max (19) (19) where SOCEV min and SOCEV max are the minimum and max- imum value of SOC [29] where SOCEV min and SOCEV max are the minimum and max- imum value of SOC [29] • If the objective function includes conﬂicted terms, then we need to treat each objective function differently. But in our case, a single-objective problem based on judg- ment number is selected. TABLE 2. The criteria for the judgment matrix. For the convenience of the description, assuming all the objectives are to be min- imized. • The ‘‘conﬂict’’ implies that there is no single solution at the same time which can fulﬁll all objectives, yet a solution set. These solutions structure the Pareto-front in the objective space, and these solutions are called Pareto-optimal solutions and structure the Pareto Set in the decision space. Moreover, these solutions ought to equally distribute on the Pareto-front. To avoid this problem, a more convenient matrix number matrix is used. 6) BATTERY OPERATION Frequent charging and discharging of the battery will severely affect the lifetime. Following are the constraints which are expressed as follows: 1) PSO PROCESS • Step 1: Start initialization of swarm with its velocity and position, coefﬁcients, and maximum iterations. • Step 2: Set the objective as a ﬁtness value. • Step 3: Calculate the ﬁtness for every swarm for personal best, while comparing with other swarms for global best. • Step 4: Modify swarm velocity and position based on Equation (24). B. ISLAND OPERATION • Step 5: Modify the personal best and global best solu- tions accordingly. The following two approaches of the scheduling strategies for economic MG operation are adopted in island mode. • Step 6: Repetitions of steps 4 and 5 until achieving the limit for maximum iterations. • Step 7: The end product is global best, personal best, and its relevant position. 1) SCHEDULING SCHEME 1 1) SCHEDULING SCHEME 1 Electric vehicles are charging in autonomous mode. The load, which includes conventional and EV charging, is supplied by DGs and the PG. Power ﬂow is in both directions.    xk+1 i,d = xk i,d + vk i,d vk i,d = ωvk i,d + c1.randk 1 . pbestk i,d −xk i,d + c2.randk 2 . gbestk d −xk i,d (24) where xk i,d, vk i,d, ω, c2, c1, pbestk i,d, gbestk d, randk 1 , and randk 2 are the position of a swarm i in k-th iteration; velocity; inertia weight factor; acceleration coefﬁcients; personal best; global best; and random numbers respectively. where xk i,d, vk i,d, ω, c2, c1, pbestk i,d, gbestk d, randk 1 , and randk 2 are the position of a swarm i in k-th iteration; velocity; inertia weight factor; acceleration coefﬁcients; personal best; global best; and random numbers respectively. 2) SCHEDULING SCHEME 2 Electric vehicles are charging and discharging in coordinated mode. During the off-peak electricity pricing hours, EVs can be charged to store energy in EV batteries for discharging them at the peak pricing hours. The load, which includes conventional and EV charging, is supplied by renewables and PG. EVs start to discharge during peak pricing hours to feed the peak load, and DGs, PG, and EVs supply the system load. EVs are further used for transportation during parity hours, and DGs and PG supply the system load which includes only the conventional. Power ﬂow is in both directions. A. BASIC ABC CONCEPT A. BASIC ABC CONCEPT In 2005, Karaboga [18] had described a bee swarm algo- rithm called an artiﬁcial bee colony (ABC) algorithm for optimizing multi-variable numerical functions. The main idea is the intelligence and behavior of honey bee movements [34]. In 2005, Karaboga [18] had described a bee swarm algo- rithm called an artiﬁcial bee colony (ABC) algorithm for optimizing multi-variable numerical functions. The main idea is the intelligence and behavior of honey bee movements [34]. Using a global search algorithm like the ABC algorithm is the best method to combat the local extremes issues [34]. There are three players in bee colony foodstuffs: i) the food sources, ii) the employed bees, and iii) the unemployed bees, which are divided into onlooker and scout bees [34]. Employed bee examines a food source and executes a negotiation dancing after returning to a colony to attract visitors to its food supply. As the length of the dancing is related to the consistency of the food supply, stronger suppliers (global optima) have a higher chance that the onlooker bees should prefer. Once a food supply is depleted, the used bee is turned into a guide bee that looks unexpectedly for a fresh supply of food. This is a crucial stage in the feeding cycle as it helps prospective food suppliers to be found at minimal cost to the colony [35] and [18]. Using a global search algorithm like the ABC algorithm is the best method to combat the local extremes issues [34]. There are three players in bee colony foodstuffs: i) the food sources, ii) the employed bees, and iii) the unemployed bees, which are divided into onlooker and scout bees [34]. Employed bee examines a food source and executes a negotiation dancing after returning to a colony to attract visitors to its food supply. A. GRID-CONNECTED OPERATION DEs ramp rate is expressed as: The following two approaches of the scheduling strategies for economic MG operation are adopted in grid-connected mode. \|PG(t) −PG(t −1)\| ≤rmax ∗1t (18) 62082 VOLUME 8, 2020 VOLUME 8, 2020 timization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Function H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi until the termination criterion is reached: until the termination criterion is reached: 1) SCHEDULING SCHEME 3 EVs are charging in autonomous mode. The system load, which is a combination of conventional and EV charging, is supplied by DGs. To avoid frequent charging and to dis- charge which will severely affect BSS life, BSS is used within a set time duration, i.e., off-peak hours from 23:00-24:00, 0:00-6:00, while peak hours from 17:00-23:00. When DGs output is not enough to meet the requirement, one portion will be shut off. VI. ABC ALGORITHM VI. ABC ALGORITHM 2) SCHEDULING SCHEME 4 EVs are charging and discharging in coordinated mode. The peak and off-peak load hours will be changed, which includes peak hours and off-peak hours. BSS is discharged 0:00-6:00 while it is charged at 17:00-24:00. The system load comprising of conventional and EV charging is supplied by distributed generators (DGS) and electric vehicles (EVs). When DGs output is not enough to meet the requirement, one portion will be shut off. A. PSO CONCEPT In the current study, the objective is to optimize the ED considering OC, PTC, and CE. The on-looker bee supervises and sends many employed bees to ﬁnd the food source (i.e., the optimal size of DGs). While the scout bee brings the same solution during each speciﬁed iterations. The on-looker bee then checks the ﬁtness (i.e., cost function) for the best solution and saves it in memory. During each iteration, an on- looker bee selects the best optimal solution out of multiple solutions after a speciﬁed number of iterations. In the second PSO is a search and intelligence-based optimization, which is suggested by Kennedy and Eberhart in 1995 [29]. The main idea is to establish a swarming group with random num- bers [33]. The position of every swarm can be represented as Xi = (xi1, xi2, . . . , xid)T , and while the velocity of every swarm is Vi = (vi1, vi2, . . . , vid)T , where i = 1, 2, . . . , n, n is the population size. Every swarm continuously adjusts its position and velocity based on the expression shown below, 62083 VOLUME 8, 2020 H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi FIGURE 2. ABC optimization technique flowchart. phase, the scout bee is instructed by an on-looker bee to ﬁnd the random food source (i.e., random solutions for DG size). For a global optimum solution, on-looker bee assigns the duty to the scout bee for a random search to avoid the trap in local minima. Therefore, ABC is a diversiﬁed algorithm that ﬁnds the global best optimal solution without trapping in the local minima, which shows its superior behavior, among other algorithms. TABLE 4. DGs parameters. TABLE 4. DGs parameters. TABLE 4. DGs parameters. (26) where Obj is the objective function, while equality and inequality constraints are represented by h Xij and g Xij respectively. The penalty factors are abbreviated λeq,m and λineq,n can be adjusted in the optimization process. glim can be deﬁned as follows: TABLE 5. Emissions parameters. glim =    Xj, if Xj,min ≤Xj ≤Xj,max Xj,min, if Xj < Xj,min Xj,max, if Xj > Xj,max (27) (27) The value of the penalty factor can be increased if one or more variables violate the limits, and the corresponding individual will, therefore, be discarded to skip the infeasible solution. The ﬂowchart of the ABC algorithm is illustrated in Fig. 2. respectively which is also mentioned in Table 3. The max- imum line capacity is 300 kW. Energy buying and selling prices are real-time values for this paper. The compensation cost for the interrupted load (LS) is 1.450 PKR/kWh, and Table 3 shows EV parameters [29], and Table 4 shows DG parameters [29]. DGs means wind, solar, diesel and fuel cells. Table 5 shows the emissions coefﬁcients and treatment costs [29]. Fig. 4 shows. Wind and solar power, load proﬁle and real electricity price [29]. Different types of EVs are considered in this research. The number of EVs ranges from a minimum value of 80 to a maximum value of 700. Regarding B. INITIAL SOLUTIONS The initial factors in the ABC algorithm are the number of food points (NFP), which is equal to the total bees. The initial population for solutions is created by random numbers, with the following relationship for random positions [18]: FIGURE 2. ABC optimization technique flowchart. Xij = Xj,min + rand × Xj,max −Xj,min , i = 1, 2, . . . NFP, j = 1, 2, . . . , J (25) (25) TABLE 3. EV parameters. where Xij is the ith population of the jth vector and NFP is set to equal 5. Xj,min and Xj,max show the minimum and maximum boundaries of the jth vector. At the same time, rand is a random number that is uniformly distributed, ranging from 0 to 1. The following relationship can represent the ﬁtness function: Fitnessi = Obj Xij + M X m=1 λeq,m h Xij 2 + N X n=1 λineq,n g Xij −glim 2 (26) TABLE 4. DGs parameters. B. COMPARISON BETWEEN PSO AND ABC Scheduling strategy-1 is chosen to be the study case to investigate the performance of PSO and ABC algorithms. Fig. 6 shows the convergence value of ﬁtness function, while Table 5 depicts parameters of PSO and proposed ABC algorithms. To ﬁnd how scalable the proposed approach is, the comparison of ﬁtness value for both algorithms (i.e., ABC and PSO) in Fig. 6 shows the fast convergence during the ABC method. Furthermore, the comparison Tables 5 also indicates the computational burden which is half in the case of the ABC method. For the computational cost of the proposed approach, Table 5 shows the simulation time of 4.098 s which is half as compared to that of PSO. Further, the computational cost is 395.01 PKR which is 2.23 % lower as compared to the PSO method. The ﬁtness function which decides the computational burden is also shown in Figure 5 which shows fast convergence of the ABC convergence curve. the speciﬁc network topology of the MG, authors have used the parameters of the base paper by keeping the same MG topology, as mentioned in the base paper. The base of the real-time price set in Fig. 4(c) is taken from an article [29] which is already mentioned in the manuscript. Fig. 3 shows the ﬂowchart of the proposed ABC and PSO optimization methodology. the speciﬁc network topology of the MG, authors have used the parameters of the base paper by keeping the same MG topology, as mentioned in the base paper. The base of the real-time price set in Fig. 4(c) is taken from an article [29] which is already mentioned in the manuscript. Fig. 3 shows the ﬂowchart of the proposed ABC and PSO optimization methodology. The current study suggests a dispatch cycle of 1 day, which is subdivided into minute steps. The population size is 5, maximum iterations are 5, and the upper bound of the acceleration coefﬁcient is set as 1. VII. STUDY SYSTEM This paper suggested an MG, including wind, solar, diesel engine, fuel cell, BSS and electric vehicles. In the grid- connected mode, the BSS is not active for scheduling due to the support from the power grid. In the islanded model, the BSS is actively participating in scheduling. There are 30 wind generators and 30 solar generators. The maximum capacity of wind and solar is 30 kW each. DEs and FCs are 60 kW each. DE ramp rate is 1 kW/min. BSS power is 150 kWh, with a power limit of 30 kW. The minimum and maximum value of SOC for BSS is 10% and 100%, 62084 VOLUME 8, 2020 VOLUME 8, 2020 U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Function FIGURE 3. Flow chart of the employed ABC and PSO optimization methodologies. FIGURE 3. Flow chart of the employed ABC and PSO optimization methodologies. 62085 VOLUME 8, 2020 H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi FIGURE 4. (a) The power output (wind & solar); (b) Load profile of MG; (c) Electricity rate. FIGURE 5. Load profile (a) autonomous mode; (b) coordinated mode. FIGURE 5. Load profile (a) autonomous mode; (b) coordinated mode. The EV charging is analyzed at 18:00-19:00, which is a busy time, as vehicle owners usually start charging. The simula- tion analysis is precisely according to the priority of vehicle owners. If a large number of vehicles are charging suddenly, it will ultimately boost the burden on the utility grid. Fig. 5b elaborates vehicle charging at off-peak hours 0:00-8:00. This is consistent with MG stability, as well as economical due to the lowest electricity price. From 8:00 to 16:00, vehi- cles are used for transportation. Vehicles are discharging at 16:00-24:00 to contribute to ED. FIGURE 4. (a) The power output (wind & solar); (b) Load profile of MG; (c) Electricity rate. A. SIMULATION ANALYSIS TABLE 6. Comparative analysis of the computational burden between two algorithms. TABLE 6. Comparative analysis of the computational burden between two algorithms. 1) DGS OUTPUT Meanwhile, when the costs of DE and FC are low as compared to PG (see Fig. 7b from 10:00-13:00), then extra power is supplied to PG for economical operation. After 16:00, the cost of DE and FC is low as compared to PG; therefore, the power grid is not used during this period. After 16:00, vehicles started to discharge and to contribute to scheduling; therefore, PG power is decreased, and vehicles began to supply energy to PG for the economical operation. After the ED of the MG, the outcome of the scheduling strategies of EVs, FCs, DEs, and PG are shown in Fig. 7. Due to the penetration maximization of renewables, the ﬁgures are not drawn with WT and PV. During the scheduling strategy-1 of the MG system, Fig. 7a shows that the power grid output is negative from 0:00 to 5:30; i.e., the MG supplies extra energy to PG. the reason is that the WT output power is higher than the require- ments of the MG. After 5:30, the renewables are ineffective in fulﬁlling the requirements; therefore, PG is used due to its lowest overall cost. Throughout the simulation period, FC is ON and is prioritized for scheduling due to a lower price. From 08:30 to 11:00, the cost of PG is more than FC; therefore, FC is added with PG to feed requirements. C. SCHEDULING ANALYSIS IN THE GRID-CONNECTED MODE 1) DGS OUTPUT A. SIMULATION ANALYSIS Fig. 5 shows the simulation analysis of the everyday load of electric vehicles in various modes. Fig. 5a shows that the higher peak load is observed during more penetration of EVs. VOLUME 8, 2020 VOLUME 8, 2020 62086 U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Function FIGURE 6. Convergence value for PSO and ABC. FIGURE 6. Convergence value for PSO and ABC. FIGURE 6. Convergence value for PSO and ABC. FIGURE 7. Economic dispatch (a) scheduling scheme 1; (b) scheduling scheme 2. FIGURE 6. Convergence value for PSO and ABC. Since ABC optimization is a heuristic search method, its optimization results have certain randomness. In all research studies, the convergence curve. normally decides the selec- tion of the most suitable solution as the authors checked PSO and ABC algorithms while all heuristic algorithms do in the same way. Hence Fig 6 shows the effectiveness of the ABC algorithm over PSO. To further select the ﬁnal solution, the authors took an average of twenty (20) optimization results, while an average of ﬁve (5) and even three (3) results were also enough in some cases where the variation trend of ran- dom numbers was negligible. FIGURE 7. Economic dispatch (a) scheduling scheme 1; (b) scheduling scheme 2. Fig. 6 elaborates that the ABC converges faster as com- pared to PSO. As depicted in Table 6, the total simulation time is also less for ABC. Based on the comparison of the convergence values of Table 6, it is veriﬁed that the proposed ABC is superior in performance as compared to PSO. From 11:00 to 24:00, DE and FC have higher prices than PG, so PG for better economics supplies load demand. When the scheduling strategy 2 of M is adopted, Fig. 7b shows that the vehicle’s load is supplied at 0:00-8:00. Overall costs of PG and DE are lower than FC, therefore both pro- vide energy with less power contribution from FC. Between 02:00-04:00 and 06:00-08:00, DE has a lower cost. During the interval starting from 8:00 to 16:00, vehicles are not active for scheduling. Before 13:00, the cost of DE is low as compared to FC and power grid, so it supplies more energy with FC and power grid. During interval 13:00-16:00, cost of the power grid and DE is low as compared to FC, therefore both supplies more energy with FC. 2) COST FUNCTION Table 7 shows the cost for individual functions as well as an overall comprehensive single objective for both strategies. Table 7 depicts average values after repeatedly computing ten times. 62087 VOLUME 8, 2020 H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi FIGURE 8. Dispatch results (a) scheduling scheme 3; (b) scheduling scheme 4. TABLE 7. Grid-connected scheme: dispatch results (pkr) under scheduling schemes 1 and 2. TABLE 7. Grid-connected scheme: dispatch results (pkr) under scheduling schemes 1 and 2. From the values of Table 7, it is observed that selecting scheduling strategy 2 with ABC algorithm decreases the cost by 18.79%, which is the single objective cost as compared to the selection of strategy 1. Further, it reduces 17.76% of the operating cost, 29.84% pollutant emissions, and 25.52% the carbon dioxide emissions. While simulation studies with the same strategies are also carried out by using the PSO algorithm, it is observed that selecting scheduling strategy 2 with the PSO algorithm decreases the cost by 18.52%, which is the single objective cost as compared to the selection of strategy 1. Further, it reduces 17.59% of the operating cost, 29.82% pollutant emissions, and 25.17% the carbon dioxide emissions. The simulation study elaborates that vehicle coor- dinated mode can reduce all three objectives costs during the grid-connected mode of the MG. This is also veriﬁed that the best results are found with the proposed ABC algorithm, as shown in Table 7. FIGURE 8. Dispatch results (a) scheduling scheme 3; (b) scheduling scheme 4. Different results obtained in Table 8 are analyzed that all the objectives are achieved with their best costs by opting proposed ABC algorithm as compared to the PSO algorithm. This concludes the observation shown in Table 8 that ABC is showing slightly better performance as compared to the PSO algorithm to minimize the comprehensive cost (1.46%), operating cost (0.97%), carbon emission cost (0.07%), and pollutant treatment cost which is 1.39%. Due to the penetration maximization of renewables, the ﬁg- ures are not drawn with WT and PV. When scheduling strategy 3 of the MG system is adopted, Fig. 8a shows that the BSS is charging during the period 0:00-6:00, because the MG load is less, and DE and FC supply power afterward. 2) COST FUNCTION From 12:00 to 13:00, FC is unable to meet the demand, and BSS is not in service so far, while power shortfall is observed, therefore part of the load needs to be shut off. During the time interval, 15:00-16:00. EVs are connected for charging, and the BSS feeds the excess power. During 16:00-22:00, more shortage is observed due to vehicle charging, and meanwhile, BSS starts to discharge energy for low peak and low power shortage. After 22:00, MG demand is reduced and wind, solar, DE, and FC are fulﬁlling the load requirements as well as charging of the BSS. TABLE 8. Comparison (pkr) of PSO and ABC under scheduling strategies 1 and 2. TABLE 8. Comparison (pkr) of PSO and ABC under scheduling strategies 1 and 2. During MG operation with scheduling strategy 4, Fig. 8b shows that vehicle charging occurred at 0:00-8:00, which drastically increases the power demand while DE and FC powers are sufﬁcient to fulﬁll this requirement, so the BSS is controlled to remain in idle state. Time duration at 8:00-16:00 is not involved in vehicle scheduling, and the power demand is supplied by both DE and FC. DE and FC power is unable to fulﬁll load requirements at 16:00-17:00, and the BSS supplies the power mismatch. After 16:00, vehicles start discharging to contribute to scheduling while charging the BSS at the same time. Further, the FC is not participating from 18:00 to 22:00 due to the high participating Based on Fig. 8a and Fig. 8b, vehicles are charging during off-peak hours, and discharging during peak hours to accom- plish the target of peaks shifting as well as to lower the cost of buying energy units from PG. Therefore, vehicles are efﬁcient in coordinated mode and are contributing to MG stability as well as cost reduction. 1) DGS OUTPUT After the ED of the MG, the outcome of the scheduling strategies of EVs, FCs, DEs, and PG are shown in Fig. 9. Due to the penetration maximization of renewables, the ﬁgures are not drawn with WT and PV. The data of Table 9 shows improvement results after adopting the scheduling strategy 4. ABC algorithm reduces the overall single objective cost by 25.17% as compared with taking the scheduling strategy 3. Further, the scheduling strategy 4 reduces the operating cost by 25.55%, reduces the pollutant emissions by 20.38%, and reduces the carbon dioxide emissions by 28.72%. When the PSO algorithm is applied with the same strategies, it is observed that it reduces the overall single objective cost by 18.59% as compared with adopting the scheduling strategy 3. Further, the scheduling strategy 4 reduces the operating cost by 19.82%, reduces the pollutant emissions by 3.59%, and reduces the carbon dioxide emissions by 18.89%. This veriﬁes the superior performance of the proposed methodology with ABC that the vehicle coordination for charging and discharging mode is reducing the operating cost, pollutant emissions, and the carbon dioxide emissions while MG is running under the standalone (islanded) mode. Vehicles charging enhances peak loads while following the vehicles (EVs) charging in autonomous mode, leading to a substantial recompense cost due to the loss of interruptible loads (LOIL), as shown in Fig. 6a. At the same time, the vehicles operating in discharging mode obviates a signiﬁcant part of LOIL while pursuing the vehicles charging and discharging in coordi- nation mode (see Fig. 6b), hence it reduces the operating cost, pollutant emissions, and the carbon dioxide emissions. By concluding the remarks of this section, the selection of the vehicle’s coordination in charging and discharging mode will surely trim the demand shortage during the MG operation in standalone mode, which will assist in getting a better operation in terms of economics. During the scheduling strategy 1 of the MG system, forced load shedding is carried out from 01:40 to 02:20, as shown in Fig. 9(a1). However, the power grid output is positive throughout the simulation time; i.e., the power grid supplies excess electricity to the MG. This is because the PV output power is zero, while wind power is insufﬁcient to feed the load requirements. So, the power grid is used due to its low- est overall objective cost. 2) COST FUNCTION TABLE 10. Comparison (pkr) of pso and abc under scheduling strategies 3 and 4. TABLE 10. Comparison (pkr) of pso and abc under scheduling strategies 3 and 4. Table 9 shows the cost for individual functions as well as an overall comprehensive single objective for both strategies. Table 9 depicts average values after repeatedly computing 10 times. TABLE 9. Island Operation: dispatch results (pkr) under scheduling schemes 3 and 4. TABLE 9. Island Operation: dispatch results (pkr) under scheduling schemes 3 and 4. D. SCHEDULING ANALYSIS IN THE ISLAND MODE After the ED of the MG, the outcome of the scheduling strategies of EVs, FCs, DEs, and PG are shown in Fig. 6. VOLUME 8, 2020 VOLUME 8, 2020 62088 H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi showing signiﬁcantly better performance to minimize the comprehensive cost (35.40%), operating cost (28.91%), and carbon emission cost (52.04%). In comparison, drastically signiﬁcant improvement with ABC algorithm is observed in reducing pollutant treatment cost which is 467.69%. showing signiﬁcantly better performance to minimize the comprehensive cost (35.40%), operating cost (28.91%), and carbon emission cost (52.04%). In comparison, drastically signiﬁcant improvement with ABC algorithm is observed in reducing pollutant treatment cost which is 467.69%. cost. After 18:00, DE and FC power-sharing are gradually decreasing due to their higher price as compared to that of V2G until FC is not involved in delivering power at 18:00 while DE reduces its output from 20:00 onwards. VOLUME 8, 2020 1) DGS OUTPUT Throughout the simulation period, DE is enabled and is prioritized to be scheduled due to its lower objective cost. From 14:00 to 22:00, the cost of PG turns lowest among all DGs, so the power grid feeds most of the power demand. From 07:00 to 24:00, DE and FC costs are higher than that of PG; hence most of the load demand is supplied by PG for improvement of the economics. Similarly, Fig. 9(a2) shows the DGs output power dur- ing load shedding from 10:00 to 11:40, while Fig. 9(a3) shows the DGs power when load shedding is executed from 16:40 to 20:00. When the scheduling strategy 2 of MG is adopted, forced load shedding is carried out from 01:40 to 02:20, which is shown in Fig. 9(b1). Fig. 9(b1) depicts the vehicle charging load at 0:00-8:00, while overall PG cost is lower than FC and DE, so the power grid provides output power together with less power contribution from FC and DE. From 8:00 to 16:00, vehicles are not participating in scheduling, while 16:00-19:00 time range shows lower DE cost as compared to the FC; therefore, it supplies power. After 16:00, vehicles follow the discharging mode, and contributing in the schedul- ing; therefore PG power is decreased, and vehicles started to provide output to PG, and the economic beneﬁts for vehicle owners are ensured. Different results obtained in Table 10 are analyzed that all the objectives are achieved with their best costs by opting proposed ABC algorithm as compared to the PSO algorithm. This concludes the observation shown in Table 12 that ABC is Similarly, Fig. 9(b2) shows the DGs output power dur- ing load shedding from 10:00 to 11:40, while Fig. 9(b3) shows the DGs power when load shedding is executed from 16:40 to 20:00. 62089 VOLUME 8, 2020 H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi FIGURE 9. Dispatch results with load shedding scenarios 1-3 (a) scheduling scheme 1; (b) scheduling scheme 2. FIGURE 9. Dispatch results with load shedding scenarios 1-3 (a) scheduling scheme 1; (b) scheduling scheme 2. It is observed from Table 11 that by selecting scheduling strategy, 2 for LSS-1 with PSO reduces by 78.29% the com- prehensive single objective cost as compared to the selection of scheduling strategy 1. 1) DGS OUTPUT While strategy-2 also reduces by 79.25% the operating cost, reduces by 57.04% pollutant emis- sions, and decreases by 90.72% the carbon dioxide emissions. Results for scheduling strategy 2 for LSS-2 and LSS-3 with PSO are also shown in Table 11. It is also observed from Table 11 that by selecting scheduling strategy, 2 for LSS-1 with ABC reduces by 79.57% the comprehensive single objective cost as compared to the selection of schedul- ing strategy 1. While strategy-2 also reduces by 80.55% the operating cost, reduces by 58.59% pollutant emissions, and decreases by 92.23% the carbon dioxide emissions. To verify the work and to validate the presented method sufﬁciently, the authors run multiple simulation results. Table 6-9 shows the average values of twenty (20) simulation results, while one solution set in the form of Figures 6-8 is selected, and almost the same DGs trend was observed in these results. Hence, more results were only skipped for the reader to avoid any repetition with more number of pages. 2) COST FUNCTION Table 11 shows the cost for individual functions as well as an overall comprehensive single objective for both strategies. Table 11 depicts average values after repeatedly computing two times. VOLUME 8, 2020 62090 H. U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Functi TABLE 11. Grid-connected Operation: dispatch results (pkr) under scheduling schemes 1 and 2. TABLE 11. Grid-connected Operation: dispatch results (pkr) under scheduling schemes 1 and 2. TABLE 12. Comparative analysis (pkr) of PSO and ABC under load shedding scenarios. TABLE 12. Comparative analysis (pkr) of PSO and ABC under load shedding scenarios. Results for scheduling strategy 2 for LSS-2 and LSS-3 with ABC are also shown in Table 11. This simulation result elaborates that the vehicle’s coordination in charging and discharging mode is reducing the operating cost, carbon emissions and pollutant emissions during the grid-connected mode of the MG. This is also veriﬁed that all the objectives are achieved with their best prices by opting proposed ABC algorithm as compared to the PSO algorithm. Contradictory performance between PSO and ABC is observed only in one case i.e., LSS-3, when PSO beats ABC in cost minimization of three objectives. In this case, PSO reduces by 53.76% the comprehensive single objective cost as compared to the selec- tion of scheduling strategy 1. While strategy-2 also reduces by 53.70% of the operating cost, reduces by 52.16% pollutant emissions, and decreases by 62.48% of the carbon dioxide emissions. units from PG. Therefore, the vehicle’s coordination in both (charging and discharging) mode under different LSSs con- tributes to the MG stability, as well as declines the compre- hensive single objective cost, the operating cost, pollutant, and the carbon dioxide emissions. Similarly, LSS-2 during load shedding from 10:00 to 11:40 while LSS-3 during load shedding duration from 16:40 to 20:00 also shows a similar trend in reducing the overall single objective function cost as well as the operating cost, pollutant and the carbon dioxide emissions. On the other hand, ABC reduces by 51.20% the compre- hensive single objective cost as compared to the selection of scheduling strategy 1. While strategy-2 also reduces by 52.03% the operating cost, reduces by 31.36% pollutant emis- sions, and decreases by 59.79% of the carbon dioxide emis- sions. 2) COST FUNCTION Different results shown in Table 12 are analyzed that all the objectives are achieved with their best costs by opting for the PSO algorithm instead of the proposed ABC algorithm. This concludes the observation shown in Table 12 that PSO is showing slightly better performance during peak load hours to minimize the comprehensive cost (4.76%), operating cost (3.11%) and carbon emission cost (4.31%) while signiﬁcant improvement with PSO is observed in reducing pollutant treatment cost which is 39.88%. VOLUME 8, 2020 REFERENCES [1] R. H. Lasseter and P. Piagi, ‘‘Microgrid: A conceptual solution,’’ in Proc. PESC, Jun. 2004, pp. 4285–4290. [23] Y. Li, X. Fan, Z. Cai, and B. Yu, ‘‘Optimal active power dispatching of microgrid and distribution network based on model predictive control,’’ Tsinghua Sci. 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Further, V2G based multi- objective ED model of the MG system is developed, and the judgment matrix methodology is used to convert it into a single objective. Keeping in view the safety constraints, the model validity, along with the algorithms, is veriﬁed through simulation of the case studies by using PSO and ABC algorithms under different scheduling strategies. With the use of the ABC algorithm, the simulation studies depict the superior performance of vehicles with charging/discharging in a coordinated mode in terms of better operation economics as compared to the autonomous charging mode. Further, the dual-mode (seamless transition) is also analyzed during load shedding hours with consideration of three different sce- narios. 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Wang, ‘‘Optimized daily dispatching strategy of building-integrated energy systems consider- ing vehicle to grid technology and room temperature control,’’ Energies, vol. 11, no. 5, p. 1287, 2018. [17] L. Polanco Vasquez, C. Carreño Meneses, A. Pizano Martínez, J. López Redondo, M. Pérez García, and J. Álvarez Hervás, ‘‘Optimal energy man- agement within a microgrid: A comparative study,’’ Energies, vol. 11, no. 8, p. 2167, 2018. [18] W.-M. Lin, C.-S. Tu, and M.-T. Tsai, ‘‘Energy management strategy for microgrids by using enhanced bee colony optimization,’’ Energies, vol. 9, no. 1, p. 5, 2015. [19] J. Ben Hmida, M. Javad Morshed, J. Lee, and T. Chambers, ‘‘Hybrid imperialist competitive and grey wolf algorithm to solve multiobjective optimal power ﬂow with wind and solar units,’’ Energies, vol. 11, no. 11, p. 2891, 2018. ACKNOWLEDGMENT REFERENCES 150, Jan. 2020, Art. no. 107040. HABIB UR RAHMAN HABIB was born in Fort Abbas, Pakistan, in 1988. He received the B.Sc. degree in electrical engineering and the M.Sc. degree in electrical power engineering from the University of Engineering and Technology at Tax- ila, Pakistan, in 2009 and 2015, respectively. He is currently pursuing the Ph.D. degree with the State Key Laboratory of Advanced Electro- magnetic Engineering and Technology, Huazhong University of Science and Technology, Wuhan, China. Since 2009, he has been with the COMSATS Institute of Informa- tion Technology, Pakistan, and the Wah Engineering College, University of Wah, Pakistan. He has been a Management Trainee Ofﬁcer (Maintenance Department) with Dynamic Packaging Pvt., Ltd., Lahore, Pakistan. He is also permanently with the Department of Electrical Engineering, University of Engineering and Technology at Taxila. His research interests include electrical planning and estimation, energy resources and planning, modeling and simulation, renewable energy technology and management, smart grid applications in power systems, distributed generation and microgrid, power electronics, power quality, artiﬁcial intelligence, deep learning, fuzzy con- trollers, heuristic algorithms design and optimization, energy economics, and model predictive control and its application in the power industry. BASHAR SAKEEN FARHAN received the B.Sc. degree in electrical and electronic engineering from the College of Engineering, Al-Mustansiriya University, Iraq, in 2004, the M.Tech. degree in power system from SHIATS, India, in 2013, and the Ph.D. degree in electrical engineering from HUST, China, 2017. He was an Engineer to Bagdad Governorate–Bagdad Governorate Direc- torate of Water, Iraq, from 2005 to 2019. He joined the Electrical Engineering Department, Engineer- ing College, Al-Iraqia University, in December 2019. KOTB M. KOTB received the B.Sc. and M.Sc. degrees in electrical engineering from Tanta Uni- versity, Egypt, in 2012 and 2017, respectively. He is currently pursuing the Ph.D. degree with the Budapest University of Technology and Eco- nomics, Budapest, Hungary. He started working as a Teaching Assistant at the Department of Electri- cal Power and Machines Engineering, Faculty of Engineering, Tanta University, in 2013, where he was promoted to the degree of Assistant Lecturer, in May 2017. His research interests include power electronics, renewable energy systems, and microgrid technologies. UMASHANKAR SUBRAMANIAM (Senior Member, IEEE) is currently an Associate Pro- fessor with the Renewable Energy Laboratory, College of Engineering, Prince Sultan University, Saudi Arabia. He has more than 15 years of teach- ing, research, and industrial R&D experience. REFERENCES Res., vol. 179, May 2020, Art. no. 106058. [28] R. Shi, S. Li, C. Sun, and K. Lee, ‘‘Adjustable robust optimization algo- rithm for residential microgrid multi-dispatch strategy with consideration of wind power and electric vehicles,’’ Energies, vol. 11, no. 8, p. 2050, 2018. [8] M. Basu, ‘‘Dynamic economic emission dispatch using nondominated sorting genetic algorithm-II,’’ Int. J. Electr. Power Energy Syst., vol. 30, no. 2, pp. 140–149, Feb. 2008. [29] H. Liu, Y. Ji, H. Zhuang, and H. Wu, ‘‘Multi-objective dynamic economic dispatch of microgrid systems including vehicle-to-grid,’’ Energies, vol. 8, no. 5, pp. 4476–4495, 2015. [9] X. Zhang, K. W. Chan, H. Wang, B. Zhou, G. Wang, and J. Qiu, ‘‘Multiple group search optimization based on decomposition for multi-objective dispatch with electric vehicle and wind power uncertainties,’’ Appl. Energy, vol. 262, Mar. 2020, Art. no. 114507. [30] D. Vyas and A. Santini. Use of National Surveys for Estimating ‘Full’ PHEV Potential For Oil Use Reduction. [Online]. Available: https://www.anl.gov/es 62092 VOLUME 8, 2020 U. R. Habib et al.: Energy Cost Optimization of Hybrid Renewables Based V2G Microgrid Considering Multi Objective Function [31] Z.-S. Xu and J. Chen, ‘‘Approach to group decision making based on interval-valued intuitionistic judgment matrices,’’ Syst. Eng.-Theory Pract., vol. 27, no. 4, pp. 126–133, Apr. 2007. ASAD WAQAR received the Electrical Engineer- ing degree from UET Taxila, in 2002, the mas- ter’s degree in electrical power engineering from RWTH Aachen, Germany, in 2011, and the Ph.D. degree in electrical engineering from the Huazhong University of Science and Technology, China, in 2016. He is currently working as an Associate Professor with the Department of Elec- trical Engineering, Bahria University, Islamabad, Pakistan. His research interests include smart grids, microgrids operation and control, power quality, power electronics, energy economics, and network reinforcement planning. [32] J. Kennedy, ‘‘Particle swarm optimization,’’ in Encyclopedia of Machine Learning and Data Mining, C. Sammut and G. I. Webb, Eds. Boston, MA, USA: Springer, 2010, pp. 760–766. [33] T. L. Saaty, ‘‘How to make a decision: The analytic hierarchy process,’’ Interfaces, vol. 24, no. 6, pp. 19–43, Dec. 1994. [34] D. Karaboga and B. Basturk, ‘‘A powerful and efﬁcient algorithm for numerical function optimization: Artiﬁcial bee colony (ABC) algorithm,’’ J. Global Optim., vol. 39, no. 3, pp. 459–471, Oct. 2007. [35] F. M. Janeiro, Y. Hu, and P. M. Ramos, ‘‘Peak factor optimization of multi- harmonic signals using artiﬁcial bee colony algorithm,’’ Measurement, vol. REFERENCES Previously, he worked as an Associate Professor and the Head of VIT, Vellore. He is a Senior R&D and a Senior Application Engineer in the ﬁeld of power electronics, renewable energy, and electrical drives. He has published more than more than 250 research articles in national and international journals and conferences. He has also authored/coauthored/contributed 12 books/chapters and 12 technical articles on power electronics applications in renewable energy and allied areas. He is a Senior Member of PES, IAS, PSES, YP, and ISTE. He is also an Associate Editor of IEEE ACCESS. He received the Danfoss Innovator Award-Mentor, from 2014 to 2015 and 2017 to 2018, the Research Award by VIT University, from 2013 to 2018, the INAE Summer Research Fellowship, in 2014. He has taken charge as the Vice-Chair of the IEEE Madras Section and the Chair of the IEEE Student Activities, from 2018 to 2020. He was an Executive Member, from 2014 to 2016, and the Vice-Chair of the IEEE MAS Young Professional by the IEEE Madras Section, from 2017 to 2019. He is an Editor of Heliyon, an Elsevier journal, and various other reputed journals. Under his guidance 24 PG students and more than 25 UG Students completed the senior design project work. Also six Ph.D. scholars completed Doctoral thesis as Research Associate. He is also involved in collaborative research projects with various international and national level organizations and research institutions. SHAORONG WANG received the B.Sc. degree in electrical engineering from Zhejiang University, Hangzhou, China, in 1984, the M.Sc. degree in electrical engineering from North China Electric Power University, China, in 1990, and the Ph.D. degree from the Huazhong University of Science and Technology, China, in 2004. He is currently a Professor with the School of Electrical and Electronics Engineering, Huazhong University of Science and Technology. His research interests include power system operation and control, power grid planning, active distribution networks, AI and big data applications in power systems, and robotic inspection in power systems. 62093 VOLUME 8, 2020 VOLUME 8, 2020
https://openalex.org/W1953489421	https://europepmc.org/articles/pmc4627274?pdf=render	English	null	Antimicrobial peptides target ribosomes	Oncotarget	2,015	cc-by	1,077	Antimicrobial peptides target ribosomes Daniel N. Wilson, Gilles Guichard and C. Axel Innis 35), a PrAMP featuring the N-terminal 35 residues of bovine cathelicidin Bac7, accumulates within Escherichia coli to high concentrations and inhibits protein synthesis by binding to the ribosome. Antimicrobial peptides (AMPs) are a diverse group of molecules that play a vital role in the innate immune response of plants and animals [1]. With an average length of thirty or so residues, AMPs often feature a net positive charge due to a high arginine, lysine and/ or histidine content. This in turn endows them with an amphiphilic character that enables them to associate with the phospholipid bilayer of bacteria, while staying clear from the eukaryotic cell membrane. It is no surprise then that many AMPs form transmembrane pores that disrupt the bacterial bilayer, causing rapid lysis and cell death. This lytic activity is a particular concern when it comes to developing AMP-based therapeutics, given that high peptide concentrations could ultimately result in unwanted cytotoxic effects on mammalian cells. Structural studies published by our groups (Seefeldt et al. 2015) [5] and by the Steitz laboratory (Roy et al. 2015) [6] further revealed the atomic details of the interactions between the bacterial ribosome and a 19-residue variant of the oncocin peptide, termed Onc112. Oncocin is produced by the milkweed bug (Oncopeltus fasciatus) and is representative of an entire family of PrAMPs, including pyrrhocorcin (firebug), metalnikowin (green shield bug), drosocin (fruit fly) and apidaecin (bee). The structures, and associated biochemical data, reveal that Onc112 interacts with the large subunit of the bacterial ribosome and blocks the binding site for an incoming aminoacyl-tRNA, thus effectively trapping the ribosome in an inactive initiation complex on the mRNA (Figure 1) [5,6]. Furthermore, this complex is itself destabilized due to probable steric clashes between the aminoacyl moiety of the initiator tRNA and the Onc112 peptide [5]. y While pore-forming peptides account for a large fraction of known AMPs, other classes of peptides inhibit microbial growth by targeting intracellular processes, rather than through damage to the bacterial membrane. Among these, proline-rich antimicrobial peptides (PrAMPs) have attracted considerable attention in recent years as a possible way to counter the rapid increase in bacterial resistance to our dwindling arsenal of effective antibiotics. Contrary to the pore-forming AMPs, PrAMPs are transported into the bacterial cytoplasm by specific transporters, such as SbmA in Gram-negative bacteria. Such transport mechanisms are absent in mammalian cells and PrAMPS cross-react only mildly with intracellular eukaryotic proteins. Editorial Editorial Oncotarget, Vol. 6, No. 19 www.impactjournals.com/oncotarget/ Antimicrobial peptides target ribosomes Daniel N. Wilson, Gilles Guichard and C. Axel Innis As a result, they are generally considered to be non-toxic [2], making them ideal scaffolds upon which to develop novel alternatives to classical antibiotics. What’s more, certain PrAMPs have been shown to cross the blood-brain barrier to selectively target brain cells, thus emphasizing their potential for tissue-specific drug delivery. The binding site of Onc112 on the large ribosomal subunit overlaps with the binding site of many Figure 1: The antimicrobial peptide oncocin inhibits the transition from the initiation to the elongation phase of protein synthesis. For many years, it was thought that insect-derived PrAMPs, such as drosocin, pyrrhocoricin or apidaecin, exert their inhibitory effects by targeting the bacterial heat shock protein DnaK and many PrAMP derivatives with increased affinity for this intracellular chaperone were developed as a result. Two recent papers, however, have challenged this view. Late in 2014, Krizsan et al. [3] showed that apidaecin and oncocin inhibit protein translation in bacteria by binding to and inactivating the 70S ribosome, a property that was shown to depend not only on cationic residues within the peptide, but also on a couple of conserved hydrophobic side chains. A month later, a study by Mardirossian et al. [4] noted that Bac7(1- Figure 1: The antimicrobial peptide oncocin inhibits the transition from the initiation to the elongation phase of protein synthesis. Figure 1: The antimicrobial peptide oncocin inhibits the transition from the initiation to the elongation phase of protein synthesis. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 16826 clinically important classes of antibiotics, such as the chloramphenicols, pleuromutilins, lincosamides and macrolides [7]. While this is very promising for the development of novel antibiotics, it is unclear whether resistance mutations that arise against currently used antibiotics will also confer cross-resistance against PrAMPs. The structural and biochemical knowledge acquired through these and further studies will therefore provide a solid framework upon which to design much- needed improved antibacterial compounds, either peptidic or peptidomimetic, that overcome multi-drug resistant pathogenic bacteria. C. Axel Innis: Institut Européen de Chimie et Biologie, Université de Bordeaux, Pessac, and INSERM U869, Bordeaux, France Correspondence to: C. Axel Innis, email axel.innis@inserm.fr Keywords: Chromosome Section, antimicrobial peptides, ri- bosome, protein synthesis, antibiotics Received: June 25, 2015 Published: July 12, 2015 clinically important classes of antibiotics, such as the chloramphenicols, pleuromutilins, lincosamides and macrolides [7]. Antimicrobial peptides target ribosomes Daniel N. Wilson, Gilles Guichard and C. Axel Innis While this is very promising for the development of novel antibiotics, it is unclear whether resistance mutations that arise against currently used antibiotics will also confer cross-resistance against PrAMPs. The structural and biochemical knowledge acquired through these and further studies will therefore provide a solid framework upon which to design much- needed improved antibacterial compounds, either peptidic or peptidomimetic, that overcome multi-drug resistant pathogenic bacteria. C. Axel Innis: Institut Européen de Chimie et Biologie, Université de Bordeaux, Pessac, and INSERM U869, Bordeaux, France C. Axel Innis: Institut Européen de Chimie et Biologie, Université de Bordeaux, Pessac, and INSERM U869, Bordeaux, France Correspondence to: C. Axel Innis, email axel.innis@inserm.fr Correspondence to: C. Axel Innis, email axel.innis@inserm.fr Keywords: Chromosome Section, antimicrobial peptides, ri- bosome, protein synthesis, antibiotics Received: June 25, 2015 Published: July 12, 2015 Keywords: Chromosome Section, antimicrobial peptides, ri- bosome, protein synthesis, antibiotics Received: June 25, 2015 Published: July 12, 2015 bosome, protein synthesis, antibiotics Published: July 12, 2015 References 1. Wang, G. et al., Pharmaceuticals. (Basel) 2015; 8:123-50. 2. Hansen, A. et al., Antimicrobial Agents and Chemotherapy. 2012; 56:5194-5201. 3. Krizsan, A. et al., Angewandte Chemie International Edition. 2014; 53:12236-12239. 4. Mardirossian, M. et al., Chem Biol. 2014; 21:1639-47. 5. Seefeldt, A.C. et al., Nat Struct Mol Biol. 2015; 22:470-5. 6. Roy, R.N. et al., Nat Struct Mol Biol. 2015; 22:466-9. 6. Roy, R.N. et al., Nat Struct Mol Biol. 2015; 22:466-9. 7. Wilson, D.N., Nat Rev Microbiol. 2014; 12:35-48. 7. Wilson, D.N., Nat Rev Microbiol. 2014; 12:35-48. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 16827
https://openalex.org/W4389388249	http://www.international-agrophysics.org/pdf-174993-97227?filename=Experimental research on.pdf	English	null	Experimental research on shear mechanical properties of tomato stem	International Agrophysics	2,023	cc-by	6,549	Int. Agrophys., 2024, 38, 13-20 doi: 10.31545/intagr/174993 Int. Agrophys., 2024, 38, 13-20 doi: 10.31545/intagr/174993 Experimental research on shear mechanical properties of tomato stem** Qimin Gao1, Lei Cheng1, Haiyang Shen1, Yongchun Chen2, Weisong Zhao1, Zhenwei Wang1, and Mingjiang Chen1* 1Nanjing Institute of Agricultural Mechanization, Ministry of Agriculture and Rural Affairs, Nanjing 210014, China 2College of Mechanical and Electrical Engineering, Xinjiang Agricultural University, Urumqi 830052, China Received May 22, 2023; accepted November 8, 2023 continued to expand (Zeng et al., 2021). After the tomato harvest period, the huge amount of residue remaining is an important biomass resource, and its development and utili- zation are increasingly valued by governments around the world (Guo et al., 2018). In the process of returning them to the field, tomato stem are very difficult to break down and decompose because the stems or vine between the plants are creeping and intertwining. It is difficult to deploy operating machinery in order to process tomato plant resi- dues. Crushing and incorporating residues into the soil may exacerbate the occurrence of diseases and pests, while hap- hazard disposal or incineration may pose a serious threat to transportation safety and cause environmental pollution (Chen et al., 2022). Therefore, it is necessary to carry out relevant research concerning the comprehensive utilization of tomato stems, with the purpose of reducing tomato stem waste, improving mechanical collection efficiency, and promoting the resource utilization of crop waste. Abstract. In order to design a tomato stem crushing and bagging machine and also optimize its operational parameters, knowledge concerning the mechanical properties of harvest-ready tomato stems is required. The mechanical shear properties of the stems were studied by conducting a single-factor experiment, tak- ing the blade angle, moisture content and sampling range as the experimental factors and the peak shear force as the evaluation indexes. The results showed that the blade angle had no signifi- cant effect on the mechanical shear properties of tomato stems, while the leaf water content and sampling location had significant effects on the mechanical shear properties of tomato stems. The largest peak force for cutting the tomato stem was observed in the middle of the sampling range, therefore a shear force greater than 411 N should be provided when designing the crushing device. At the same time, the machine should be used to harvest the tomato stalks as early as possible. Corresponding author e-mail: chenmingjiang@caas.cn *This work was supported by the National key research and de- velopment program (Grant No. 2022YFD2002103) (2022-2026), Institute-level basic scientific research business expenses project of the Chinese Academy of Agricultural Sciences (Grant No. S202210) (2022-2023). The study will provide the necessary experimental data and theoretical basis for the development of an optimized tomato stem crushing and bagging machine. Keywords: tomato stem, maximum shear force, straight knife © 2024 Institute of Agrophysics, Polish Academy of Sciences © 2024 Institute of Agrophysics, Polish Academy of Sciences 1. INTRODUCTION The treatment methods for the resource utilization of tomato stems in China are summarized in this study. Stalk cutting is closely related to the efficient and low damage harvesting of crops and also to the comprehensive utili- zation of stalk resources (Wu and Song, 2022). After the tomato harvest period, the huge amount of residues remain- ing is an important biomass resource, and the tomato stem must be collected in order to be used for resource utiliza- tion, an exception to this process is in situ stem recycling technology. In order to optimize the machinery related to harvesting, knowledge concerning the physical character- istics of tomato stems is required (Kovács and Kerényi, 2019). Tomato straw may be utilized in various ways in The tomato is one of the world’s commercial crops and occupies an important position in the global food trade (Zhang et al., 2023; Wu and Song, 2022). With the increas- ing demand for tomatoes, the scale of the world's total tomato production will continue to expand (Wang et al., 2019). China produces more tomatoes than any other coun- try. In 2019, China's tomato harvest area was 1 086 800 ha, and the annual output was 62 869 500 t, accounting for 21.60 and 34.78% of the world's total harvest area and annual out- put, respectively. The industrial scale of this production has Q. GAO et al. 14 China. The available options include a direct return to the fields, their use as a fertilizer or a substrate, or else such residues may be used for the production of edible mush- rooms, biogas and animal feed (Zhang et al., 2023). Geng et al. (2016) employed fermented tomato straw compost mixed with vermiculite to grow tomato seedlings, and their results revealed that the seedlings performed well. Liu et al. (2014) and Zhang J. et al. (2013) studied the compost- ing performance and fertilizer efficiency of tomato straw compost using composting fungicides and conditioners, respectively. The results showed that the different effects of different composting additives on the composting effi- ciency of tomato straw compost had a certain impact on the application of tomato straw waste. concluded that topping should be completed as early as pos- sible within 100 to 120 days after planting (Aydın and Arslan, 2018). 1. INTRODUCTION The abovementioned studies concerning the shear force of tomato stems are relatively rare, but the research methods followed and the mechanical properties of these crop stems provide a theoretical support for future research, and also, this study may point to future directions in research concerning the mechanical properties of tomato stems. Information relating to the physical and mechanical properties of the tomato stem is limited based on the exist- ing foundational research. Therefore, this paper takes the tomato stem as a research subject, and studies the shear mechanical properties of tomato stems and also the fac- tors affecting the magnitude of its shear force, including the blade angle, shearing speed, moisture content, and sam- pling range of the tomato stem. It is proposed that this study will provide basic data and a theoretical basis for the mech- anization of tomato stalk collection and crushing, such as the degree of cutting force required for crushing and the optimal harvesting time period, this will be accomplished through the shear performance test of tomato stalks. The primary step in the comprehensive utilization of tomato stems is stem cutting. Extensive research has been conducted by scholars in China and abroad concerning the mechanical properties of crop stems. Guo et al. (2014) explored the energy consumption of cutting mature tomato and eggplant stems using a 40° rake angle sawtooth blade, it was found that the cutting energy consumption was low, the cutting efficiency was high, and the cutting effect was good. Shahbazi et al. (2012) studied the relationship between the shear stress and shear energy consumption of safflower and also the moisture content and stem position, and found that the shear stress and shear energy consumption increased with increasing moisture content and also increased from the top to the bottom of the stem. The maximum shear stress and shear energy consumption were 11.04 MPa and 938.33 MJ, respec- tively, at the lower part of the stem, with a moisture content of 37.16%. Zhang X. et al. (2013) found that the average elastic modulus of root cucumber vine stems was 280.58 MPa, which was much higher than the average elastic modulus of 198.81 and 137.22 MPa in the middle and head sections, respectively. 1. INTRODUCTION The maximum shear force and the critical across-compression failure force were found to be 419.1 and 409.0 N, respec- tively, with a water content of 10%, the sampling location was the lower part of dwarf-dense-early major cotton variety stalks, and the loading speed was 40 mm min–1 (Liang et al., 2020). The maximum shearing force and shearing energy of the Glycyrrhiza glabra stalks were found to be 174.03 N and 0.48 J. With increasing water content, the shearing force first decreased and then fluctuated slightly (Wen et al., 2021). The maximum shear forces at different diameters (7, 8, 9, 10, and 11 mm) of wild chrysanthemum stem within the cutting range were determined to be 120.0, 159.2, 213.8, 300.0, and 378.2 N, respectively, through the use of a biomechanical testing machine and a custom-made shear blade (Wang et al., 2022). It was shown that at a distance of 0-15 cm (zone A) and 16-30 cm (zone B) from the top of the cotton plant, the maximum shear force required is higher in zone B (121 N) as compared to zone A (73 N), which suggests that topping should be carried out as close as possible to the ground and that the topping process can be carried out with lower energy consumption. In terms of cutting energy requirements, it was EXPERIMENT RESEARCH ON SHEAR MECHANICAL PROPERTIES OF TOMATO STEM EXPERIMENT RESEARCH ON SHEAR MECHANICAL PROPERTIES OF TOMATO STEM 15 Fig. 3. Different blade angles. Fig. 3. Different blade angles. Fig. 2. Tomato stem shear test bench: 1 – display, 2 – computer, 3 – WDW–20 microcomputer–controlled electronic universal test- ing machine, 4 – block, 5 – press plate, 6 – moving knife, 7 – tool chuck, 8 – stem sample, 9 – fixed knife, 10 – pad block adjustment track. Fig. 3. Different blade angles. The moisture of the tomato stem was determined by drying each sample in a forced-air oven at 103°C for 24 h (ASABE Standard S358.2, 2008). The samples were dried naturally, sampling occurred every 24 h to measure the mois- ture content. The moisture content was reported in percentage terms based on the dry material. After the completion of the equilibrium period, the stem moisture content (SMC) of the samples for each level of humidity was calculated according to the ASABE S358.2 (2008) method as follows: Fig. 2. Tomato stem shear test bench: 1 – display, 2 – computer, 3 – WDW–20 microcomputer–controlled electronic universal test- ing machine, 4 – block, 5 – press plate, 6 – moving knife, 7 – tool chuck, 8 – stem sample, 9 – fixed knife, 10 – pad block adjustment track. The diameter of each sample (average diameter at the midpoint) was measured using an electronic digital cal- liper (GUANGLU, China) with a resolution of 0.01 mm. The WDW-20 microcomputer-controlled electronic universal testing machine (the maximum load of the machine is 10 000 N, its precision is ±0.01 N) from Jinan Chuanbai Instrument Equipment was used for the stem shearing test. The tomato stem shear device consisted of a display, computer, WDW-20 microcomputer-controlled electronic universal testing machine, block, press plate, moving knife, tool chuck, stem sample, fixed knife, and a pad block adjustment track. The experimental diagram and process are shown in Fig. 2. The force, displacement of the contact point, deformation, and loading speed may be recorded in real time during the test process. The test data and report may be saved at the end of the test. The shearing force FSmax (N) of the test was measured using a universal testing machine. Prior to the test, the cushion block 1 and the fixed blade 6 are placed on the guide rail 7. 2. MATERIALS AND METHODS The research was carried out using cherry tomato (Lycopersicon esculentum var. Cerasiforme A. Gray) stems from a vegetable production facility in Guli Street, Jiangning District, Nanjing City, Jiangsu Province, on July 7, 2022. Fresh plants that had grown well and were free from disease and insect pests were selected, also mechanical damage to the stems was avoided. The stems were pulled out completely while the tomatoes were still on the hanging vines (Kovács and Kerényi, 2019). The test was carried out immediately after the sample was collected. During the test, 80 samples were selected, the average height of the whole plant stem was 2 100 mm, the leaves and side branches were manually removed, and the stem was equally divided into three regions: top, middle, and bottom (Fig. 1) (Aydın and Arslan, 2018). Fig. 1. Sample of tomato stem. Fig. 1. Sample of tomato stem. EXPERIMENT RESEARCH ON SHEAR MECHANICAL PROPERTIES OF TOMATO STEM The shear- ing blade 3 is mounted on the tool clamp 4 and locked with a bolt. The tomato stem is placed on the fixed blade 6 and the two cushion blocks 1, and the stem ends are pressed with a pressure plate 2 in order to enhance the stem's bending resistance and ensure a favourable shearing performance. The thickness of the shearing blade is 15 mm, and there are three different blade angles α, where α equals 20, 30, or 40° (Fig. 3). Studies have shown that the angle of the cutting edge has a significant effect on the cutting forces, and that the preferred cutting edge angle of a tool is in the range of 40-45° (Qian et al., 2023). The higher the cutting angle, the higher the cutting power consumption and peak cutting force, it was found that the cutting power consump- tion and peak cutting force with an edge angle of 20-25° were the lowest for cutting sesame stalks (He et al., 2021). SMC(dry base %) = \|W1 −W2\| W2 100% , where: W1 is the weight of the biomass at the point at which a constant weight (g) is achieved and W2 is the dry matter of the biomass (g). In order to study the mechanical shear properties of the tomato stem, the peak shear force was taken as the target value, and the influence of the shearing blade angle, moisture content, and sampling range on the target value was analysed. Based on the test results of moisture content determination (Fig. 4), which revealed a level of around 50% on the 5th day, there is a window of 5-15 days of crop rotation in greenhouse planting. Therefore, the average moisture content of tomato stems needs to be reduced to three different levels: 50, 30, and 10%. In order to ensure the controllability of the mois- ture content of tomato stem, the samples need to be placed in a chamber with a constant temperature and humidity before the experiment, this has the effect of standardizing their mois- ture content. In order to minimize the influence of moisture content regulation on the internal structure of the stems, the Fig. 4. Changes in moisture content of tomato vines every 24 h. Moisture content (%) Moisture content (%) Fig. 4. Changes in moisture content of tomato vines every 24 h. Q. GAO et al. 3. RESULTS AND DISCUSSION The results clearly showed that different water content levels and sampling locations had significantly different effects on the physical properties and mechanical behaviour of the tomato stems. Therefore, this study could be used to determine the optimal harvesting time and cutting location of tomato stems. The changes in the moisture contents of the tomato stem samples every 24 h are shown in Fig. 4. The moisture content in the middle part of the stem decreased at the slowest rate, and the moisture content in the lower part of the stem decreased at the fastest rate. The attenuation rate of the moisture content of the whole plant was 5.26%, of which, the attenuation rate in the middle of the stem was 3.83% and the attenuation rate in the lower part of the stem was 7.18%.l The single factor test method was applied in order to study the influence of blade angle on the mechanical shear properties of tomato stem. This was necessary to ensure that other test conditions were consistent, therefore the water content was set at 30% and the sampling range was set at 1 400 mm to study the influence of the blade angle at 20, 30, and 40° on the experimental indexes. The test was repeated three times at each level. The single factor test method was applied to studying the influence of water content on the shear mechanical properties of tomato stems. This was necessary to ensure that the other test conditions were consistent, therefore the blade angle was set at 30° and the sampling position was set at 1400 mm to study the influence of leaf water content at 10, 30, and 50% on the experimental indexes. The test was repeated three times at each level. The test results of the study concerning the influence of the sampling position on the mechanical shear properties of tomato stem are shown in Table 2. At a significance level of α=0.05, IBM SPSS Statistics 24 software was used to conduct a p-value test on the sampling locations of the leaves. The variance analysis is shown in Table 3. The results show that the mechanical shear properties of the sampling locations of the leaves on tomato stems were 0.01<p<0.05. Therefore, the sampling location of the shear leaves has a significant effect on the mechanical shear properties of tomato stems. EXPERIMENT RESEARCH ON SHEAR MECHANICAL PROPERTIES OF TOMATO STEM The attenuation rate o the moisture content of the whole plant was 5.26%, of which the attenuation rate in the middle of the stem was 3.83% an the attenuation rate in the lower part of the stem was 7.18% The test results of the study concerning the influence o the sampling position on the mechanical shear properties o tomato stem are shown in Table 2. At a significance level o α=0.05, IBM SPSS Statistics 24 software was used to condu a p-value test on the sampling locations of the leaves. Th variance analysis is shown in Table 3. The results show th the mechanical shear properties of the sampling locations o the leaves on tomato stems were 0.01<p<0.05. Therefore, th sampling location of the shear leaves has a significant effe on the mechanical shear properties of tomato stems. Table 2. Effect of sampling position on shear mechanical prop erties of tomato stem Sampling range (mm) Peak shear force 700 181 700 205.2 700 260.6 1 400 287.8 1 400 243.8 1 400 294.6 2 100 188.6 2 100 179.6 2 100 146.2 Table 3. Variance analysis of the effect of sampling position o shear mechanical properties of tomato stem Sources Sum of squares Degree of freedom Mean square F Significance EXPERIMENT RESEARCH ON SHEAR MECHANICAL PROPERTIES OF TOMATO STEM 16 parameters of the constant temperature and humidity chamber are adjusted to a temperature of 30°C and a relative humidity of 95% (Theerarattananoon et al., 2011). Before the experi- ment, the tomato stem samples were taken out of the constant temperature and humidity chamber and dried at a low tem- perature of 40°C. The moisture content was measured using a rapid moisture analyser and adjusted to the desired level (Yu et al., 2012). Samples were taken from the lower, middle, and upper parts of the stem, that is, 700, 1 400 and 2 100 mm, with a random 100 mm section being taken from each sampling range for testing. Table 1 shows the factor level table of a sin- gle factor experimental design. and the sampling position was set at 1 400 mm to investigate the effect of blade moisture content at 10, 30 and 50%. In the sampling position test, the moisture content was set at 30% and the blade angle was set at 30° to examine the effect of the blade sampling position on the test metrics at 700, 1 400 and 2 100 mm. All tests were repeated three times at each level. ty ri- nt m- ng Yu nd th ng n- to ar re he as at as ng al re he et at as ng al to he °, at as to d. re ri- ns In nd te m- 0° the effect of blade moisture content at 10, 30 and 50%. In th sampling position test, the moisture content was set at 30% and the blade angle was set at 30° to examine the effect of th blade sampling position on the test metrics at 700, 1 400 an 2 100 mm. All tests were repeated three times at each level. 3. RESULTS AND DISCUSSION The results clearly showed that different water conten levels and sampling locations had significantly differen effects on the physical properties and mechanical behaviou of the tomato stems. Therefore, this study could be used t determine the optimal harvesting time and cutting locatio of tomato stems. The changes in the moisture contents of th tomato stem samples every 24 h are shown in Fig. 4. Th moisture content in the middle part of the stem decreased the slowest rate, and the moisture content in the lower part o the stem decreased at the fastest rate. 3. RESULTS AND DISCUSSION The single factor test method was applied to studying the influence of the sampling position on the mechanical shear properties of tomato stem. This was necessary to ensure the consistency of the other test conditions, so the water content was set at 30%, the blade angle was set at 30°, and the degree of influence of the leaf sampling position at 700, 1 400, and 2 100 mm on the experimental indexes was studied. The test was repeated three times at each level.f Table 2. Effect of sampling position on shear mechanical prop- erties of tomato stem Table 2. Effect of sampling position on shear mechanical prop- erties of tomato stem Sampling range (mm) Peak shear force 700 181 700 205.2 700 260.6 1 400 287.8 1 400 243.8 1 400 294.6 2 100 188.6 2 100 179.6 2 100 146.2 The factors affecting the mechanical properties of tomato stem shear were investigated using a one-way test method. The influences considered included the blade angle, moisture content and sampling location, and a total of three factori- al tests were set up. The consistency of the test conditions beyond the factors studied had to be ensured for each trial. In the blade angle test, the moisture content was set at 30% and the sampling range was set at 1 400 mm in order to investigate the effect of 20, 30 and 40° blade angles on the test param- eters. In the water content test, the blade angle was set at 30° Table 1. Factor level table of single factor experiment Levels Factor Blade angle (°) Moisture content (%) Sampling range (mm) 1 20 50 700 2 30 30 1 400 3 40 10 2 100 Table 1. Factor level table of single factor experiment Factor Table 1. Factor level table of single factor experiment Table 3. Variance analysis of the effect of sampling position on shear mechanical properties of tomato stem Table 3. Variance analysis of the effect of sampling position on shear mechanical properties of tomato stem Sources Sum of squares Degree of freedom Mean square F Significance Intra-class 16 325.929 2 8 162.964 8.373 0.018 Interblock 516 849.387 6 974.898 Total 22 175.316 8 EXPERIMENT RESEARCH ON SHEAR MECHANICAL PROPERTIES OF TOMATO STEM EXPERIMENT RESEARCH ON SHEAR MECHANICAL PROPERTIES OF TOMATO STEM 17 l Fig. 5. The influence of various factors on shearing characteristics: (a) curves of shearing force and displacement under sampling rang (b) effect of the sampling range on the shearing force, (c) curves of shearing force and displacement under moisture content, (d) effe of the moisture content on the shearing force, (e) curves of shearing force and displacement under blade angle, (f) effect of the bla angle on the shearing force. Fig. 5. The influence of various factors on shearing characteristics: (a) curves of shearing force and displacement under sampling range, (b) effect of the sampling range on the shearing force, (c) curves of shearing force and displacement under moisture content, (d) effect of the moisture content on the shearing force, (e) curves of shearing force and displacement under blade angle, (f) effect of the blade angle on the shearing force. The influence of the sampling range of the shearing force was examined when the stem had a moisture content of 30% and a blade angle of 30°. The displacement curve was drawn with Origin 9.1 (Origin Lab) using real-time data from force variation with displacement produced under three sampling ranges, as shown in Fig. 5a. The highest peak shear force of 294.6 N was observed at the middle sampling range, while the lowest peak shear force of 146.2 N was observed at the top sampling range. As shown in Fig. 5b, the shearing force increased and then dropped as the sampling position moved from the bottom to the top according to the results. The sam- pling range and shearing force of the tomato stems were Q. GAO et al. 18 18 analysed using variance analysis and curve fitting under the same water content. The mean diameters of the top, middle, and bottom regions were 6.40, 9.82, and 7.5 mm, and the standard deviations were 0.53, 0.63, and 0.26. Therefore, it was inferred from the results that the shearing force increased with increases in the tomato stem diameter. Previous stud- ies have shown that the fibre fraction forms the main part of the mechanical properties of the stalk (Réquilé et al., 2018). EXPERIMENT RESEARCH ON SHEAR MECHANICAL PROPERTIES OF TOMATO STEM The influence of different water content levels on the shearing force in the middle part of the stem was studied. The real-time data of force variation with displacement under three water contents were obtained, and the result- ing displacement curve was drawn using Origin 9.1 (Origin Lab), as shown in Fig. 5c. The maximum peak shear force of 360.6 N was observed at a moisture content of 50%, while the minimum peak shear force of 275.6 N was observed at a moisture content of 10%. As the moisture content decreased, the shearing force increased rapidly, and there was a slight fluctuation when it reached 30%, as shown in Fig. 5d. A decrease in the shearing energy with the increas- du is sh th tie th pe ex cu th en Th a s fo 20 w 4. w ter ab ni ste Ta tie Ta sh So Int Int To Table 4. Effect of moisture content on shear mechanical proper- ties of tomato stem Moisture content (%) Peak shear force 50 94 50 158.6 50 145.91 30 243.8 30 294.6 30 287.8 10 217 10 219.5 10 369.4 Table 5. Variance analysis of effects of moisture content on shear mechanical properties of tomato stem Sources Sum of squares Degree of freedom Mean square F Significance Intra-class 38 810.826 2 19 405.413 6.097 0.036 Interblock 19 097.915 6 3 182.986 Total 57908.741 8 The test results of the study concerning the effect of water content on the mechanical shear properties of tomato stem are shown in Table 4. At a significance level of α=0.05, IBM SPSS Statistics 24 software was used to conduct a p-value test on the water content. The variance analysis is shown in Table 5. The results showed that the water content was 0.01<p<0.05 regarding the shear mechanical properties of tomato stems. Therefore, leaf moisture content has a significant effect on the mechanical shear properties of tomato stems.lf The influence of different water content levels on the shearing force in the middle part of the stem was studied. The real-time data of force variation with displacement under three water contents were obtained, and the result- ing displacement curve was drawn using Origin 9.1 (Origin Lab), as shown in Fig. 5c. EXPERIMENT RESEARCH ON SHEAR MECHANICAL PROPERTIES OF TOMATO STEM The maximum peak shear force of 360.6 N was observed at a moisture content of 50%, while the minimum peak shear force of 275.6 N was observed at a moisture content of 10%. As the moisture content decreased, the shearing force increased rapidly, and there was a slight fluctuation when it reached 30%, as shown in Fig. 5d. A decrease in the shearing energy with the increas- 4. CONCLUSIONS In this paper, the mechanical properties of tomato stems were studied based on an analysis of the physical charac- teristics of stems of crops grown booth in China and both abroad. The results showed that the blade angle had no sig- nificant effect on the mechanical shear properties of tomato stems, while the leaf water content and sampling location Table 6. Effect of shear blade angle on shear mechanical proper- ties of tomato stem Blade angle (°) Peak shear force 20 207.6 20 287.8 20 136.6 30 243.8 30 287.8 30 294.6 40 324.8 40 238.4 40 411 Table 7. Variance analysis of the effect of blade angle on the shear mechanical properties of tomato stem Sources Sum of squares Degree of freedom Mean square F Significance Intra-class 19 635.387 2 9 817.693 2.114 0.202 Interblock 27 861.173 6 4 643.529 Total 47 496.560 8 Table 4. Effect of moisture content on shear mechanical proper- ties of tomato stem Moisture content (%) Peak shear force 50 94 50 158.6 50 145.91 30 243.8 30 294.6 30 287.8 10 217 10 219.5 10 369.4 Table 5. Variance analysis of effects of moisture content on shear mechanical properties of tomato stem Sources Sum of squares Degree of freedom Mean square F Significance Intra-class 38 810.826 2 19 405.413 6.097 0.036 Interblock 19 097.915 6 3 182.986 Total 57908.741 8 Table 6. Effect of shear blade angle on shear mechanical proper- ties of tomato stem Blade angle (°) Peak shear force 20 207.6 20 287.8 20 136.6 30 243.8 30 287.8 30 294.6 40 324.8 40 238.4 40 411 Table 7. Variance analysis of the effect of blade angle on the shear mechanical properties of tomato stem Table 4. Effect of moisture content on shear mechanical proper- ties of tomato stem Moisture content (%) Peak shear force 50 94 50 158.6 50 145.91 30 243.8 30 294.6 30 287.8 10 217 10 219.5 10 369.4 Table 5. Variance analysis of effects of moisture content on shear mechanical properties of tomato stem Table 4. Effect of moisture content on shear mechanical proper- ties of tomato stem Table 6. Effect of shear blade angle on shear mechanical proper- ties of tomato stem Table 7. Variance analysis of the effect of blade angle on the shear mechanical properties of tomato stem Table 5. EXPERIMENT RESEARCH ON SHEAR MECHANICAL PROPERTIES OF TOMATO STEM Based on this finding, it may be concluded that the fibre frac- tion is the primary cause of the increase in shear force.f ing stem moisture content of the maize was reported because of the positive relationship between the shearing force and the cellulose and lignin content (Chen et al., 2007).l The test results of the study concerning the influence of the blade angle on the mechanical shear properties of tomato stem are shown in Table 6. At a significance level of α=0.05, IBM SPSS Statistics 24 software was used to con- duct a p-value test on the blade angle. The variance analysis is shown in Table 7. The results showed that the mechanical shear properties of tomato stem were p > 0.1, so the effect of the blade angle (20°~ 40) on the mechanical shear proper- ties of tomato stem was not significant. It was not shown that the blade angle had no significant effect on the shearing performance of tomato stems. In fact, the blade angle had an extremely significant effect on the peak force of sesame stalk cutting (He et al., 2021). In the middle of the stem where the moisture content was 30%, the influence of the differ- ent blade angles on the shearing force is shown in Fig. 5e. The maximum peak shear force of 411 N was observed at a shearing blade angle of 40°, while the minimum peak shear force of 207.6 N was observed at a shearing blade angle of 20°. The results indicate that the shearing force increased with an increase in the blade angle, as shown in Fig. 5f. the mechanical properties of the stalk (Réquilé et al., 2018). Based on this finding, it may be concluded that the fibre frac- tion is the primary cause of the increase in shear force. The test results of the study concerning the effect of water content on the mechanical shear properties of tomato stem are shown in Table 4. At a significance level of α=0.05, IBM SPSS Statistics 24 software was used to conduct a p-value test on the water content. The variance analysis is shown in Table 5. The results showed that the water content was 0.01<p<0.05 regarding the shear mechanical properties of tomato stems. Therefore, leaf moisture content has a significant effect on the mechanical shear properties of tomato stems. 4. CONCLUSIONS Variance analysis of effects of moisture content on shear mechanical properties of tomato stem Sources Sum of squares Degree of freedom Mean square F Significance Intra-class 19 635.387 2 9 817.693 2.114 0.202 Interblock 27 861.173 6 4 643.529 Total 47 496.560 8 EXPERIMENT RESEARCH ON SHEAR MECHANICAL PROPERTIES OF TOMATO STEM 19 Guo Q., Zhang X., Xu Y., Li P., Chen C., and Wu S., 2014. Design and experiment of cutting blade for cane straw. Trans. Chin. Soc. Agric. Engin., 30, 47-53.f had significant effects on the mechanical shear properties of tomato stems. The effect of the sampling location was mainly due to the cross-sectional diameters of the different parts of the tomato stalks, and also the moisture content had an effect on the shear force by influencing certain changes in the levels of cellulose and lignin in the stalks. He Z., Ding H., and Li Z., 2021. Effect of cutting parameters on cutting energy of sesame stalk. J. Henan Univ. Sci. Technol. (Natural Sci.), 42(4), 28-34, https://doi.org/10.15926/j. cnki.issn1672-6871.2021.04.005 Á 1. The sampling range of the tomato stems had a great influence on the shearing force. When the sampling range was between 0 and 2 100 mm, the largest peak force which occurred during the cutting of the tomato stem was observed in the middle of the sampling range. 1. The sampling range of the tomato stems had a great influence on the shearing force. When the sampling range was between 0 and 2 100 mm, the largest peak force which occurred during the cutting of the tomato stem was observed in the middle of the sampling range. Kovács Á. and Kerényi G., 2019. Physical characteristics and mechanical behaviour of maize stalks for machine develop- ment. Int. Agrophys., 33, 427-436, https://doi.org/10.31545/ intagr/113335 Liang R., Chen X., Zhang B., Peng X., Meng H., Jiang P., Li J., Zhou P., and Kan Z., 2020. Tests and analyses on mechanical characteristics of dwarf-dense-early major cotton variety stalks. Int. Agrophys., 34, 333-342, https://doi.org/10.31545/ intagr/122575f 2. Through the moisture content test and mechani- cal experiments it was established that the shearing force increased when the moisture content increased. Therefore, the machine should harvest as early as possible. Liu W., Zhang J., Li B., and Wang S., 2014. Effect of microorgan- ism agents on tomato straw compost and changes of the NPK element forms. Soil Fertil. Sci. 4. CONCLUSIONS Chin., 03, 88-92, htt- ps://doi org/10 11838/sfsc 20140318 3. Upon analysing all experimental data, it was found that the maximum peak shearing force recorded was 411 N. Therefore, the design of the crushing device should be capable of providing a force greater than 411 N. Qian J., Ma S., and Xu Y., 2023. Design and test of sliding shear combined sugarcane basecutter. Trans. Chin. Soc. Agric. Engin., 39(10), 37-47, https://doi.org/10.11975/j.issn.1002-6819.202302124 This study was aimed at establishing a foundation for understanding the mechanical properties of tomato stems. It will provide the necessary experimental data and a sound theoretical basis for developing efficient crushing and bag- ging machines for tomato stems. By taking into account the maximum shear force required for tomato stalks as derived from this study, energy wastage can be avoided. Additionally, the study suggests that the machine should operate within 1-5 days after the tomato harvesting period to ensure optimum processing time. Réquilé S., Goudenhooft C., Bourmaud A., Le Duigou A., and Baley C., 2018. Exploring the link between flexural behaviour of hemp and flax stems and fibre stiffness. Ind. Crops Prod., 113, 179-186, https://doi.org/10.1016/j.indcrop.2018.01.035 Shahbazi F. and Galedar M., 2012. Bending and shearing proper- ties of safflower stalk. J. Agric. Sci. Technol., 14, 743-754. Theerarattananoon K., Xu F., Wilson J., Ballard R., Mckinney L., Staggenborg S., Vadlani P., Pei Z.J., and Wang D., 2011. Physical properties of pellets made from sorghum stalk, corn stover, wheat straw, and big bluestem. Ind. Crops Prod., 33, 325-332, https://doi.org/10.1016/j.indcrop.2010.11.014f Conflicts of Interest: The authors declare that there is no conflict of interest regarding the publication of this paper. Wang L., Jiang P., Zou M., and Liu D., 2019. The effect of power ultrasound on tomato peeling. J. Chin. Inst. Food Sci. Technol., 19(02), 185-191. Wang T., Liu Z., Yan X., Mi G., Liu S., Chen K., Zhang Shilin., Wang X., Zhang Shuo., and Wu X., 2022. Finite element model construction and cutting parameter calibration of wild chrysanthemum stem. Agriculture, 12, 894, https://doi. org/10.3390/agriculture12060894 5. REFERENCES ASABE Standard S358.2, 2008. Moisture measurement-forages. ASAE. St. Joseph, MI. Wen B.-Q., Li Y., Kan Z., Li J., Li L., Ge J., Ding L., Wang K., and Shi Y., 2021. Experimental study on microstructure and mechanical properties of stalk for Glycyrrhiza Glabra. J. Biomech., 118, 110-198, https://doi.org/10.1016/j. jbiomech.2020.110198 Aydın İ. and Arslan S., 2018. Mechanical properties of cotton shoots for topping. Ind. Crops Prod., 112, 396-401, https:// doi.org/10.1016/j.indcrop.2017.12.036 Chen S., Huang D., Zhang L., Guo X., Zhang S., and Cao X., 2022. The effects of straw incorporation on physicochemical prop- erties of soil: A Review. J. Irrig. Drain., 41(06), 1-11.f Wu K. and Song Y., 2022. Research progress analysis of crop stalk cutting theory and method. Trans. Chin. Soc. Agric. Mach., 53(06), 1-20. Chen Y., Chen J., Zhang Y., and Zhou D., 2007. Effect of harvest date on shearing force of maize stems. Livestock Sci., 111, 33-44. https://doi.org/10.1016/j.livsci.2006.11.013 Yu Y., Lin Y., Mao M., Wang W., Tian F., Pan J., and Ying Y., 2012. Experimental study on tensile properties of corn straw. Trans. Chin. Soc. Agric. Engin., 28, 70-76, https:// doi.org/10.3969/j.issn.1002-6819.2012.06.012 Geng F., Li R., Gao B., Cai H., Wang D., Ge M., and Yang Z., 2016. Composting of tomato residues and cow dung under the aerobic condition and its recyclable potential in tomato nursery seedling. Soil Fertil. Sci. Chin., 01, 102-106, htt- ps://doi.org/10.11838/sfsc.20160118 Zeng X., Zhang C., He Y., Zhou Q., Li D., Zheng L., and Zhang Z., 2021. Analysis of world tomato production based on FAO Data from 1980 to 2019. Hunan Agric. Sci., 11, 104-108. Guo Q., Xu Y., Li P., and Chen C., 2018. Study on microstructure and mechanical properties of eggplant stalk. Jiangsu Agric. Sci., 46(06), 189-192. Zhang F., Zhou L., Zhou X., Shen W., Li Y., Chen L., and Zhao G., 2023. Research progress on resource utilization of tomato straw waste in China. Vegetables, 28-32. 20 Zhang X., Sun X., Xu Y., Li P., Guo Q., and Zhang S., 2013. Mechanical characteristics and microstructure of cucumber vine straw. J. Agric. Mech. Res., 36, 9133-9135. Zhang J., Liu W., and Li B., 2013. Screening of microorganism agents during tomato straw and chicken manure composting. J. Agric. Univ. Hebei, 36, 76-81, https://doi.org/10.13320/j. cnki.jauh.2013.05.016 Q. GAO et al. Zhang X., Sun X., Xu Y., Li P., Guo Q., and Zhang S., 2013. Mechanical characteristics and microstructure of cucumber vine straw. J. Agric. Mech. Res., 36, 9133-9135. Zhang J., Liu W., and Li B., 2013. Screening of microorganism agents during tomato straw and chicken manure composting. J. Agric. Univ. Hebei, 36, 76-81, https://doi.org/10.13320/j. cnki.jauh.2013.05.016
https://openalex.org/W2782443149	https://www.nature.com/articles/s41598-017-18770-4.pdf	English	null	The effect of maternal vitamin D deficiency during pregnancy on body fat and adipogenesis in rat offspring	Scientific reports	2,018	cc-by	6,918	The effect of maternal vitamin D deficiency during pregnancy on body fat and adipogenesis in rat offspring Juan Wen1,2,3, Qin Hong2,3, Xingyun Wang1,2,3, Lijun Zhu1,2,3, Tianqi Wu2,3, Pengfei Xu 1,3, Z Fu1,3, Lianghui You1,3, Xing Wang1,3, Chenbo Ji1,2,3,4 & Xirong Guo1,2,3,4 Received: 25 April 2017 Accepted: 18 December 2017 Published: xx xx xxxx Received: 25 April 2017 Accepted: 18 December 2017 Published: xx xx xxxx To evaluate the effects of maternal vitamin D deficiency on body fat and adipogenesis in offspring rats, and explore the potential mechanism, we constructed a vitamin D deficient rat model and performed metabolic activity evaluation, body fat monitoring, biochemical analysis, adipogenesis assay, methylation microarray and RNA-seq for their offspring rats. We found the weight of vitamin D deficient (VDD) offspring was gradually higher than that of control (CLT) offspring, and the difference was significant since 10 weeks old. When compared with CTL offspring, the 24 h heat production, peak blood glucose, adipose tissue volume and blood lipid indexes were significantly increased in VDD offspring at 14 weeks old. Moreover, a significant increase in proliferation rate and number of lipid droplets for pre-adipocytes was also observed in VDD offspring group. DNA methylation profiling showed that compared to CTL group, 608 promoters and 204 CpG islands were differentially methylated in the VDD group, involving 305 genes. When combined with the results of RNA-seq, 141 genes of the methylated genes were differentially expressed. In conclusion, vitamin D deficiency during pregnancy may promote the proliferation and differentiation of pre-adipocytes, which may be associated with methylation alterations of genes, ultimately leading to offspring obesity. Overweight and obesity is an important and increasingly prevalent public health problem worldwide1,2. When compared to non-obese people, obese people are at greater risk of diabetes, hypertension, dyslipidemia, and other cardiovascular disease3. Thus, obesity and its serious comorbidities exert a heavy toll in both human and economic terms3. The occurrence and development of obesity is a very complicated process involving heredity, environment, diet structure and many other factors. There is more and more evidence that non-proper nutrient in early life (including during pregnancy and lactation) may directly contribute to the epidemic of adiposity in childhood, adolescence and adulthood4,5. Due to the presence of an abundance of vitamin D receptors (VDR) on adipocytes, the association between vitamin D and obesity has been the focus of attention6,7. p y y Vitamin D is a fat-soluble secosteroid which is transported to the liver for hydroxylation to 25-hydroxyvitamin D [25(OH)D], the main circulating form of vitamin D and the best measure of vitamin D status. The second hydroxylation to the active form 1,25-hydroxyvitamin D [1,25(OH)D] occurs mostly in the kidneys. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 25 April 2017 Accepted: 18 December 2017 Published: xx xx xxxx The effect of maternal vitamin D deficiency during pregnancy on body fat and adipogenesis in rat offspring Juan Wen1,2,3, Qin Hong2,3, Xingyun Wang1,2,3, Lijun Zhu1,2,3, Tianqi Wu2,3, Pengfei Xu 1,3, Z Fu1,3, Lianghui You1,3, Xing Wang1,3, Chenbo Ji1,2,3,4 & Xirong Guo1,2,3,4 Then, vita- min D binds to VDR and participates in a wide array of activities including calcium and phosphorus metabolism, embryonic tissue development and mature, immune function, cellular growth and differentiation, and glucose metabolism8–10. Many studies have suggested that the vitamin D level is significantly lower in obese populations than in non-obese populations. A survey of the 1958 British birth cohort from 7,198 Caucasian subjects showed that serum 25(OH)D concentrations decreased with increasing body mass index (BMI)11. Even though in the 1Nanjing Maternity and Child Health Care Institute, Nanjing Maternity and Child Health Care Hospital, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing, 210004, China. 2Department of Children Health Care, Nanjing Maternity and Child Health Care Hospital, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing, 210004, China. 3State key Laboratory of Reproductive Medicine, Nanjing Maternity and Child Health Care Hospital, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing, 210004, China. 4Institute of Pediatrics, Nanjing Medical University, Nanjing, 210029, China. Juan Wen, Qin Hong and Xingyun Wang contributed equally to this work. Correspondence and requests for materials should be addressed to C.J. (email: chenboji@njmu.edu.cn) or X.G. (email: xrguo@njmu.edu.cn) SCIeNtIfIC Reports \| (2018) 8:365 \| DOI:10.1038/s41598-017-18770-4 1 www.nature.com/scientificreports/ Figure 1. The vitamin D deficient rat model was constructed successfully and the effects of maternal vitamin D deficiency on weight, blood lipid indexes and blood glucose levels for offspring. (a,b) The 25(OH)D concentrations of the VDD and CLT dams and their offspring. (c) The weight of the VDD and CLT offspring. (d) The plasma concentrations of TC, TG, HDL, and LDL of VDD and CTL offspring. (e) Blood glucose levels after the glucose injection (at 0, 15, 30, 60, 120 min) of VDD and CTL offspring at six and fourteen week of age. VDD, vitamin D deficient group; CTL, control; TC, total cholesterol; TG, triglycerides; HDL, high density lipoprotein; LDL, low density lipoprotein; Data are represented as mean ± SE; P < 0.05. Figure 1. The vitamin D deficient rat model was constructed successfully and the effects of maternal vitamin D deficiency on weight, blood lipid indexes and blood glucose levels for offspring. (a,b) The 25(OH)D concentrations of the VDD and CLT dams and their offspring. (c) The weight of the VDD and CLT offspring. (d) The plasma concentrations of TC, TG, HDL, and LDL of VDD and CTL offspring. The effect of maternal vitamin D deficiency during pregnancy on body fat and adipogenesis in rat offspring Juan Wen1,2,3, Qin Hong2,3, Xingyun Wang1,2,3, Lijun Zhu1,2,3, Tianqi Wu2,3, Pengfei Xu 1,3, Z Fu1,3, Lianghui You1,3, Xing Wang1,3, Chenbo Ji1,2,3,4 & Xirong Guo1,2,3,4 (e) Blood glucose levels after the glucose injection (at 0, 15, 30, 60, 120 min) of VDD and CTL offspring at six and fourteen week of age. VDD, vitamin D deficient group; CTL, control; TC, total cholesterol; TG, triglycerides; HDL, high density lipoprotein; LDL, low density lipoprotein; Data are represented as mean ± SE; P < 0.05. non-obese populations, BMI and percentage body fat content were inversely related to the serum 25(OH)D con- centrations12,13. Moreover, women and children with vitamin D deficiency had higher rates of obesity than those with non-deficient vitamin D level14. In a randomized controlled trial for calcium plus vitamin D supplementa- tion, a significantly greater decrease in fat mass loss was observed in the calcium plus vitamin D supplementation group than in the control group, although there was no significant difference in body weight change between groups15.i g p Vitamin D deficiency in pregnant women has become a very common phenomenon16,17. An observational study including 977 pregnant women showed lower maternal vitamin D status was associated with greater fat mass at ages 4 and 6 y18. And a Spain cohort also yielded similar results and concluded that vitamin D deficiency in pregnancy may increase the risk of prenatal and early postnatal overweight in offspring19. In our previous large cohort study, we found women with 25(OH)D < 37.5 nmol/L had infants with higher birth weight in a linear regression model, and women with 25(OH)D < 50.0 nmol/L had increased risk of macrosomia, when compared with women with 25(OH)D concentrations from 50.0 to 74.9 nmol/L20. Thus, the effects of maternal vitamin D deficiency on birth weight or body fat of offspring could not be ignored. Furthermore, evidence has shown that the effects of maternal nutrition on offspring obesity may be related with epigenetic mechanisms21,22. And data are now emerging suggesting that alterations in DNA methylation is central to the processes of developmental plasticity and also support the relationships between early life effects and later metabolic status23,24. Therefore, we constructed a vitamin D deficient rat model and performed metabolic activity evaluation, body fat monitoring, biochemical analysis, adipogenesis assay, methylation microarray and RNA-seq for their offspring rats to evaluate the effects of vitamin D deficiency during pregnancy on body fat and adipogenesis in offspring rats. Results Removal of dietary vitamin D prior to conception and throughout gestation resulted in total depletion of 25(OH) D in the vitamin D deficient (VDD) dams, when compared with the control (CLT) dams (Fig. 1a). There was no significant difference in weight and pup numbers between the CTL and VDD dams. Although the 25(OH)D concentrations of the VDD and CLT offspring at 1 week old largely reflected the levels in their respective dams (Fig. 1b), the VDD and CLT offspring in birth weight did not exist significant difference (P > 0.05). However, we found the weight of VDD offspring was gradually higher than that of CTL offspring, and the difference was significant since the offspring at 10 weeks old (Fig. 1c).f gif p g ( g ) Biochemical analysis for offspring at 14 weeks old showed that the plasma concentrations of TC, TG, HDL, and LDL were significantly increased in VDD offspring when compared with CTL offspring (P < 0.05) (Fig. 1d). We also measured blood glucose for VDD and CTL offspring at each week of age, and found the blood glucose of VDD offspring was significantly higher than that of CTL offspring at 60 min after glucose injection at 6 weeks old, and at 15 and 30 min after glucose injection at 14 weeks old. However, the blood glucose of the two groups reached the same level at 120 min after injection (Fig. 1e). SCIeNtIfIC Reports \| (2018) 8:365 \| DOI:10.1038/s41598-017-18770-4 2 www.nature.com/scientificreports/ Figure 2. The feeding behavior, locomotor activity and energy expenditure of the VDD and CLT offspring. (a–e) Feeding behavior (water and food intake) and locomotor activity (horizontal, vertical and fine motor activity) of VDD and CTL offspring. (f–h) The 24 h VO2, VCO2, RER and heat production of VDD and CTL offspring. VDD, vitamin D deficient group; CTL, control; VO2, oxygen consumption; VCO2, carbon dioxide production; RER, respiratory exchange rate; Data are represented as mean ± SE; P < 0.05. Figure 2. The feeding behavior, locomotor activity and energy expenditure of the VDD and CLT offspring. (a–e) Feeding behavior (water and food intake) and locomotor activity (horizontal, vertical and fine motor activity) of VDD and CTL offspring. (f–h) The 24 h VO2, VCO2, RER and heat production of VDD and CTL offspring. Results The Vldlr (Very low density lipoprotein receptor) gene encodes a lipoprotein receptor and plays important roles in VLDL-triglyceride metabolism and the reelin Further body fat monitoring for offspring at 14 weeks old showed that TV, AV and AV/TV were significantly increased in VDD offspring when compared with CTL offspring (P < 0.05) (Figs 3a,b and 4). To gain further insights into the effect of vitamin D deficiency on adipogenesis, xCELLigence assays and oil red O staining were conducted on the pre-adipocytes isolated from offspring retroperitoneal adipose tissue, and showed a significant increase in proliferation rate and number of lipid droplets in VDD group compared with CTL group (Fig. 3c,d).fi g g g To explore the potential mechanism for the effect of vitamin D deficiency on adipose adipogenesis, we per- formed a comprehensive DNA methylation profiling and RNA-seq analysis, and found that compared to CTL group, 608 promoters and 204 CpG islands (CGIs) were differentially methylated in the VDD group, involving 305 genes. When combined with the results of RNA-seq, 141 genes of the methylated genes were differentially expressed. Then we validated 7 obesity or lipid metabolism related genes (Hif1a, Spon1, H19, Adrb2, Vldlr, Zfp36, Smad3), and found that compared to CTL group, Vldlr was hypermethylated and low expressed, and Hif1α was demethylated and high expressed in the VDD group (Fig. 5). The Vldlr (Very low density lipoprotein receptor) gene encodes a lipoprotein receptor and plays important roles in VLDL-triglyceride metabolism and the reelin signaling pathway, whereas the Hif1α (Hypoxia inducible factor 1 alpha subunit) gene was involved in energy metabolism, angiogenesis, apoptosis, and other genes which could increase oxygen delivery or facilitate metabolic adaptation to hypoxia. SCIeNtIfIC Reports \| (2018) 8:365 \| DOI:10.1038/s41598-017-18770-4 Results VDD, vitamin D deficient group; CTL, control; VO2, oxygen consumption; VCO2, carbon dioxide production; RER, respiratory exchange rate; Data are represented as mean ± SE; P < 0.05. For the offspring at 14 weeks old, feeding behavior, locomotor activity, and the energy expenditure were meas- ured using the PhenoMaster system. Removal of dietary vitamin D prior to conception and throughout gestation did not cause any alterations of feeding behavior (water and food intake) and locomotor activity (horizontal, vertical and fine motor activity) (P > 0.05) (Fig. 2a–e). However, the 24 h VO2, VCO2, RER and heat production of VDD offspring were significantly higher than that of CLT offspring (P < 0.05) (Fig. 2f–h).fi For the offspring at 14 weeks old, feeding behavior, locomotor activity, and the energy expenditure were meas- ured using the PhenoMaster system. Removal of dietary vitamin D prior to conception and throughout gestation did not cause any alterations of feeding behavior (water and food intake) and locomotor activity (horizontal, vertical and fine motor activity) (P > 0.05) (Fig. 2a–e). However, the 24 h VO2, VCO2, RER and heat production of VDD offspring were significantly higher than that of CLT offspring (P < 0.05) (Fig. 2f–h).fi fif Further body fat monitoring for offspring at 14 weeks old showed that TV, AV and AV/TV were significantly increased in VDD offspring when compared with CTL offspring (P < 0.05) (Figs 3a,b and 4). To gain further insights into the effect of vitamin D deficiency on adipogenesis, xCELLigence assays and oil red O staining were conducted on the pre-adipocytes isolated from offspring retroperitoneal adipose tissue, and showed a significant increase in proliferation rate and number of lipid droplets in VDD group compared with CTL group (Fig. 3c,d). To explore the potential mechanism for the effect of vitamin D deficiency on adipose adipogenesis, we per- formed a comprehensive DNA methylation profiling and RNA-seq analysis, and found that compared to CTL group, 608 promoters and 204 CpG islands (CGIs) were differentially methylated in the VDD group, involving 305 genes. When combined with the results of RNA-seq, 141 genes of the methylated genes were differentially expressed. Then we validated 7 obesity or lipid metabolism related genes (Hif1a, Spon1, H19, Adrb2, Vldlr, Zfp36, Smad3), and found that compared to CTL group, Vldlr was hypermethylated and low expressed, and Hif1α was demethylated and high expressed in the VDD group (Fig. 5). Discussion d VDD, vitamin D deficient group; CTL, control; TV, total volume; AV, adipose tissue volume; C1–C6, six samples from CTL offspring; D1–D6, six samples from VDD offspring; Data are represented as mean ± SE; P < 0.05. Figure 3. The effects of vitamin D deficiency on adipose tissue volume and adipogenesis. (a,b) The TV, AV, AV/ TV of VDD and CTL offspring. (c,d) xCELLigence assays and oil red O staining showed a significant increase in proliferation rate and number of lipid droplets of pre-adipocytes in VDD group compared with CTL group. VDD, vitamin D deficient group; CTL, control; TV, total volume; AV, adipose tissue volume; C1–C6, six samples from CTL offspring; D1–D6, six samples from VDD offspring; Data are represented as mean ± SE; P < 0.05. there were no differences in body mass at birth, but at six months, the vitamin D restricted groups were signifi- cantly heavier than their controls25, which was similar to the results of this study. More importantly, our results firstly suggested that maternal vitamin D deficiency could result in body fat increase of offspring. Large-scale, population-based studies showed that the negative correlation between serum vitamin D level and body fat was more prominent than that between vitamin D level and BMI26. Moreover, the plasma concentrations of TC, TG, HDL, and LDL were significantly increased in VDD offspring when compared with CTL offspring at 14 weeks old, suggesting lipid metabolism disorder was appeared in adulthood of offspring for maternal vitamin D deficiency. Population-based studies have shown that vitamin D level was closely related with lipid metabolism. For example, a randomized trial of 51 adults found a significant negative correlation between 25(OH)D and the ratio of LDL to HDL27. In our study, there was no significant difference in serum 25(OH)D between the CTL and VDD adult offspring, so we speculated that maternal vitamin D deficiency laid the foundations for lipid metabolism disorder in adulthood of offspring. Blood glucose is closely bound up with lipid metabolism. In the 2016 year’s European Association for the Study of Diabetes (EASD) meeting, vitamin D was indicated to improve insulin sensitivity and reduce the accumulation of fat in muscles though a mice model of diet-induced insulin resistance, which was constructed by Dr. Elisa Benetti and colleagues from the University of Turin, Italy (www. easdvirtualmeeting.org). Discussion d Our study comprehensively evaluated the effect of maternal vitamin D deficiency on the body weight, body fat, glucose and lipid metabolism and metabolic activity of the offspring, and found maternal vitamin D deficiency not only affected body weight, body fat, glucose and lipid metabolism in adulthood of offspring, but also affected proliferation and differentiation of the pre-adipocytes, suggesting non-proper nutrient in early life may directly contribute to the epidemic of adiposity in the future life. In order to better explain the mechanism behind the above phenomenon, we investigated the effect of maternal vitamin D deficiency on the alterations of DNA meth- ylation in the adipose tissue of its offspring. We put forward the conclusion that vitamin D deficiency during pregnancy may promote the proliferation and differentiation of pre-adipocytes, which may be associated with methylation alterations of genes, such as Vldlr and Hif1α, ultimately leading to offspring obesity. The findings may provide some important clues for further mechanisms exploration.f p p p In the animal experiments, we found the weight of VDD offspring was gradually higher than that of CTL offspring, but the birth weight between the VDD and CLT offspring did not exist significant difference. In a study for investigating the effects of maternal vitamin D deficiency on offspring kidney development, they found SCIeNtIfIC Reports \| (2018) 8:365 \| DOI:10.1038/s41598-017-18770-4 3 www.nature.com/scientificreports/ Figure 3. The effects of vitamin D deficiency on adipose tissue volume and adipogenesis. (a,b) The TV, AV, AV/ TV of VDD and CTL offspring. (c,d) xCELLigence assays and oil red O staining showed a significant increase in proliferation rate and number of lipid droplets of pre-adipocytes in VDD group compared with CTL group. VDD, vitamin D deficient group; CTL, control; TV, total volume; AV, adipose tissue volume; C1–C6, six samples from CTL offspring; D1–D6, six samples from VDD offspring; Data are represented as mean ± SE; P < 0.05. Figure 3. The effects of vitamin D deficiency on adipose tissue volume and adipogenesis. (a,b) The TV, AV, AV/ TV of VDD and CTL offspring. (c,d) xCELLigence assays and oil red O staining showed a significant increase in proliferation rate and number of lipid droplets of pre-adipocytes in VDD group compared with CTL group. Discussion d And human studies have suggested a strong link between vitamin D deficiency and risk of diabetes regardless of age28. In addition, multiple studies and meta-analysis have shown that women with low 25(OH)D concentrations in pregnancy had significantly increased risks of gestational diabetes29,30. In summary, maternal vitamin D deficiency could result in changes of weight, body fat and metabolism in offspring. However, these changes were not related with vitamin D deficiency themselves. Thus, understanding the role of maternal nutrition in offspring outcomes merits further exploration.h there were no differences in body mass at birth, but at six months, the vitamin D restricted groups were signifi- cantly heavier than their controls25, which was similar to the results of this study. More importantly, our results firstly suggested that maternal vitamin D deficiency could result in body fat increase of offspring. Large-scale, population-based studies showed that the negative correlation between serum vitamin D level and body fat was more prominent than that between vitamin D level and BMI26. Moreover, the plasma concentrations of TC, TG, HDL, and LDL were significantly increased in VDD offspring when compared with CTL offspring at 14 weeks old, suggesting lipid metabolism disorder was appeared in adulthood of offspring for maternal vitamin D deficiency. Population-based studies have shown that vitamin D level was closely related with lipid metabolism. For example, a randomized trial of 51 adults found a significant negative correlation between 25(OH)D and the ratio of LDL to HDL27. In our study, there was no significant difference in serum 25(OH)D between the CTL and VDD adult offspring, so we speculated that maternal vitamin D deficiency laid the foundations for lipid metabolism disorder in adulthood of offspring. Blood glucose is closely bound up with lipid metabolism. In the 2016 year’s European Association for the Study of Diabetes (EASD) meeting, vitamin D was indicated to improve insulin sensitivity and reduce the accumulation of fat in muscles though a mice model of diet-induced insulin resistance, which was constructed by Dr. Elisa Benetti and colleagues from the University of Turin, Italy (www. easdvirtualmeeting.org). And human studies have suggested a strong link between vitamin D deficiency and risk of diabetes regardless of age28. In addition, multiple studies and meta-analysis have shown that women with low 25(OH)D concentrations in pregnancy had significantly increased risks of gestational diabetes29,30. In summary, maternal vitamin D deficiency could result in changes of weight, body fat and metabolism in offspring. Discussion d However, these changes were not related with vitamin D deficiency themselves. Thus, understanding the role of maternal nutrition in offspring outcomes merits further exploration.h f p g p The association of low vitamin D status with obesity has been well established31. It is reported that serum 25(OH)D < 50 nmol/L was significantly associated with new-onset obesity32. Recently, Wang et al. performed a genome-wide association study (GWAS) of the gut microbiota and discovered a significant association of the VDR gene (encoding vitamin D receptor) with gut microbial characteristics, which is essential for bile acid and fatty acid metabolism33. In vitro studies have demonstrated that 25(OH)D could inhibit adipogenesis and induce adipocyte apoptosis34. Assumptions exist that the active form of vitamin D manifests the ability to inhibit differ- entiation of pre-adipocytes by alteration of activity of the adipogenic transcription factors35. Moreover, reports on the favorable effect of vitamin D supplementation on the process of reduction in adipose tissue were also appeared36. Although these findings substantially support our study, it still remains unresolved to what extent vitamin D deficiency represents the cause of obesity. i y p y Furthermore, several studies have shown that nutrition supply could induce alterations in DNA methylation, and then result in changes in biological phenotype21. A recent study found that maternal vitamin D deficiency during pregnancy resulted in insulin resistance in rat offspring, which was associated with inflammation and Iκbα methylation37. However, alterations of methylation may more often than not be a phenomenon that affects SCIeNtIfIC Reports \| (2018) 8:365 \| DOI:10.1038/s41598-017-18770-4 4 www.nature.com/scientificreports/ Figure 4. Transverse micro-CT images of the waist of offspring rats for body fat monitoring. C1–C6, six samples from CTL offspring; D1–D6, six samples from VDD offspring. Figure 4. Transverse micro-CT images of the waist of offspring rats for body fat monitoring. C1–C6, six samples from CTL offspring; D1–D6, six samples from VDD offspring. Figure 4. Transverse micro-CT images of the waist of offspring rats for body fat monitoring. C1–C6, six samples from CTL offspring; D1–D6, six samples from VDD offspring. Figure 5. Methylation levels of Vldlr and HIF1α in promoter, and expression levels of Vldlr and HIF1α. VDD, vitamin D deficient group; CTL, control; Data are represented as mean ± SE; P < 0.05. igure 5. Methylation levels of Vldlr and HIF1α in promoter, and expression levels of Vldlr and HIF1α. Methodsh The offspring rats of the two groups were individually placed in the PhenoMaster/LabMaster device (TSE Systems, Germany). Water intake, food intake, locomotor activity, 24 h oxygen consumption (VO2), carbon dioxide production (VCO2), and heat production were recorded over 24 h at 40 min intervals. The respiratory exchange rate (RER) was estimated by calculating the ratio of VCO2/VO2. Body fat monitoring for offspring rats. Six offspring rats from each group were selected for body fat monitoring and fasted 24 h prior to anesthetization with ether. Transverse micro-CT images of the waist from L3 to L6 were scanned using a Micro-CT scanner (SkyScan 1176, SkyScan Co., Belgium) with resolution of 35 μm, voltage of 100 kV, current of 100 μA, exposure of 474 ms, and rotation step (degree) of 0.500. Analysis of micro-CT images was performed using Nrecon software (SkyScan Co.). Total volume (TV) and adipose tissue volume (AV) of abdomen were detected, and the ratio of AV/TV was calculated. Pre-adipocyte preparation and adipogenesis Assay. The offspring rats aged 7 days were anesthetized with ether and retroperitoneal adipose tissue was excised through a sterile laparotomy procedure. The tissue sample was sliced into small pieces and washed with phosphate-buffered saline (PBS). The tissue pieces were then digested in PBS containing 1 mg/mL collagenase for 60 min at 37 °C. The cellular pellet was isolated via centrifu- gation (1000 rpm for 15 min) and suspended in DMEM medium supplemented with 10% FBS. The pre-adipocytes were plated in culture flask and incubated in a humidified 5% CO2 incubator. To evaluate the effects of vitamin D deficiency on adipogenesis, a real-time cell proliferation assay and oil red O staining were conducted to evaluate the capacity of proliferation and differentiation, respectively.h ® p y pf p y The real-time cell proliferation assay was conducted using the xCELLigence® impedance-based, label-free, real time cellular analysis system (RTCA) (ACEA Biosciences, San Diego CA, USA) according to the manufac- turer’s instructions. The pre-adipocytes containing 6 × 104 cells were seeded in each well of E-Plate L8. The plate was then inserted into the RTCA machine caged in incubator and the cell growth was continuously monitored for at least 72 h in 1 h intervals. Data was analyzed using the RTCA Software 1.2 program (Roche Diagnostics). All data is presented as the mean normalized cellular index over time. Methodsh The study was approved by the institutional review board of Nanjing Maternity and Child Health Care Institute nd the methods were carried out in accordance with the approved guidelines. g A total of 43 pregnant Sprague-Dawley rats were used. All breeding animals were housed in incandescent light devoid of ultraviolet B radiation on a 12 h light/dark cycle (lights on 0600 h), at a constant temperature of 21 ± 2 °C and 60% relative humidity, with food and water provided ad libitum. Four week-old female rats were allocated to two dietary groups, with each group matched for body weight. One group received a vitamin D replete diet (1000 I.U/kg, Dyets Inc., PA, USA) throughout the study (Control group). The another group was fed a vitamin D deficient diet (0 I.U/kg, Dyets Inc., PA, USA) from 4 weeks of age until birth. From birth, all rats and their offspring consumed the vitamin D replete diet. All pregnant rats were weighed weekly, and serum samples were collected every two weeks in order to measure the 25(OH)D concentrations. Male offspring were selected for the following experiments. They were weighed weekly before 6 weeks of age, and every two weeks after 6 weeks of age. Vitamin D measurement. Serum concentrations of 25(OH)D were measured by using an in vitro diag- nostic enzyme immunoassay kit OCTEIA 25-Hydroxy Vitamin D (Immunodiagnostic Systems, Boldon, United Kingdom) according to the manufacturer’s instructions. The reported analytic sensitivity of the immunoassay was 6.8–380 nmol/L. Biochemical analysis and metabolic activity evaluation for offspring rats. Six offspring rats at 14 weeks old were randomly selected from each group. Plasma concentrations of total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL), and low density lipoprotein (LDL) were measured using commercial kits (Asan Pharmaco Co., Seoul, Korea) after fasting for 12 h.f t Other six offspring rats were randomly selected from each group for intraperitoneal glucose tolerance test (IPGTT) at each week of age. Rats were fasted for 16 h and injected intraperitoneally with a 20% glucose solution prepared in saline (2 mg/g body weight). Blood was drawn from the tail vein before and after the glucose injection (at 0, 15, 30, 60, 120 min) and blood glucose was measured using an Arkray glucometer (GLUCOCARD II Series Test Meter, Japan).fiff Other six offspring rats were randomly selected from each group when a significant difference in the offspring weight between the two groups appeared. Discussion d VDD, itamin D deficient group; CTL, control; Data are represented as mean ± SE; *P < 0.05. multiple genomic loci. The influence of aging and environmental influences such as nutrition supply seems to be on global methylation patterns, in turn exerting local effects on groups of genes38. Hence, the gradually accumu- lated methylation alterations may help to interpret the phenotypic difference appeared in adulthood of offspring for maternal vitamin D deficiency. SCIeNtIfIC Reports \| (2018) 8:365 \| DOI:10.1038/s41598-017-18770-4 5 www.nature.com/scientificreports/ In summary, through the animal experiments, cell experiments, and further mechanism exploration based on epigenetics, we concluded that vitamin D deficiency during pregnancy may promote the proliferation and differentiation of pre-adipocytes, which may be associated with methylation alterations of genes, such as Vldlr and Hif1α, ultimately leading to offspring obesity. Further studies are warranted to validate our hypothesis and extend our findings. Methodsh All xCELLigence experiments were performed in triplicates.h The pre-adipocytes were cultured in 6-well plates and maturation was induced. For oil red O staining, the culture medium was removed and cells were washed twice with PBS. Cells were fixed with 4% formalin/PBS for 30 min at room temperature. The fixation cells were washed with PBS again and stained with 0.5% Oil Red-O/ isopropyl alcohol solution for 60 min at room temperature. After washing three times, cells were observed using an optical microscope and photographed. SCIeNtIfIC Reports \| (2018) 8:365 \| DOI:10.1038/s41598-017-18770-4 6 www.nature.com/scientificreports/ DNA methylation profiling. For VDD offspring and CTL offspring at 14 weeks old, a total of six DNA samples from adipose tissue were used to perform comprehensive DNA methylation profiling of gene promot- ers and CpG islands to determine the differentially methylated genes using methylated DNA immunoprecipi- tation followed by hybridization to the NimbleGen DNA Methylation Promoter Plus CpG Island Microarray. And RNA-seq was used to determine the differentially expressed genes. For further validate the methylation and expression levels of the differentially methylated and expressed genes, Sequenom EpiTYPER® and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used in another six DNA samples from adipose tissue of the two groups. The significant differentially methylated regions were defined as at least one CpG site showing absolute difference in methylation level of ≥50% between groups at a significance level of P-value ≤ 0.05. A FDR (False Discovery Rate) correction was also applied to correct for multiple hypothesis testing. Statistical analysis. All the data were presented as mean ± standard error (SE). The statistical significances between VDD and CTL group were performed using Student’s t-test. P ≤ 0.05 in a two-sided test was considered statistically significant and indicated in the figures. Statistical analysis. All the data were presented as mean ± standard error (SE). The statistical significances between VDD and CTL group were performed using Student’s t-test. P ≤ 0.05 in a two-sided test was considered statistically significant and indicated in the figures. statistically significant and indicated in the figures. References Body size and serum 25 hydroxy vitamin D response to oral supplements in healthy older adults. J Am Coll Nutr 27, 274–279 (2008).hi 4. Robinson, C., Chiang, M., Thompson, S. N. & Sondike, S. B. 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J.W., Q.H., X.W., L.Z., T.W., P.X., Z.F., L.Y. and X.W. collected the data. J.W., C.J. and X.G. conducted the statistical analysis and interpretation. J.W. and Q.H. wrote the report. C.J. and X.G. revised the report. All the authors reviewed the report and approved the final version. Acknowledgementsh g This work was supported in part by the National Key Basic Research Program of China (2013CB530604), the Key project of the National Natural Science Foundation of China (81330067), the National Natural Science Foundation of China (81270928, 81200642, 81301173, 81300683, 81500649, 81600685, 81600687), the Natural Science Foundation of Jiangsu Province (BK20140086, BK20160141, BE2016619) and the Medical Science and technology development Foundation (JQX13012). 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https://openalex.org/W4312887506	http://journal3.um.ac.id/index.php/fip/article/download/2630/1770	Indonesian	null	Hubungan antara Stres Akademik dan Prokrastinasi Akademik dengan Prestasi Belajar Siswa Kelas X Sekolah Menengah Atas	Jurnal Pembelajaran, Bimbingan, Dan Pengelolaan Pendidikan	2,022	cc-by-sa	5,224	Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 ISSN: 2797-3174 (online) DOI: 10.17977/um065v2i52022p447-459 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 ISSN: 2797-3174 (online) DOI: 10.17977/um065v2i52022p447-459 Abstract The purpose of this study is to determine the relationship between academic stress and academic procrastination with learning achievement. This study used a correlational research design. The population of this research was the X graders of SMA Brawijaya Smart School Malang with a total of 194 students, the research sample totaling 100 students obtained using the Slovin formula with an error tolerance of 7 percent. The sampling using simple random sampling method. This study used research instrument in the form of scales. First, "Student Life Stress Inventory" scale which was adapted in Indonesian by (Rohana, 2017). Second, the academic procrastination scale developed by the author based on the characteristics of academic procrastination according to (Ferrari et al., 1995). Both instruments had been tested for validity and reliability. The measurement of learning achievement used the score of the mid-semester exam in the odd semester of 2021/2022. The results showed that academic stress and academic procrastination had a negative relationship with learning achievement and together academic stress and academic procrastination affected learning achievement. Researchers hope the results of this study can be information and considerations for school counselors or BK teachers in providing services to students. In addition, researchers hope that further researchers can continue development research that produces a product that can help students related to problems of academic stress, academic procrastination and learning achievement. Keywords: academic stress; academic procrastination; learning achievement Hubungan antara Stres Akademik dan Prokrastinasi Akademik dengan Prestasi Belajar Siswa Kelas X Sekolah Menengah Atas Lia Artika Sari, Djoko Budi Santoso, Ella Faridati Zen Universitas Negeri Malang, Jl. Semarang No. 5 Malang, Jawa Timur, Indonesia Penulis korespondensi, Surel: lia.artika.1801116@students.um.ac.id Lia Artika Sari, Djoko Budi Santoso, Ella Faridati Zen Universitas Negeri Malang, Jl. Semarang No. 5 Malang, Jawa Timur, Indonesia Penulis korespondensi, Surel: lia.artika.1801116@students.um.ac.id Lia Artika Sari, Djoko Budi Santoso, Ella Faridati Zen Universitas Negeri Malang, Jl. Semarang No. 5 Malang, Jawa Timur, Indonesia Penulis korespondensi, Surel: lia.artika.1801116@students.um.ac.id Paper received: 2-5-2022; revised: 20-5-2022; accepted: 27-5-2022 Paper received: 2-5-2022; revised: 20-5-2022; accepted: 27-5-2022 Paper received: 2-5-2022; revised: 20-5-2022; accepted: 27-5-2022 This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. 1. Pendahuluan Salah satu jalur pendidikan di Indonesia adalah jalur pendidikan formal, pada jalur pendidikan formal terdapat salah satu jenjang yaitu sekolah menengah atas. Sekolah menengah merupakan tempat dimana siswa mengembangkan kemampuan kognitif, emosional dan psikomotoriknya melalui tugas sekolah. Rentang usia siswa yang duduk pada jenjang Sekolah Menengah Atas adalah umur 15-18 tahun, yang mana pada usia tersebut terhitung sebagai usia remaja. Piaget (Mu’min, 2013) menyebutkan bahwa pada usia remaja tahap perkembangan kognitif siswa telah mencapai taraf operasional formal, yang artinya seluruh aktifitas dan perilaku siswa SMA adalah hasil dari kematangan berpikir logis. Hal tersebut sejalan dengan Standar Kompetensi Kemandirian Peserta didik atau SKKPD siswa SMA yaitu pada aspek kematangan intelektual. Kemampuan seseorang dalam berfikir rasional dan berperilaku efektif dalam adaptasi dengan lingkungan disebut dengan kematangan intelektual. Sehingga pada sudut pandang akademik kematangan intelektual sendiri dapat membantu siswa dalam memperoleh capaian belajar yang maksimal. Capaian belajar yang didapatkan pelajar sepanjang ia di sekolah dapat disebut dengan prestasi belajar, yang diartikan sebagai hasil yang didapatkan pelajar berupa kesan dan bisa membawa perubahan diri individu sebagai hasil dari kegiatan belajar itu sendiri. Prestasi belajar merupakan penilaian guru terhadap murid-muridnya dalam bentk simbol, angka, huruf maupun kalimat setelah melakukan pembelajaran selama waktu tertentu (Mahardika, 2019; Rosyid et al., 2019). Selain itu prestasi belajar diartikan sebagai tingkat kesempurnaan yang telah dicapai siswa yang meliputi ranah kognitif, afektif dan psikomotorik S. Nasution (Hamdu & Agustina, 2017). Jika sesorang belum mampu dalam memenuhi target pada ketia aspek tersebut maka prestasi belajar dikatakan kurang memuaskan. Prestasi belajar di Indonesia direpresentasikan dalam bentuk nilai UH, nilai raport, nilai UN dan lain sebagainya. Pemerintah di Indonesia mengharapkan siswa dapat memperolah capaian belajar yang baik dan bermutu, agar mereka bisa menjadi lulusan yang berintelektual, kreatif serta menjadi pribadi yang berkualitas. Namun pada kenyataannya masih banyak fenomena prestasi belajar rendah pada siswa di indonesia. Salah satu fenomena permasalahan terkait prestasi belajar adalah menurunnya nilai UN siswa SMA tahun 2018. Kementerian Pendidikan dan Kebudayaan (Kemendikud) mengakui adanya penurunan hasil nilai Ujian Nasional (UN) siswa pada jenjang sekolah menengah atas/ sederajat tahun 2018. Penurunan nilai terjadi terutama pada mata pelajaran matematika, kimia dan fisika. Menurut Kementrian Pendidikan dan Kebudayaan (kemendikbud) faktor internal dan ekternal individu turut mempengaruhi rendahnya nilai UN siswa SMA. Berdasarkan studi pendahuluan yang dilakukan peneliti diperoleh data bahwa di SMA Brawijaya Smart School terdapat siswa yang mendapatkan nilai dibawah kriteria ketuntasan minimal atau dikenal dengan istilah KKM. Abstrak Tujuan pada penelitian ini adalah untuk mengetahui hubungan antara stres akademik dan prokrastinasi akademik dengan prestasi belajar. Penelitian ini menggunakan rancangan penelitian korelasional. Populasi penelitian adalah siswa kelas X SMA Brawijaya Smart School Malang sejumlah 194 siswa, sampel penelitian berjumlah 100 siswa yang diperoleh menggunakan rumus slovin dengan tingkat kelonggaran 7% dan pengambilan sampel menggunakan metode simple random sampling. Penelitian ini menggunakan alat ukur atau intrumen penelitian berupa “Student Life Stress Inventory” yang diadaptasi dalam bahasa indonesia oleh (Rohana, 2017) dan skala kedua adalah skala prokrastinasi akademik yang dikembangkan penulis berdasar pada ciri prokrastinasi akademik menurut (Ferrari et al., 1995). Kedua instrumen tersebut telah teruji validitas dan reliabilitasnya. Pengukuran prestasi belajar menggunakan nilai Ujian tengah Semester pada semester ganjil 2021/2022. Hasil penelitian menunjukkan bahwa stres akademik dan prokrastinasi akademik memiliki hubungan negatif dengan prestasi belajar dan secara bersama-sama stres akademik dan prokrastinasi akademik mempengaruhi prestasi belajar. Harapan peneliti hasil penelitian ini dapat menjadi informasi dan pertimbangan untuk konselor sekolah atau guru BK dalam membeikan layanan kepada siswa. Selain itu, peneliti berharap untuk peneliti selanjutnya bisa melanjutkan penelitian pengembangan yang menghasilkan sebuah produk yang dapat membantu siswa terkait permasalahan stres akademik, prokrastinasi akademik dan prestasi belajar. Kata kunci: stres akademik; prokrastinasi akademik; prestasi belajar This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 mengalami stres dikarenakan adanya tuntutan tugas yang terlampau banyak sehingga menjadi tekanan tersendiri bagi siswa. Stres yang disebabkan karena tekanna atau tuntutan akademik disebut dengan stres akademik. Stres akademik diartikan sebagai respon atau reaksi individu yang muncul karena banyaknya tuntutan tugas akademik yang harus diselesaikan oleh siswa atau pelajar (Alvin, 2007; Desmita, 2010; Gadzella et al., 2005). Dampak buruk yang bisa ditimbulkan akibat adanya stress adalah meningkatnya kelelahan sehingga mengakibatkan menurunnya produktifitas dalam belajar. (Oon, 2007) menyatakan stres akademik apabila terjadi secara berkepanjangan pada siswa dapat berakibat pada daya tubuh menurun sehingga siswa mudah jatuh sakit. Stres akademik siswa membawa pengaruh negatif pada kesehatan fisik dan mental, sehingga dapat mengganggu kinerja siswa dan berujung pada penurunan prestasi belajarnya di sekolah (Akgun & Ciarrochi, 2003). Hal lain yang sering dikaitkan dengan prestasi belajar siswa di sekolah adalah perilaku prokrastinasi akademik. Prokrastinasi akademik menurut (Atmoko & Hidayah, 2014; Ferrari et al., 1995) adalah penundaan yang dilakukan pada tugas formal yang berkaitan pada akademik. Salah satu dampak perilaku prokrastinasi sendiri adalah terjadinya penurunan prestasi belajar siswa. Moris dan Catherin berpendapat bahwa perilaku prokrastinasi akademik sendiri menyebabkan kerugian besar pada kinerja siswa (Mahardika, 2019). Sejalan dengan pendapat tersebut penelitian (Choi & Sarah, 2009) menyebutkan bahwa prokrastinasi akademik sendiri dianggap sebagai perilaku negatif dengan indikasi malas yang dapat menyebabkan prestasi yang buruk. Hasil penelitian menunjukkan bahwa prokrastinasi akademik mempengaruhi prestasi akademik secara signifikan (Zahra & Hernawati, 2015). Selama ini belum ada penelitian di Indonesia terkait hubungan antara stres akademik dan prokrastinasi akademik dengan prestasi belajar siswa. Namun hasil studi pendahuluan dan elaborasi dari berbagai penelitian terdahulu didapatkan hasil bahwa stres akademik dan prokrastinasi memiliki hubungan signifikan dengan prestasi belajar siswa. Oleh karena itu perlu dilakukan pembuktian secara empiris terkait “Hubungan Antara Stres Akademik dan Prokrastinasi Akademik dengan Prestasi Belajar Siswa Kelas X di SMA Brawijaya Smart School”. Tujuan dari penelitian ini ialah: (1) untuk mengetahui hubungan antara stres akademik dengan prestasi belajar, (2) untuk mengetahui hubungan antara prokrastinasi akademik dengan prestasi belajar, (3) untuk mengetahui hubungan antara stres akademik dan prokrastinasi akademik dengan prestasi belajar. 1. Pendahuluan Pada kelas X sendiri terdapat kurang lebih 50 siswa yang mendapatkan nilai dibawah KKM pada beberapa mata pelajaran. Berdasarkan hasil wawancara dengan guru BK SMA Brawijaya Smart School didapatkan hasil bahwa salah satu tantangan dalam menghadapi pembelajaran daring dan luring terbatas di era pandemi covid-19 adalah kondisi siswa yang cenderung menunda-nunda pekerjaan yang berakibat pada kondisi psikologisnya. Hasil wawancara yang dilakukan peneliti dengan lima siswa kelas X SMA Brawijaya Smart School yang menyatakan bahwa mereka cenderung 448 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 sebesar 100 siswa. Pengambilan sampel menggunakan jenis probability sampling dengan teknik simple random sampling. sebesar 100 siswa. Pengambilan sampel menggunakan jenis probability sampling dengan teknik simple random sampling. Tahap penenutuan sampel meliputi: (1) jumlah sampel ditentukan dengan menggunakan rumus slovin dengan batas ketelitian 0,07 sehingga jumlah sampel 100 siswa, (2) teknik yang digunakan simple random sampling pengambilan sampel secara acak (3) menyebar intrument di jurusan IPA dan IPS. Pengumpulan data tentang stres akademik menggunakan alat ukur “Student Life Stress Inventory” oleh Gadzella yang kemudian diadaptasi oleh (Rohana, 2017). Alat ukur ini memiliki jumlah item 34 pernyataan. Skala ini kemudian di uji kembali validitas dan reliabilitasnya. Data prokrastinasi akademik didapatkan dengan instrumen penelitian berupa skala yang dikembangkan oleh peneliti beracuan pada ciri-ciri prokrastinasi akademik menurut (Ferrari et al., 1998). Skala prokrastinasi akademik yang disusun peneliti memiliki 25 item pernyataan dan pilihan jawaban yang digunakan adalah tidak setuju, kurang setuju, setuju dan sangat setuju. Sedangkan data mengenai prestasi belajar diperoleh dengan melakukan studi dokumentasi pada nilai ulangan tengah semester (UTS) pada semester ganjil 2021/2022 yang diperoleh dari konselor sekolah. Sebelum melakukan pengukuran menggunakan intrumen, sebelumnya dilakukan uji validitas dan reliabilitas intrumen. Intrumen yang akan di uji adalah “Student Life Stress Inventory” dan skala prokrastinasi akademik. Validitas dilakukan untuk mengetahui kelayakan instrumen dan reliabilitas dilakukan untuk mengetahui dapat dipercayakah alat ukur ini untuk mengukur variabel yang akan diukur (Sugiyono, 2013). Uji coba intrumen dilakukan di kelas X-2 di SMA Brawijaya Smart School dengan menggunakan google formulir. Pengujian ini dilakukan dengan uji validitas dan reliabilitas dengan menggunakan SPSS 25 for windows. Berdasarkan uji validitas pada instrument “Student Life Stress Inventory” diperoleh hasil bahwa 34 item dinyatakan valid karena memiliki nilai rhitung > 0,349 dan memiliki koefisien alpha sebesar 0,826 yang berarti skala “Student Life Stress Inventory” memiliki reabilitas sangat kuat. Sedangkan skala prokrastinasi akademik setelah dilakukan uji validitas terdapat 5 item gugur karena memiliki nilai rhitung < 0,349, item yang gugur meliputi item nomor 5,10,21,24 dan 25. Skala prokrastinasi akademik memiliki nilai koefisien alpha sebesar 0,920 yang berarti memiliki reliabilitas yang sangat kuat. Setelah melalui uji validitas dan reliabilitas pada instrument “Student Life Stress Inventory” dan skala prokrastinasi akademik, kemudian dilakukan pengumpulan data kepada siswa. Data penelitian selanjutnya digunakan untuk menjawab hipotesis penelitiaan. Analisis data yang digunakan adalah analisis deskriptif dan statistik. Analisis korelasional sebelum dilakukan setelah melalui uji prasyarat kemudian dilanjutkan dengan analisis korelasional. 2. Metode Penelitian korelasional digunakan dalam penelitian ini (Sugiyono, 2013). Penelitian guna menguji teori-teori tertentu untuk mencari tahu hubungan antar variabel dengan menggunakan intrumnet penelitian sebagai alat ukur disebut penelitian korelasional (Creswell, 2012). Penelitian ini meneliti hubungan tiga variabel, yaitu variabel bebas (stres akademik (X1) dan prokrastinasi akademik (X2)) dan variabel terikat (prestasi belajar (Y)). Sampel penelitian berjumlah 100 siswa dari keseluruhan populasi yang berjumlah 194 siswa, yang merupakan seluruh siswa kelas X SMA Brawijaya Smart School. Penentuan jumlah sampel menggunakan perhitungan rumus slovin sehingga diperoleh jumlah sampel penelitian 449 3.1.1. Hasil Uji Deskriptif Tabel 1. Kategori Variabel Stres akademik No. Kategori Frekuensi Persentase Interpretasi 1 Tinggi 8 8 % Sangat sedikit 2 Sedang 64 64% Banyak 3 Rendah 28 28% Sedikit Prokrastinasi akademik 1 Tinggi 34 34 % Sedikit 2 Sedang 61 61% Banyak 3 Rendah 5 5% Sangat sedikit Prestasi belajar 1 Tinggi 25 25 % Sedikit 2 Sedang 59 59% Cukup banyak 3 Rendah 16 16% Sangat sedikit Tabel 1. Kategori Variabel Hasil analisis deskriptif disajikan pada tabel 2. Kategori variabel. Pada tabel diatas dijabarkan bahwa sebagian besar siswa memiliki tingkat stres akademik dalam dalam kategoru tinggi sebanyak 8%, kategori sedang 64%, dalam kategori rendah 28%. Sehingga dapat dikatakan bahwa sebagian besar siswa memiliki tingkat stres dalam kategori sedang. Untuk variabel prokrastinasi akademik sebagian besar siswa berada pada tingkat kategori tinggi sebanyak 34%, dalam kategori sedang 61% dan kategori rendah 5%.. Sedangkan untuk prestasi siswa yang berada pada tingkat tinggi sebanyak 25%, dalam kategori sedang 59% dan rendah sebanyak 16%. 3.1. Hasil Penelitian ini menghasilkan data deskriptif dan analisis statistik. Analisis deskriptif hasilnya berupa gambaran tentang ketiga variabel penelitian yaitu stres akademik, 450 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 prokrastinasi akademik dan prestasi belajar. Sedangkan hasil analisis statistik yang dilakukan meliputi uji prasyarat, uji korelasi dan uji hipotesis penelitian. 3.1.1. Hasil Uji Deskriptif 3.1.2. Hasil Uji Prasyarat Uji normalitas dilakukan dengan menggunakan Kolmogrov Smirnov (1- Sample K-S) yang menunjukkan hasil bahwa variabel stres akademik (X1) memiliki nilai signifikansi sebesar 0,140, variabel prokrastinasi akademik (X2) memiliki nilai signifikansi 0,064 dan variabel prestasi belajar (Y) memiliki nilai signifikansi sebesar 0,113. Berdasarkan nilai sig pada ketiga variabel, dapat dikatakan bahwa ketiga variabel memiliki nilai signifikansi > 0,05, maka bisa dapat disimpulkan bahwa data stres akademik, prokrastinasi akademik dan prestasi belajar memiliki sebaran terdistribusi normal. Uji linearitas dengan menggunakan Test of Linearity. Dalam uji linearitas beracuan pada nilai Deviation from Linearity > 0,05 maka terdapat hubungan linear antara variabel independen dengan variabel dependen. Hasil uji linearitas pada penelitian ini menunjukkan bahwa nilai Deviation from Linearity stres akademik dengan prestasi belajar 0.506>0,05 dan nilai Deviation from Linearity prokrastinasi akademik dengan prestasi belajar 0.429>0,05 yang artinya stress akademik dan prestasi belajar memiliki hubungan linear, begitu pula prokrastinasi akademik dan prestasi belajar memiliki hubungan linear. 451 3.1.3. Hasil Analisis Korelasi 3.1.3. Hasil Analisis Korelasi Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 Dasar pengambilan keputusan dalam uji multikolinearitas apabila nilai tolerance < 0,10 atau nilai VIF > 10 maka terjadi multikolinearitas. Diketahui nilai tolerance variabel stres akademik dan prokrastinasi akademik 0,919 > 0,1- dan VIF 1,089<10 sehingga tidak terjadi masalah multikolinearitas pada penelitian ini. Hasil uji heteroskedastisitas dilakukan dengan berpatokan dengan melihat titik-titik pada grafik scatterplot analisis regresi. Dasar keputusan uji heteroskedastisitas apabila titik- titik menyebar diatas dan dibawah angak 0, penyebaran titik tidak membentuk pola bergelombang dan titik-titik tidak membentuk pola tertentu. Hasil uji heteroskedastisitas bisa dilihat pada gambar 1. Grafik scatter plot yang menunjukkan titik-titik menyebar diatas dan dibawah angka 0 pada sumbu Y, menyebar merata dan tidak membentuk pola tertentu sehingga dapat disimpulkan data penelitian tidak terjadi heteroskedastisitas. Gambar 1. Hasil Uji Heteroskedastisitas Gambar 1. Hasil Uji Heteroskedastisitas 3.1.3.1. Hubungan antara stres akademik (X1) dengan prestasi belajar (Y) 3.1.3.1. Hubungan antara stres akademik (X1) dengan prestasi belajar (Y) Tabel 2. Uji Korelasi Dalam mencari hubungan antara variabel independen (stres akademik) dengan variabel dependen (prestasi belajar) dilakukan dengan SPSS 25 for windows menggunakan teknik uji korelasi pearson product moment. Pengambilan keputusan berdasar pada penyataan apabila rhitung > rtabel maka terdapat hubungan antar variabel. Nilai rtabel dilihat pada tabel distribusi r tabel dengan nilai N= 100 dan nilai signifikansi 5% maka dapat diketahui nilai rtabel 0.195. Nilai 3.1.3.1. Hubungan antara stres akademik (X1) dengan prestasi belajar (Y Tabel 2. Uji Korelasi Tabel 2. Uji Korelasi Tabel 2. Uji Korelasi Dalam mencari hubungan antara variabel independen (stres akademik) dengan variabel dependen (prestasi belajar) dilakukan dengan SPSS 25 for windows menggunakan teknik uji korelasi pearson product moment. Pengambilan keputusan berdasar pada penyataan apabila rhitung > rtabel maka terdapat hubungan antar variabel. Nilai rtabel dilihat pada tabel distribusi r tabel dengan nilai N= 100 dan nilai signifikansi 5% maka dapat diketahui nilai rtabel 0.195. Nilai 452 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 rhitung -0,467 > rtabel 0.195 yang artinya terdapat hubungan antara stres akademik dengan prestasi belajar. Berdasarkan pada tabel diatas diketahui nilai rhitung -0,467 dan nilai sig 0,00. Nilai rhitung yang menunjukkan angka negatif yaitu -0,467 yang artinya stres akademik dengan prestasi belajar memiliki hubungan yang negatif sehingga dapat diartikan jika stres akademik tinggi maka prestasi belajar rendah dan berlaku sebaliknya. rhitung -0,467 > rtabel 0.195 yang artinya terdapat hubungan antara stres akademik dengan prestasi belajar. Berdasarkan pada tabel diatas diketahui nilai rhitung -0,467 dan nilai sig 0,00. Nilai rhitung yang menunjukkan angka negatif yaitu -0,467 yang artinya stres akademik dengan prestasi belajar memiliki hubungan yang negatif sehingga dapat diartikan jika stres akademik tinggi maka prestasi belajar rendah dan berlaku sebaliknya. 3.1.4.1. Hubungan antara X1 (stres akademik) dengan Y (prestasi belajar) Hasil pengujian hipotesis dilakukan dengan melihat nilai sig pada tabel hasil uji korelasi. Peneletian ini menjukkan bahwa terdapat hubungan negatif antara stres akademik dengan prestasi belajar. Nilai sig 0,00< 0,05 artinya Ha diterima dan Ho ditolak. Hal ini berarti terdapat hubungan signifikan antara stres akademik dengan prestasi belajar siswa kelas X SMA Brawijaya Smart School Malang. 3.1.3.3. Hubungan antara stres akademi (X1) dan prokrastinasi akademik (X2) dengan prestasi belajar (Y) 3.1.3.3. Hubungan antara stres akademi (X1) dan prokrastinasi akademik (X2) dengan prestasi belajar (Y) 3.1.3.3. Hubungan antara stres akademi (X1) dan prokrastinasi akademik (X2) dengan prestasi belajar (Y) Tabel 4. Korelasi ganda Tabel 4. Korelasi ganda Proses analisis korelasi ganda dilakukan dengan SPSS 25 for windows. Dasar dalam pengambilan keputusan pada analisis korelasi ganda ini apabila nilai Fhitung > Ftabel. Berdasarkan hasil uji korelasi ganda diperoleh nilai Fhitung 46.681 > Ftabel. Penentuan Ftabel dengan menggunakan rumus Ftabel = F (k; n-k) dengan k=2 dan n= 100 kemudian bisa ditentukan nilai Ftabel pada tabel distribusi nilai F sehingga diketahui nilai Ftabel sebesar 3.090. Sehingga berdasarkan uji korelasi ganda nilai Fhitung 46.681 > Ftabel 3.090 yang artinya terdapat hubungan antara stres akademik dan prokrastinasi akademik dengan prestasi belajar. Dapat diketahui pula nilai R Square sebesar 0.490 dan nilai sig. Fchange 0,00. Kontribusi stres akademik dan prokrastinasi akademik dengan prestasi belajar 49% (R Square x 100), sedangkan 51% dipengaruhi oleh faktor lain yang tidak ikut diteliti. 3.1.3.2. Hubungan antara prokrastinasi akademik (X2) dengan prestasi belajar (Y) (Y) Tabel 3. Uji Korelasi Tabel 3. Uji Korelasi Tabel 3. Uji Korelasi Tabel 3. Uji Korelasi Uji korelasi dengan bantuan SPSS 25 for windows dan menggunakan teknik uji korelasi pearson product moment. Pengambilan keputusan berdasar pada pernyataan apabila rhitung > rtabel maka terdapat hubungan antara prokrastinasi akademik dengn prestasi belajar. Nilai rtabel dapat diketahui dengan melihat pada tabel distribusi r tabel dengan nilai N= 100 dan nilai signifikansi 5% maka diketahui nilai rtabel 0.195. Nilai rhitung -0,634 > rtabel 0.195 sehingga diartikan bahwa terdapat hubungan antara stres akademik dengan prestasi belajar. Berdasarkan pada tabel diatas diketahui nilai rhitung sebesar -0,634 dan nilai sig 0,00. Nilai rhitung menunjukkan angka negatif yaitu -0,634 yang artinya terdapat hubungan negatif stres akademik dengan prestasi belajar. Hal ini dapat diartikan jika stres akademik tinggi maka prestasi belajar rendah dan berlaku sebaliknya. 453 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 3.1.3.3. Hubungan antara stres akademi (X1) dan prokrastinasi akademik (X2) dengan prestasi belajar (Y) Tabel 4. Korelasi ganda 3.1.4.3. Hubungan antara X1 (stres akademik) dan X2 (prokrastinasi akademik) dengan Y (prestasi belajar) Berdasarkan uji korelasi ganda diperoleh hasil yang menyatakan bahwa stres akademik dan prokrastinasi akademik memiliki hubungan dengan prestasi belajar. Pengambilan keputusan dalam uji hipotesis berdasar pada pernyataan apabila nilai sig Fchange < 0,05 maka Ha diterima dan Ho ditolak. Diketahui nilai sig Fchange 0,00 < 0,05 yang artinya Ha diterima dan Ho ditolak sehingga bisa dikatakan bahwa terdapat hubungan signifikan antara stres akademik dan prokrastinasi akademik dengan prestasi belajar. 3.1.4.2. Hubungan antara X2 (prokrastinasi akademik) dengan Y (prestasi belajar) Hasil pengujian hipotesis dilakukan dengan melihat nilai sig pada tabel hasil uji korelasi. Penelitian ini menjukkan bahwa terdapat hubungan antara prokrastinasi akademik dengan prestasi belajar. Nilai sig 0,00< 0,05 yang artinya Ha diterima dan Ho ditolak, sehingga dengan kata lain terdapat hubungan signifikan antara prokrastinasi akademik dengan prestasi belajar siswa kelas X SMA Brawijaya Smart School Malang. 454 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 3.2.1. Hubungan antara Stres Akademik dengan Prestasi Belajar Berdasarkan hasil uji hipotesis didapatkan keputusan bahwa Ha diterima dan Ho ditolakk, sehingga dapat diartikan bahwa terdapat hubungan signifikan antara stres akademik dengan prestasi belajar dengan nilai sig 0,000 dan nilai pearson correlation atau rhitung -0,467. Nilai pearson correlations -0,467 menunjukkan bahwa stres akademik dan prestasi belajar memiliki hubungan negatif, maksudnya disini semakin tinggi stres akademik seseorang maka semakin rendah prestasi belajar yang diperolehnya dan berlaku sebaliknya. Hal ini sejalan dengan penelitian (Mentari, 2018) dimana hasil penlitian ini menyatakan bahwa hubungan yang terjadi antara stres akademik dengan indeks prestasi mahasiswa berupa hubungan negatif. Penelitian ini sejalan dengan (Mulyani, 2013) bahwa stres akademik bisa mengakibatkan fisik menjadi tegang dan psikologis terganggu yang dapat merubah perilaku dan mempengaruhi prestasi belajar siswa secara kognitif. Sehingga dapat dikatakan bahwa dampak dari stress akademik sendiri dapat menurunkan prestasi belajar siswa. (Gadzella et al., 2005) menyatakan fisik yang bermasalah seperti sakit kepala dan nyeri punggung akan menganggu kinerja kognitif siswa dalam berfikir, padahal proses belajar tidak bisa lepas dari kegiatan berfikir. Sehingga dapat dikatakan bahwa stres akademik sebagai salah satu hambatan siswa dalam menunjukkan kinerja terbaik dalam belajar di sekolah. Sejalan dengan hal tersebut Johnson (dalam Desmita, 2010) terdapat 10% sampai dengan 30% pelajar yang menderita stres akademik juga mengalami penurunan hasil belajar. Siswa kelas X SMA Brawijaya Smart School memiliki tingkat stress akademik sedang-tinggi, sebaiknya siswa mampu mengelola stres akademik yang muncul karena adanya berbagai tekanan dan juga beban tugas sekolah. Dengan mampu mengelola stres akademik yang dialami maka siswa akan lebih bisa meningkatkan prestasi belajarnya disekolah. Sebagaimana hasil dari penelitian ini yang menyatakan bahwa stress akademik dapat mempengaruhi prestasi belajar siswa. Stres akademik tentu bukanlah hal yang mudah untuk dikendalikan dengan cepat oleh siswa. Keterkaitannya dengan guru bimbingan dan konseling adalah guru BK di sekolah dapat memberikan layanan kepada siswa untuk mengelola stres yang dialami. pengelolaan stress akademik yang tepat secara tidak langsung juga membantu siswa dalam meningkatkan prestasi belajarnya. 455 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 3.2.2. Hubungan antara Prokrastinasi Akademik dengan Prestasi Belajar Berdasarkan hasil uji hipotesis diketahui bahwa Ha diterima dan Ho ditolak, artinya terdapat hubungan signifikan antara prokrastinasi akademik dengan prestasi belajar dengan nilai sig 0,000 dan nilai pearson correlation atau rhitung -0,634. Nilai pearson correlations -0,634 menunjukkan bahwa stres akademik dan prestasi belajar memiliki hubungan yang negatif, artinya semakin rendah stres akademik maka semakin tinggi prestasi belajarnya dan berlaku sebaliknya. Sejalan dengan hal tersebut penelitan (Yuhan & Yamleam, 2021) menunjukkan bahwa prokrastinasi akademik dengan prestasi belajar memiliki hubungan negatif dan signifikan Penelitian ini mendukung teori yang disampaikan (Suhadianto & Pratitis, 2020) menyatakan salah satu dampak dari prokrastinasi akademik adalah menurunnya prestasi belajar. Moris dan Catherin menyatakan bahwa perilaku prokrastinasi akademik sendiri menyebabkan kerugian besar pada kinerja siswa (Mahardika, 2019). Keberhasilan prestasi belajar ditentukan dengan ketepatan siswa dalam menyelesaikan tugas akademiknya. Maka bisa ditarik benang merah antara prokrastinasi akademik dengan prestasi belajar siswa, saat siswa diberikan tugas akademik dan mempunyai kecenderungan menghindar atau menunda penyelesaian tugas yang diberikan sehingga bisa berdampak pada hasil belajar atau prestasi belajar siswa. Sebagian besar siswa kelas X SMA Brawijaya Smart School memiliki tingkat prokrastinasi akademik sedang, sebaiknya siswa mampu mengurangi perilaku prokrastinasi akademik untuk mencapai prestasi belajar yang maksimal. Dengan tingkat prokrastinais akademik yang rendah bisa meningkatkan prestasi belajar. Sebagaimana hasil dari penelitian ini yang menyatakan bahwa prokrastinasi akademik memiliki hubungan yang negatif dengan prestasi belajar, yang artinya siswa dengan prokrastinasi akademik rendah akan mendapatkan prestasi yang cenderung tinggi. Peran konselor sekolah atau guru BK dalam membantu siswa mengatasi prokrastinasi akademik sangat diperlukan dalam hal ini. Pasalnya prokrastinasi akademik pada siswa kelas X SMA Brawijaya Smart School tergolong dalam golongan sedang-tinggi. Alangkah baiknya apabila guru BK bisa mengembangkan layanan pada bidang pribadi dan belajar dalam membantu siswa mengatasi prokrastinasi akademik. Guru BK dapat mempertimbangkan penggunaan panduan sinema edukasi untuk menurunkan prokrastinasi siswa (Dinata, Rahman, & Indreswari, 2021) Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 Berdasarkan uji hipotesis penelitian menyatakan bahwa Ha diterima dengan begitu Ho ditolak yang artinya terdapat hubungan signifikan antara stress akademik dan prokrastinasi akademik dengan prestasi belajar. Diketahui nilai sig. Fchange sebesar 0,000 dan nilai Fhitung sebesar 46.681. Diketahui pula nilai R Square bebesar 0.49 yang artinya stres akademik dan prokrastinasi akademik mempengaruhi prestasi belajar sebesar 49% dan sisanya dipengaruhi oleh variabel lain diluar penelitian ini. Stres akademik dan prokrastinasi akademik menjadi salah satu hambatan siswa dalam menunjukkan kinerja terbaik dalam belajar di sekolah. Stres akademik yang dialami siswa bisa membawa dampak pada kondisi fisik dan psikologis siswa, yang berujung pada terganggunya kinerja siswa dalam belajar. Salah satu dampak stres akademik menurut (Mulyani, 2013) bahwa stres akademik bisa mengakibatkan fisik menjadi tegang dan psikologis terganggu yang dapat merubah perilaku dan mempengaruhi prestasi belajar siswa secara kognitif. Sehingga dapat dikatakan bahwa dampak dari stress akademik sendiri dapat menurunkan prestasi belajar siswa. proses belajar tidak bisa lepas dari kegiatan berfikir. Prokrastinasi akademik secara umum adalah tindakan merugikan bagi siswa. Prokrastinasi membuat tugas akademik menjadi kian menumpuk dan membuat waktu pengerjaan tugas semakin sempit. Dampak prokrastinasi akademik pada bidang akademik siswa meliputi pekerjaan atau tugas tertunda, nilai atau prestasi belajar menurun, tugas menumpuk (Suhadianto & Pratitis, 2020). Sejalan dengan hal tersebut (Choi & Sarah, 2009) menyebutkan bahwa prokrastinasi dianggap sebagai perilaku negatif dengan indikasi malas yang dapat menyebabkan prestasi yang buruk. Berdasarkan pernyataan sebelumnya dapat ditarik kesimpulan bahwa stress akademik dan prokrastinasi akademik dapat membawa dampak negatif bagi prestasi belajar siswa. Sejalan dengan hasil temuan pada penelitian ini yang menyatakan bahwa stres akademik dan prokrastinasi akademik memiliki hubungan negatif dengan prestasi belajar. Dimana saat siswa memiliki tingkat stres akademik dan tingkat prokrastinasi akademik rendah maka prestasi belajarnya tinggi. Hasil penelitian menunjukkan bahwa stres akademik dan prokrastinasi akademik keduanya mempengaruhi prestasi belajar sebesar 49%, sedangkan 51% dipengaruhi oleh faktor-faktor lain diluar penelitian. Menurut Dalyono (2015) bagaimana seseorang belajar juga turrut mempengaruhi hasil belajar. Belajar tanpa menggunakan teknik atau cara yang tepat tidak bisa membawa hasil yang maksimal. Selain itu, kondisi sekolah juga turut mempengaruhi hasil belajar siswa. Kondisi sekolah disini meliputi kualitas guru, fasilitas/perlengkapan sekolah, pelaksanaan tat tertib dan jumlah siswa. Salah satu factor yang turut berpengaruh pada prestasi belajar adalah motivasi belajar. Menurut Sardiman (2003) motivasi belajar diartikan sebagai motif dari siswa yang dapat memberikan arah kegiatan belajar, sehingga tujuan belajar dapat terpenuhi. Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 Berdasarkan paparan tersebut dapat disimpulkan bahwa prestasi belajar selain dipengaruhi oleh stres akademik dan prokrastinasi akademik terdapat faktor lain di luar penelitian ini. 3.2.3. Hubungan antara Stres Akademik dan Prokrastinasi Akademik dengan Prestasi Belajar Prestasi belajar menjadi topik yang paling sering diperbincangkan oleh pendidik, pemerintah bahkan oleh para orang tua. Prestasi belajar dapat direpresentasikan dengan simbol, angka, huruf dan kalimat, yang dapat mencerminkan hasil yang telah diperoleh tiap- tiap siswa pada jangka waktu tertentu (Rosyid et al., 2019. Setiap siswa tentu berharap prestasi belajarnya di sekolah dalam predikat tinggo, namun dalam proses pencapainnya terdapat faktor-faktor yang turut mempengaruhinya. 456 Daftar Rujukan j Akgun, S., & Ciarrochi, J. (2003). Learned Resourcefulness Moderates the Relationship Between Academic Stress and Academic Performance.Pdf. Educational Psychology, 23(3). Akgun, S., & Ciarrochi, J. (2003). Learned Resourcefulness Moderates the Relationship Between Academic Stress and Academic Performance.Pdf. Educational Psychology, 23(3). Alvin. (2007). Self efficacy: The exercise of a control. W.H. Freeman & Company. Alvin. (2007). Self efficacy: The exercise of a control. W.H. Freeman & Company. Atmoko, A., & Hidayah, N. (2014). Landasan Sosial Budaya dan Psikologis Pendidikan (1st ed.). Gunung Samudra. https://books.google.co.id/books?id=h0MyDwAAQBAJ&lpg=PP1&ots=0NBaywcRMt&dq=Atmoko %26 Hidayah (2014) &lr&hl=id&pg=PR2#v=onepage&q=Atmoko & Hidayah (2014)&f=false Choi, J. ., & Sarah, V. . (2009). Why Not Procrastinate? Development and Validation of a New Active Procrastination Scale. Journal of Social Psychology, 2. Creswell, J. W. (2012). Research Design: Pendekatan Kualitatif, Kuantitatif dan Mixed, terj. Achmad Fawaid. Pustaka Pelajar. Desmita. (2010). Psikologi Perkembangan Peserta Didik. PT. Remaja Roskakarya. Dinata, W. C., Rahman, D. H., & Indreswari, H. (2021). Pengembangan Panduan Sinema Edukasi dengan Model Blended Learning untuk Menurunkan Prokrastinasi Akademik Siswa Sekolah Menengah Atas. Buletin Konseling Inovatif, 1(2), 83–89. doi: 10.17977/um059v1i22021p83-89 Ferrari, J. ., Johnson, J. ., & Mc Cown, W. . (1995). Procrastination and task avoidance: Theory, research, and treatment. Springer Science & Business Media. https://books.google.co.id/books?id=Lu4r0H_wcVcC&lpg=PA1&ots=-iYau7DrfP&dq=ferrari 1995&lr&hl=id&pg=PA1#v=onepage&q=ferrari 1995&f=false Ferrari, J. R., Keane, S. M., Wolfe, R. N., & Beck, B. L. (1998). The antecedents and consequences of academic excuse-making: Examining individual differences in procrastination. Research in Higher Education, 39(2). Gadzella, B. M., Baloglu, M., Masten, W. G., & Wang, Q. (2005). Evaluation of the Student Life-stress Inventory- Revised. Journal of Instructional Psychology, 39(January 2012). Hamdu, G., & Agustina, L. (2017). Motivasi1.Pdf. Pengaruh Motivasi Belajar Siswa Terhadap Pestasi Belajar Ip Di Sekolah Dasar, 12(1), 90–96. Mahardika, I. (2019). 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Simpulan Kesimpulan dalam penelitian ini menyatakan bahwa: (1) terdapat hubungan negatif antara stres akademik dengan prestasi belajar pada siswa kelas X SMA Brawijaya Smart School, (2) terdapat hubungan negatif antara prokrastinasi akademik dengan dengan prestasi belajar pada siswa kelas X SMA Brawijaya Smart School (3) konsep stres akademik dan prokrastinasi 457 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 Jurnal Pembelajaran, Bimbingan, dan Pengelolaan Pendidikan, 2(5), 2022, 447–459 akademik secara serentak (simultan) berpengaruh negatif terhadap prestasi belajar siswa, dari data analisis diantara kedua variabel tersebut yang paling besar mempengaruhi prestasi belajar adalah prokrastinasi akademik. Hubungan negatif disini diartikan bahwa semakin tinggi stres akademik dan prokrastinasi akademik siswa maka semakin rendah prestasi belajar yang diperoleh. Zahra, Y., & Hernawati, N. (2015). 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https://openalex.org/W4389212669	https://journalsofznu.zp.ua/index.php/psych/article/download/3939/3762	Ukrainian	null	THE ROLE OF MORAL BELIEFS IN THE FORMATION OF PUBLIC READINESS TO FIGHT CORRUPTION	Žurnal sučasnoï psihologìï	2,023	cc-by	6,213	23 23 23 УДК 159.923.32-057 DOI https://doi.org/10.26661/2310-4368/2023-4-3 УДК 159.923.32-057 DOI https://doi.org/10.26661/2310-4368/2023-4-3 Key words: corruption, moral beliefs, public readiness, anti-corruption culture, social ethics, psychological factors, anti-corruption strategies. THE ROLE OF MORAL BELIEFS IN THE FORMATION OF PUBLIC READINESS TO FIGHT CORRUPTION Zarichanskyi O. A. Candidate of Pedagogical Sciences, Associate Professor, Senior Researcher at the Department of Problems of Information and Psychological Struggl of the Research Center Military Institute of Taras Shevchenko National University of Kyiv Mykhailo Lomonosov str., 81, Kyiv, Ukraine orcid.org/0000-0002-6934-9521 olegzarvin69@gmail.com Zarichanskyi O. A. Candidate of Pedagogical Sciences, Associate Professor, Senior Researcher at the Department of Problems of Information and Psychological Struggle of the Research Center Military Institute of Taras Shevchenko National University of Kyiv Mykhailo Lomonosov str., 81, Kyiv, Ukraine orcid.org/0000-0002-6934-9521 olegzarvin69@gmail.com This scientific article is devoted to the study of the role of moral beliefs in the formation of public readiness to fight corruption. Corruption remains a serious problem in many countries and has a wide range of negative social, economic and political consequences. Understanding the psychological factors underlying people’s attitudes toward corruption is critical to developing effective strategies to combat this problem. This article analyzes an important aspect of the modern social phenomenon – corruption, and determines the role of moral beliefs in the formation of public readiness to fight it. Using an interdisciplinary approach, including elements of psychology and sociology, the article examines the influence of moral values and beliefs on the attitude of citizens to corrupt practices.i The article also defines which moral beliefs are key to creating a positive attitude toward anti-corruption measures and initiatives. The author uses empirical data from surveys and psychological experiments to analyze the attitude of individuals towards corruption and their willingness to oppose it. In this study, we examine in detail the moral factors that can support or inhibit a society’s willingness to fight corruption. We use a mixed research method, including qualitative and quantitative data analysis. A survey is conducted among a representative sample of citizens using structured questionnaires. We include questions about moral values and willingness to participate in anti-corruption initiatives in the questionnaires. Group interviews were also conducted with participants to gain a deeper understanding of their moral and ethical beliefs and their attitudes towards corruption. The results of the study reveal key psychological factors that influence attitudes toward corruption, including the role of moral beliefs in shaping positive or negative perceptions of corruption. The author of the article come to the conclusion that moral beliefs have a huge impact on public readiness to fight corruption and are a key factor in the formation of effective anti-corruption strategies. РОЛЬ МОРАЛЬНИХ ПЕРЕКОНАНЬ У ФОРМУВАННІ ГРОМАДСЬКОЇ ГОТОВНОСТІ ДО БОРОТЬБИ З КОРУПЦІЄЮ Зарічанський О. А. кандидат педагогічних наук, доцент, старший науковий співробітник відділу проблем інформаційно-психологічного протиборства Науково-дослідного центру Військовий інститут Київського національного університету імені Тараса Шевченка вул. Михайла Ломоносова, 81, Київ, Україна orcid.org/0000-0002-6934-9521 olegzarvin69@gmail.com Зарічанський О. А. кандидат педагогічних наук, доцент, старший науковий співробітник відділу проблем інформаційно-психологічного протиборства Науково-дослідного центру Військовий інститут Київського національного університету імені Тараса Шевченка вул. Михайла Ломоносова, 81, Київ, Україна orcid.org/0000-0002-6934-9521 olegzarvin69@gmail.com Ключові слова: корупція, моральні переконання, громадська готовність, антикорупційна культура, соціальна етика, психологічні чинники, антикорупційні стратегії. Ключові слова: корупція, моральні переконання, громадська готовність, антикорупційна культура, соціальна етика, психологічні чинники, антикорупційні стратегії. Робота присвячена вивченню ролі моральних переконань у формуванні громадської готовності до боротьби з корупцією. Корупція залишається серйозною проблемою в багатьох країнах і має широкий спектр негативних соціальних, економічних та політичних наслідків. Розуміння психологічних чинників, що лежать в основі ставлення людини до корупції, є критичним для розробки ефективних стратегій боротьби з цією проблемою. Ця стаття аналізує важливий аспект сучасного суспільного явища – корупцію – та визначає роль моральних переконань у формуванні громадської готовності до боротьби з нею. За допомогою інтердисциплінарного підходу, включаючи елементи психології та соціології, стаття розглядає вплив моральних цінностей та переконань на ставлення громадян до корупційних практик. Також у статті визначено, які саме моральні переконання є ключовими для створення позитивного ставлення до антикорупційних заходів та ініціатив. Використано емпіричні дані з опитувань і психологічних експериментів для аналізу ставлення індивідів до корупції та їхньої готовності виступати проти неї. У цьому дослідженні ми детально розглядаємо моральні фактори, які можуть підтримувати або пригнічувати готовність суспільства до боротьби з корупцією. Використано змішаний метод дослідження, який включає якісний та кількісний аналіз даних. Проводиться опитування серед репрезентативної вибірки громадян з використанням структурованих анкет. До анкет включено питання про моральні цінності, а також про готовність брати участь в антикорупційних ініціативах. Також проводились групові інтерв’ю з учасниками, щоб отримати більш глибоке розуміння їхніх моральних та етичних переконань та їхнього ставлення до корупції. Результати дослідження дозволяють виявити ключові психологічні чинники, що впливають на ставлення до корупції, включаючи роль моральних переконань у формуванні позитивного або негативного сприйняття корупції. р ру Автор статті доходить висновку, що моральні переконання мають величезний вплив на громадську готовність до боротьби з корупцією і є ключовим фактором у формуванні ефективних антикорупційних стратегій. Розуміння ролі моральних цінностей може сприяти розробці програм та заходів для підвищення громадської свідомості та готовності діяти в інтересах боротьби з корупцією. Journal of modern psychology № 4 (31), 2023 ISSN 2310-4368 24 Журнал сучасної психології № 4 (31), 2023 THE ROLE OF MORAL BELIEFS IN THE FORMATION OF PUBLIC READINESS TO FIGHT CORRUPTION Слід зазначити, що у науковій літературі пропонується безліч різних термінів для опису практично одного і того ж психологічного утворення [6, c. 70]. При- вертає увагу неясність і розмитість меж пропоно- ваних понять і визначень і навіть деяка внутрішня суперечливість з точки зору психології запропоно- ваних концепцій. Спроба подивитися на проблему корупції з іншого боку, висуваючи постулат про те, що для того, щоб перемогти корупцію, досить при- вчити населення України ніколи не давати хабарів. Західні вчені (К. Dupuy, S. Neset, 2018) зазначають, що «є безсилими і нездатними протистояти коруп- ції і зупинити корупційну поведінку людей, наділе- них владою» [11, c. 18]. Здається цілком зрозумілим той факт, що коли перед звичайним громадянином постає альтернатива: дати хабар і отримати бажане благо або не давати хабара і блага не отримати, швидше за все, громадянин України цей хабар дасть. Людина не може жити поза епохою. Перерахуємо терміни, що найчастіше трапля- ються в наукових джерелах. І.О. Луговий (2019) та О.О. Островерх (2013) говорять про «антико- рупційний світогляд», маючи на увазі «систему антикорупційних ідей, поглядів, принципів, у яких відбивається негативне ставлення особи- стості, соціальних груп та всього товариства до корупційної діяльності». Багато дослідників (С.О. Філліпов (2014), О.О. Євдокімова (2020), І.В. Клименко (2019), В.В. Нонік (2019)) гово- рять про «антикорупційну спрямованість особи- стості». На думку Н. Рожанської (2017), що роз- глядає цю проблему в педагогічному аспекті, це «інтегративна якість особистості, яка є підструк- турою професійної спрямованості та забезпечує неухильність дотримання антикорупційних норм у соціально-професійній діяльності за рахунок діяльнісно-мотиваційної установки на відтво- рення морально-правової стійкості до проявів корупції» [12, c. 202]. Дослідниками прогнозується формування анти- корупційної спрямованості особистості в резуль- таті активної роботи з «реалізації антикорупцій- ної освіти». Вже протягом кількох років у країні затверджуються спеціальні програми антикоруп- ційної освіти, що передбачають заходи, спрямо- вані на виховання нетерпимості до корупційних правопорушень [7, c. 320]. Однак, як свідчить практика, ці програми відрізняються певною декларативністю та вимагають науково обґрунто- ваного психологічного супроводу. На нашу думку, одним з недоліків цих програм є те, що вони пев- ною мірою ігнорують реалії повсякденного життя та конкретну соціокультурну ситуацію, що скла- лася у сучасній Україні, тому у разі практичної реалізації вони можуть зіткнутися з об’єктивно зумовленими труднощами [13, c. 134]. Так, наприклад, І.В. Співак (2019) заявляє, що «антикорупційна етика повинна ґрунтуватися на добровільному, усвідомленому виборі особи- стістю стратегії своєї поведінки, де корупційна поведінка має, зрештою, стати «непристойною» на ціннісно-орієнтаційному рівні. Однак ще 2005 року Джон М. THE ROLE OF MORAL BELIEFS IN THE FORMATION OF PUBLIC READINESS TO FIGHT CORRUPTION Understanding this role of moral values can contribute to the development of programs and activities to increase public awareness and willingness to act in the interests of fighting corruption. Однак, незважаючи на широке обговорення та заходи щодо боротьби з корупцією, вона зали- шається актуальною проблемою в багатьох краї- нах. В контексті цієї проблеми виникає важливе питання про моральні переконання у формуванні готовності громадян вступати в активну боротьбу з корупцією та сприяти створенню більш чесного і справедливого суспільства. Недостатнє розу- Постановка проблеми. Сучасне суспільство стикається з поширеною проблемою корупції, яка має серйозний вплив на економічний розви- ток, демократичні інституції та соціальну спра- ведливість. Розуміння психологічних факторів, що впливають на готовність індивідів боротися з корупцією, є надзвичайно важливим для розробки ефективних антикорупційних стратегій [8, c. 77]. ISSN 2310-4368 Журнал сучасної психології № 4 (31), 2023 25 міння цієї взаємодії ускладнює розробку і реалі- зацію ефективних стратегій та політик для подо- лання корупції у сучасних суспільствах [3, c. 213]. міння цієї взаємодії ускладнює розробку і реалі- зацію ефективних стратегій та політик для подо- лання корупції у сучасних суспільствах [3, c. 213]. психологічний портрет корупціонера та виділити окрему межу, яка робить цю людину «злочин- ною особистістю», на кшталт Ч. Ломброзо, поки не принесли відчутних практичних результатів. Як правило, підсумковий портрет містить яко- сті, властиві абсолютно нормальному у психо- логічному плані індивідууму: «Люди, схильні до корупції, здебільшого активні, комунікабельні, стресостійкі, мають вищу освіту і перебувають у шлюбі» [1, c. 182]. «Найчастіше це чоловіки віком від 40 років, які не зловживають алкоголем і не схильні до порушення громадського порядку», – наприклад, такого висновку дійшли автори дослід- ження соціального портрета корупціонера, прове- деного у 2018 році [9, c. 681]. лання корупції у сучасних суспільствах [3, c. 213]. Тому цілком зрозумілий стійкий інтерес вітчиз- няних учених до цієї проблематики, що демон- струється ними протягом двох останніх десяти- літь. Природа корупції, її причини та наслідки, антикорупційні заходи є предметом жвавої науко- вої дискусії. Наявність великої кількості наукових праць (А.А. Вознюк (2019), О.О. Акімов (2014), А.О. Чхаідзе (2015), О.І. Кудерміна (2018), Л.І. Каз- міренко (2018), М.Д. Данчук (2007) і т. д.) дозволяє говорити про формування нової галузі психоло- гічного дослідження – психології корупції. Вчені пропонують цілу низку заходів щодо профілактики корупції [5, с. 70]. Багато дослідників одним із прі- оритетних напрямів такої профілактики називають цілеспрямовану діяльність соціальних інститутів щодо формування у громадян специфічної якості особистості – здатності протистояти корупції. Journal of modern psychology № 4 (31), 2023 THE ROLE OF MORAL BELIEFS IN THE FORMATION OF PUBLIC READINESS TO FIGHT CORRUPTION Дарлі, відомий представник соціальної психології США, професор Прінстон- ського університету, писав: «Люди, які ініціюють корупцію, не аналізують ці дії з етичного погляду. Як не дивно, вони не вважають їх неетичними» [4, c. 35]. Професор Роберт Клітгард, один із провід- них світових фахівців із боротьби з корупцією, ще 1988 року запропонував класичну формулу корупції: «Корупція = Монополія + Свобода дій – Відповідальність», яка означає, що люди, наділені владою та не стримувані високим рівнем відпо- відальності за свої діяння, схильні зловживати цією владою та здійснювати корупційні право- порушення [10, c. 135]. На нашу думку, численні дослідження, що проводяться з метою скласти Journal of modern psychology № 4 (31), 2023 ISSN 2310-4368 26 Як бачимо, наявні відомі відмінності у тлу- маченні поняття та доборі адекватної терміно- логії, що описує сутність одного психологічного утворення – здатності особистості протистояти всім формам корупційної поведінки. Більшість із вищезгаданих авторів розглядають його в рамках категоріального апарату психології та педагогіки, а численні пропоновані дефініції потребують удо- сконалення. Все це свідчить не лише про теоре- тичну, але і практичну значущість поглибленого дослідження феномену антикорупційної спрямо- ваності особистості, оскільки необхідність фор- мування такого світоглядного складника у грома- дян України не підлягає сумніву, а антикорупційна освіта та виховання є важливим елементом сис- теми протидії корупції у нашій країні [2, с. 132]. [Введите текст] суспільний тиск, можуть впливати на готовність боротьби з корупцією. 4. Розробка рекомендацій: на розроблені рекомендації для урядових інших зацікавлених сторін щодо спо і б б і Це включає аналіз ступеня суворості моральних позицій та їх впливу на бажання діяти проти корупції. у р ру 3. Вивчення впливу зовнішніх факторів: дослі- дження спрямоване на визначення, яким чином зовнішні фактори, такі як освіта, медіа, релігія або суспільний тиск, можуть впливати на формування моральних переконань та готовність боротьби з корупцією. ормування моральних переконань та 4. Розробка рекомендацій: на основі результа- тів дослідження розроблені рекомендації для уря- дових установ, громадських організацій та інших зацікавлених сторін щодо способів підвищення рівня громадської готовності до боротьби з коруп- цією на основі моральних переконань. основі результатів дослідження установ, громадських організацій та бі і і Загалом, мета емпіричного дослідження на цю тему полягає в розумінні зв’язку між мораль- ними переконаннями та готовністю громадян діяти проти корупції та виходити з рекоменда- ціями, які можуть сприяти підвищенню громад- ської готовності та ефективності заходів проти корупції. обів підвищення рівня громадської снові моральних переконань. на цю тему полягає в розумінні зв’язку між мадян діяти проти корупції та виходити з Мета статті. THE ROLE OF MORAL BELIEFS IN THE FORMATION OF PUBLIC READINESS TO FIGHT CORRUPTION Метою цього дослідження є розкриття взаємозв’язку між моральними цінно- стями, етичними переконаннями та готовністю і здатністю громадян боротися з корупцією. Дослі- дження спрямоване на встановлення факторів, що впливають на формування моральної основи для антикорупційних дій, та розробку рекомендацій для підвищення ефективності антикорупційних програм шляхом впливу на моральні цінності та етичні погляди громадян. Емпіричне дослідження включає такі аспекти, як: готовності до боротьби з корупцією на Загалом, мета емпіричного дослідженн моральними переконаннями та готовністю гро рекомендаціями, які можуть сприяти підвищен заходів проти корупції. Результати дослідження. Вибіркова су соціально-демографічного складу генеральної Результати дослідження. Вибіркова сукупність враховувала особливості соціально-демографіч- ного складу генеральної сукупності. Основними для визначення вибіркової сукупності були такі критерії, як: адміністративно-територіальний поділ, стать, вік, співвідношення міського та сіль- ського населення. Обсяг вибірки та спосіб обробки первинного матеріалу забезпечують стандартну похибку вимірювання, що не перевищує 5%. ню громадської готовності та ефективності купність враховувала особливості сукупності. Основними для визначення міністративно-територіальний поділ, стать, Об бі іб б б 1. Оцінка ефективності програм та ініціатив: дослідження включає аналіз ефективності різних програм і ініціатив, спрямованих на підвищення громадської готовності до боротьби з корупцією. Це може допомогти з’ясувати, наскільки успішно такі програми впливають на моральні переко- нання та поведінку. соціально-демографічного складу генеральної вибіркової сукупності були такі критерії, як: а вік, співвідношення міського та сільського нас первинного матеріалу забезпечують стандартн 5% 1. Оцінка ефективності програм та ініціатив: дослідження включає аналіз ефективності різних програм і ініціатив, спрямованих на підвищення громадської готовності до боротьби з корупцією. Це може допомогти з’ясувати, наскільки успішно такі програми впливають на моральні переко- нання та поведінку. соціально-демографічного складу генеральної вибіркової сукупності були такі критерії, як: а вік, співвідношення міського та сільського нас первинного матеріалу забезпечують стандартн 5% У рамках проведеного дослідження був опита- ний 601 респондент (див. рис. 1). лення. Обсяг вибірки та спосіб обробки у похибку вимірювання що не перевищує Освітній склад респондентів виглядає так: 56% досліджуваних мають вищу освіту різних рівнів та неповну вищу освіту, 34% респондентів мають середню спеціальну освіту. І лише 9% мають початкову освіту. у похибку вимірювання, що не перевищує опитаний 601 респондент (див. рис. 1). у 2. Оцінка рівня моральних переконань: дослі- дження спрямоване на визначення, наскільки істот- ними є моральні переконання людей щодо корупції. 5%. У рамках проведеного дослідження був 2. Оцінка рівня моральних переконань: дослі- дження спрямоване на визначення, наскільки істот- ними є моральні переконання людей щодо корупції. 5%. У рамках проведеного дослідження був Журнал сучасної психології №4 (31) 2023 ISSN 2310-4368 Рис. 1. Віковий склад респондентів Рис. THE ROLE OF MORAL BELIEFS IN THE FORMATION OF PUBLIC READINESS TO FIGHT CORRUPTION 1. Віковий склад респондентів Рис. 1. Віковий склад респондентів Рис. 1. Віковий склад респондентів ис 1 Віковий склад респондентів Рис. 1. Віковий склад респондентів Журнал сучасної психології № 4 (31), 2023 Журнал сучасної психології № 4 (31), 2023 ISSN 2310-4368 27 На середньому рівні своє матеріальне стано- вище оцінили 35% респондентів. Ще 38% вва- жають, що їхній достаток нижчий за середній. Високо оцінила себе за цим показником менша частка – 19% досліджуваних (див. рис. 2). [Введите текст] У результаті всього 16,1% опитаних респонден- тів такі заходи відомі, вони постійно стежать за ними. 28,3% стверджують, що про заходи протидії корупції їм відомо, але спеціально вони не стежать за ними. 13,1% опитаних респондентів нічого не знають про ці заходи. Третина опитаних (34,6%) щось чула, але нічого певного згадати про ці заходи не можуть. Такі показники свідчать про високий рівень правового нігілізму населення України, недо- віри до органів державної та муніципальної влади, а також відсутності активної громадянської позиції у питаннях протидії корупції. ановище оцінили 35% респондентів. Ще редній. Високо оцінила себе за цим новище оцінили 35% респондентів. Ще едній. Високо оцінила себе за цим (див. рис. 2). За видами діяльності розподіл вибірки такий (див. рис. 3). На середньому рівні своє матеріальне ст 38% ї ій й Рівень поінформованості населення про про- блеми корупції та заходи, які влада приймає для протидії корупції, є важливим компонентом, що визначає загальне сприйняття громадянами сту- пеня корумпованості органів влади. У ході дослі- дження респондентам ставилося питання щодо рівня обізнаності з приводу заходів влади для про- тидії корупції (див. рис. 4). [Введите текст] На середньому рівні своє матеріальне ст 38% вважають, що їхній достаток нижчий за се показником менша частка – 19% досліджувани 38% вважають, що їхній достаток нижчий за се показником менша частка – 19% досліджувани Також на респондентах була проведена оцінка інваріантності шкали довіри Ямагіші. Представ- лено статистики погодження для конфігураційної, редній. Високо оцінила себе за цим (див. рис. 2). Journal of modern psychology № 4 (31), 2023 ISSN 2310-4368 Рис. 2. Матеріальне становище респондентів За видами діяльності розподіл вибірки такий (див. рис. 3). Рис. 2. Матеріальне становище респондентів Рис. 2. Матеріальне становище респондентів За видами діяльності розподіл вибірки такий (див. рис. 3). Рис. 3. Види діяльності респондентів Рівень поінформованості населення про проблеми корупції та заходи, які влада Рис. 3. Види діяльності респондентів Рис. 2. Матеріальне становище респондентів Рис. 2. Матеріальне становище респондентів Рис. 2. Матеріальне становище респондентів За видами діяльності розподіл вибірки такий (див. рис. 3). THE ROLE OF MORAL BELIEFS IN THE FORMATION OF PUBLIC READINESS TO FIGHT CORRUPTION ис 2 Матеріальне становище респондентів Рис. 2. Матеріальне становище респондентів ості розподіл вибірки такий (див. рис. 3). За видами діяльності розподіл вибірки такий (див. рис. 3). Рис. 3. Види діяльності респондентів Рис. 3. Види діяльності респондентів За видами діяльності розподіл вибірки такий (див. рис. 3). Рис. 3. Види діяльності респондентів Рис. 3. Види діяльності респондентів с. 3. Види діяльності респондентів Рис. 3. Види діяльності респондентів Journal of modern psychology № 4 (31), 2023 ISSN 2310-4368 Рівень поінформованості населення про проблеми корупції та заходи, які влада ISSN 2310-4368 влада Journal of modern psychology № 4 (31), 2023 Рівень поінформованості на 28 метричної та скалярної інваріантності. З табл. 1 можна бачити, що підсумкова модель має досить хороші показники конфігураційної інваріантності, а також високі показники метричної (CFI < 0,01) і скалярної інваріантності (CFI < 0,01). Це дозво- ляє нам порівнювати середні значення показників індексу генералізованої довіри, отримані у різних групах. Проведено мультигруповий аналіз впливу цінностей та довіри на ставлення особистості до корупції. У табл. 2 представлені статистики пого- дження для конфігураційної та метричної інварі- антностей моделі впливу цінностей та довіри на прийнятність корупції у чотирьох вибірках. метричної та скалярної інваріантності. З табл. 1 можна бачити, що підсумкова модель має досить хороші показники конфігураційної інваріантності, а також високі показники метричної (CFI < 0,01) і скалярної інваріантності (CFI < 0,01). Це дозво- ляє нам порівнювати середні значення показників індексу генералізованої довіри, отримані у різних групах. Проведено мультигруповий аналіз впливу цінностей та довіри на ставлення особистості до корупції. У табл. 2 представлені статистики пого- дження для конфігураційної та метричної інварі- антностей моделі впливу цінностей та довіри на прийнятність корупції у чотирьох вибірках. активної громадянської позиції у питаннях про- тидії корупції. Також на респондентах була проведена оцінка інваріантності шкали довіри Ямагіші. Пред- ставлено статистики погодження для конфігу- раційної, метричної та скалярної інваріантності. З табл. 1 можна бачити, що підсумкова модель має досить хороші показники конфігурацій- ної інваріантності, а також високі показники метричної (CFI < 0,01) і скалярної інваріантно- сті (CFI < 0,01). Це дозволяє нам порівнювати середні значення показників індексу генералізо- ваної довіри, отримані у різних групах. Прове- дено мультигруповий аналіз впливу цінностей та довіри на ставлення особистості до корупції. У табл. 2 представлені статистики погодження для конфігураційної та метричної інваріантно- стей моделі впливу цінностей та довіри на при- йнятність корупції у чотирьох вибірках. У результаті всього 16,1% опитаних респон- дентів такі заходи відомі, вони постійно стежать за ними. THE ROLE OF MORAL BELIEFS IN THE FORMATION OF PUBLIC READINESS TO FIGHT CORRUPTION 28,3% стверджують, що про заходи про- тидії корупції їм відомо, але спеціально вони не стежать за ними. 13,1% опитаних респондентів нічого не знають про ці заходи. Третина опита- них (34,6%) щось чула, але нічого певного зга- дати про ці заходи не можуть. Такі показники свідчать про високий рівень правового нігілізму населення України, недовіри до органів держав- ної та муніципальної влади, а також відсутності Введите текст] респондентам ставилося питання щодо рівня обі протидії корупції (див. рис. 4). наності з приводу заходів влади для З табл. 2 можна бачити, що підсумкова модель має досить хороші показники конфігураційної інваріантності, а також задовільні показники метричної (∆CFI = 0,01) інваріантності. Це доз- воляє нам порівнювати стандартизовані регре- Журнал сучасної психології № 4 (31), 2023 ISSN 2310-4368 Рис. 4. Рівні обізнаності респондентів протидії корупції У результаті всього 16,1% опитаних респондентів такі заходи відомі, вони постійно стежать за ними. 28,3% стверджують, що про заходи протидії корупції їм відомо, але спеціально вони не стежать за ними. 13,1% опитаних респондентів нічого не знають про ці заходи. Третина опитаних (34,6%) щось чула, але нічого певного згадати про ці заходи не можуть. Такі показники свідчать про високий рівень правового нігілізму населення України, недовіри до органів державної та муніципальної влади, а також відсутності активної громадянської позиції у питаннях протидії корупції. Також на респондентах була проведена оцінка інваріантності шкали довіри Ямагіші Представлено статистики погодження для конфігураційної метричної та Рис. 4. Рівні обізнаності респондентів протидії корупції Таблиця 1 Статистика погодження моделей мультигрупового конфірматорного факторного аналізу для шкали довіри Модель інваріантності CFI ∆CFI Значення RMSEA Верхня межа 90% довірчого інтервалу RMSEA AIC X-квадрат Ступені свободи Конфігураційна 0,965 – 0,055 0,068 197,3 101,4 36 Метрична 0,967 0,002 0,045 0,056 179,4 113,4 51 Скалярна 0,967 0,0 0,044 0,055 276,4 116,4 54 Журнал сучасної психології № 4 (31), 2023 ISSN 2310-4368 Рис. 4. Рівні обізнаності респондентів протидії корупції У результаті всього 16,1% опитаних респондентів такі заходи відомі, вони постійно стежать за ними. 28,3% стверджують, що про заходи протидії корупції їм відомо, але спеціально вони не стежать за ними. 13,1% опитаних респондентів нічого не знають про ці заходи. Третина опитаних (34,6%) щось чула, але нічого певного згадати про ці заходи не можуть. Такі показники свідчать про високий рівень правового нігілізму населення України, недовіри до органів державної та муніципальної влади, а також відсутності активної громадянської позиції у питаннях протидії корупції. Також на респондентах була проведена оцінка інваріантності шкали довіри Я і і П фі ій ї ї Рис. 4. Таблиця 2 Статистика погодження моделей мультигрупового конфірматорного факторного аналізу для моделі впливу цінностей та довіри на прийнятність корупції Модель інваріантності CFI ∆CFI Значення RMSEA Верхня межа 90% довірчого інтервалу RMSEA AIC X-квадрат Ступені свободи Конфігураційна 0,999 – 0,022 0,068 429,2 212 4 Метрична 0,988 0,011 0,025 0,043 415,0 188 28 сійні коефіцієнти, що характеризують вплив цінностей та довіри на підсумковий показник прийнятності корупції у чотирьох групах, що розглядаються. У табл. 3 представлені стандар- тизовані коефіцієнти регресії, які є універсаль- ними для всіх чотирьох груп. За підсумками мультигрупового аналізу ми можемо сказати, що такі цінності, як «Влада домінування» та «Влада- ресурси» (на рівні тенденції), є універсальними факторами, що сприяють підвищенню прийнят- ності корупції для особистості. Таким чином, цін- ність «Влади» є єдиною цінністю, що підвищує прийнятність корупції для особи. До цінностей, які є культурно-універсальними факторами зни- ження прийнятності корупції для особистості, належать «Конформізм-правила» та «Універса- лізм-турбота» про інших. Тепер ми можемо ствер- джувати універсальність цього впливу. У процесі мультигрупового аналізу несподівано було отри- мано статистично значимий негативний зв’язок цінностей «Досягнення» за підсумковим показни- ком шкали прийнятності корупції. Ми очікували, що зв’язок цієї цінності зі схваленням корупції буде позитивним, припускаючи, що чим найбільш орієнтована людина на досягнення, тим менш вона схильна озиратися на наявні правила та стандарти і може заради досягнення своїх цілей відійти від правил. Проте, за теорією Ш. Шварца, цінність «Досягнення» характеризує мотивацію досяг- нення успіху відповідно до соціальних стандартів (норм). Цим пояснюється її негативний вплив на прийнятність корупції для особистості. В процесі аналізу зв’язків в окремих групах вплив цієї цін- ності не досягав необхідного рівня значущості, але у мультигруповому аналізі ефективність цієї цінності виявилася все-таки статистично значу- щою. Таким чином, статистично значущий зв’я- зок був виявлений, але він виявився протилежним припущенню, сформульованому в гіпотезі. ності не досягав необхідного рівня значущості, але у мультигруповому аналізі ефективність цієї цінності виявилася все-таки статистично значу- щою. Таким чином, статистично значущий зв’я- зок був виявлений, але він виявився протилежним припущенню, сформульованому в гіпотезі. «Генералізована довіра» так само, як і на окре- мих вибірках, не продемонструвала зв’язку з при- йнятністю корупції. На додаток до всіх вищеописаних результа- тів зазначимо, що ми також оцінювали вплив на прийнятність корупції соціально-демографічних характеристик респондентів: статі, віку та освіти. У жодній із груп статистично значущого впливу демографічних змінних на прийнятність корупції не виявлено. THE ROLE OF MORAL BELIEFS IN THE FORMATION OF PUBLIC READINESS TO FIGHT CORRUPTION Рівні обізнаності респондентів протидії корупції Таблиця 1 Статистика погодження моделей мультигрупового конфірматорного факторного аналізу для шкали довіри Модель інваріантності CFI ∆CFI Значення RMSEA Верхня межа 90% довірчого інтервалу RMSEA AIC X-квадрат Ступені свободи Конфігураційна 0,965 – 0,055 0,068 197,3 101,4 36 Метрична 0,967 0,002 0,045 0,056 179,4 113,4 51 Скалярна 0,967 0,0 0,044 0,055 276,4 116,4 54 Рис. 4. Рівні обізнаності респондентів протидії корупції Рис. 4. Рівні обізнаності респондентів протидії корупції . 4. Рівні обізнаності респондентів протидії корупції Рис. 4. Рівні обізнаності респондентів протидії корупції Журнал сучасної психології № 4 (31), 2023 ISSN 2310-4368 У результаті всього 16,1% опитаних респондентів такі заходи відомі, вони постійно стежать за ними. 28,3% стверджують, що про заходи протидії корупції їм відомо, але спеціально вони не стежать за ними. 13,1% опитаних респондентів нічого не знають про ці заходи. Третина опитаних (34,6%) щось чула, але нічого певного згадати про ці заходи не можуть. Такі показники свідчать про високий рівень правового нігілізму населення України, недовіри до органів державної та муніципальної влади, а також відсутності активної громадянської позиції у питаннях протидії корупції. Також на респондентах була проведена оцінка інваріантності шкали довіри ц Статистика погодження моделей мультигрупового конфірматорного факторного аналізу для шкали довіри Модель інваріантності CFI ∆CFI Значення RMSEA Верхня межа 90% довірчого інтервалу RMSEA AIC X-квадрат Ступені свободи Конфігураційна 0,965 – 0,055 0,068 197,3 101,4 36 Метрична 0,967 0,002 0,045 0,056 179,4 113,4 51 Скалярна 0,967 0,0 0,044 0,055 276,4 116,4 54 У результаті всього 16,1% опитаних респондентів такі заходи відомі, вони п жать за ними. 28,3% стверджують, що про заходи протидії корупції їм відомо, а Статистика погодження моделей мультигрупового конфірматорного факторног для шкали довіри 29 Таблиця 2 Ці результати говорять про те, що при- йнятність корупції для особистості перебуває під впливом більш складних і глибинних психологіч- них факторів, зокрема, як було показано в дослід- женні, низки цінностей. Підвищення рівня громадської готовності до боротьби з корупцією на основі моральних пере- конань вимагає комплексного підходу та співп- раці між урядом, громадянським суспільством та іншими зацікавленими сторонами. Ось деякі рекомендації для досягнення цієї мети на основі досягнутих результатів: 1. Освіта та інформаційна робота: і – впровадження антикорупційних тем у шкільну та університетську програми для формування анти- корупційної свідомості у молоді; – впровадження антикорупційних тем у шкільну та університетську програми для формування анти- корупційної свідомості у молоді; – забезпечення доступу до об’єктивної інформа- ції про наслідки корупції та методи її запобігання. Таблиця 3 ц Універсальні зв’язки цінностей та генералізованої довіри з прийнятністю корупції Модель інваріантності Стандартизований коефіцієнт регресії Рівень значущості Комформізм – правила -0,236 0,001 Самостійність – вчинки -0,01 0,798 Універсалізм – турбота про інших -0,175 0,001 Влада – домінування 0,147 0,001 Безпека – особиста 0,034 0,348 Влада – ресурси 0,087 0,064 Генералізована довіра 0,007 0,849 ні зв’язки цінностей та генералізованої довіри з прийнятністю корупції Journal of modern psychology № 4 (31), 2023 Journal of modern psychology № 4 (31), 2023 ISSN 2310-4368 30 2. Зміцнення правової бази: 2. Зміцнення правової бази: цією, заснованої на моральних переконаннях та громадській готовності. Важливо враховувати, що успіх у цьому напрямі вимагає тісної співпраці всіх зацікавлених сторін та постійного моніто- рингу та вдосконалення заходів. цією, заснованої на моральних переконаннях та громадській готовності. Важливо враховувати, що успіх у цьому напрямі вимагає тісної співпраці всіх зацікавлених сторін та постійного моніто- рингу та вдосконалення заходів. – підтримка та вдосконалення антикорупцій- ного законодавства; – забезпечення незалежного судового процесу та належного покарання корупціонерів. р у Висновки. Моральні переконання відіграють важливу роль у формуванні громадської готов- ності до боротьби з корупцією. Проведене дослі- дження вивчало вплив моральних цінностей та переконань на ставлення громадян до корупції та їхню готовність діяти проти цього злочинного явища. Дослідження показало, що існують тісні зв’язки між моральними переконаннями і став- ленням до корупції. Громадяни, які вважають моральні цінності важливими у своєму житті, частіше схильні вважати корупцію неприйнятною і виявляти бажання боротися з нею. Це свідчить про те, що національні програми та ініціативи, спрямовані на формування моральних цінностей та усвідомлення шкідливості корупції, можуть мати значущий вплив на громадську готовність до боротьби з цією проблемою. 3. Розвиток антикорупційних інституцій: – зміцнення ролі та повноважень антикорупцій- них органів і служб, таких як антикорупційне бюро; – забезпечення їх фінансової незалежності та надання їм необхідних ресурсів. 4. Підвищення участі громадян: – залучення громадських організацій та акти- вістів до антикорупційних ініціатив та контролю за владою; – створення механізмів для звернень громадян щодо корупційних випадків та їхнього розсліду- вання. 5. Співпраця з бізнес-спільнотою: – запровадження етичних стандартів та прак- тик у бізнесі з метою зменшення корупції в комер- ційних угодах. у 6. Прозорість та відкритість: Також дослідження підкреслило важливість освіти, релігії та культурних особливостей як зовнішніх факторів, які можуть впливати на фор- мування моральних переконань. Освіта та інфор- мованість суспільства про корупцію можуть підсилити готовність громадян боротися з цим явищем. Таблиця 3 – забезпечення відкритості бюджетного про- цесу та витрат державних коштів; – публікація інформації про діяльність дер- жавних органів та результати аудитів. р р у у 7. Нагородження та відзначення героїв антико- рупційної боротьби: – встановлення нагород та почесних звань для осіб, які активно борються з корупцією та виявля- ють моральну стійкість. Узагальнюючи результати, ми можемо визнати, що розуміння ролі моральних пере- конань у формуванні громадської готовності до боротьби з корупцією є важливим кроком у розробці більш ефективних антикорупційних стратегій і програм. Збереження та посилення моральних цінностей у суспільстві може спри- яти більш широкому прийняттю антикорупцій- них заходів та сприяти створенню чеснішого та більш прозорого суспільства. р у 8. Міжнародне співробітництво: – залучення до міжнародних ініціатив та обмін досвідом у сфері антикорупційних заходів. у ф р ру 9. Моніторинг та оцінка: – проведення регулярного моніторингу та оцінки ефективності антикорупційних заходів та програм. ф ру р р Ці рекомендації можуть стати основою для розвитку комплексної стратегії боротьби з коруп- ф ру р р Ці рекомендації можуть стати основою для розвитку комплексної стратегії боротьби з коруп- Журнал сучасної психології № 4 (31), 2023 ЛІТЕРАТУРА 1. Акімов О. О. Психологічні аспекти проявів корупції в державному управлінні. Державне управ- ління: теорія та практика. 2014. № 1. С. 181–188. 2. Клименко І. В. Формування антикорупційної культури особистості майбутнього правоохоронця в системі вищих навчальних закладів МВС України. 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https://openalex.org/W4285394929	https://vbn.aau.dk/ws/files/481217689/Hangh_j_2022.pdf	English	null	Teachers’ framing and dialogic facilitation of Minecraft in the L1 classroom	L1 educational studies in languages and literature	2,022	cc-by	17,270	Aalborg Universitet Aalborg Universitet Hanghøj, Thorkild Published in: L1-Educational Studies in Language and Literature DOI (link to publication from Publisher): 10.21248/l1esll.2022.22.2.364 Citation for published version (APA): Hanghøj, T. (2022). Teachers’ framing and dialogic facilitation of Minecraft in the L1 classroom. 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Take down policy If you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access to the work immediately and investigate your claim. Citation for published version (APA): Hanghøj, T. (2022). Teachers’ framing and dialogic facilitation of Minecraft in the L1 classroom. L1-Educational Studies in Language and Literature, 22(2), 1-31. https://doi.org/10.21248/l1esll.2022.22.2.364 Teachers’ framing and dialogic facilitation of Minecraft in the L1 classroom Hanghøj, Thorkild Downloaded from vbn.aau.dk on: October 24, 2024 Downloaded from vbn.aau.dk on: October 24, 2024 1 Hanghøj, T. (2022). Teachers’ framing and dialogic facilitation of Minecraft in the L1 class- room. Contribution to a special issue Gaming and Literacies, edited by Thorkild Hanghøj and Sandra Abrams. L1-Educational Studies in Language and Literature, 22, 1-31. https://doi.org/10.21248/l1esll.2022.22.2.364 Corresponding author: Thorkild Hanghøj, Department of Communication and Psychology, Uni- versity of Aalborg, email: thorkild@hum.aau.dk © 2022 International Association for Research in L1-Education. Keywords: digital games, dialogic education, educational framing, teacher facilitation, Minecraft © 2022 International Association for Research in L1-Education. Abstract Key examples include students analysing and interpreting games as fic- tional texts (Berger & McDougal, 2013); students searching for, reading, and produc- ing various types of game-related paratexts, such as game journalism (Hanghøj et al., 2020) or imaginary expansion packs to existing games (Beavis, 2007); students de- signing their own game concepts by combining programming skills with narrative ideas (Burn, 2007); or students developing critical literacy by producing game- related Let’s Play videos on YouTube and gaining insight into the commercial aspects of the business models surrounding games (Burwell, 2017). As the variety of these examples suggests, digital games represent complex texts and flexible tools that can be linked to the curricular aims of L1 education in multiple ways. It is a well-known fact that children all over the world spend a considerable amount of time playing digital games outside school – often more than two hours each day (WHO, 2016). Moreover, it is often argued that the multimodal communication that takes place during videogame play also extends beyond the actual game activities (e.g., Enslinn et al., 2019; Gee, 2003). In this way, it is not surprising if teachers choose to use commercial digital games in L1 education to engage students in mean- ingful literacy activities, which build on and extend their out-of-school game experi- ences in relation to curricular aims. As the two reviews by Bacalja (2022) and by Hanghøj et al. (2022) in this special issue find, several case studies have shown how the use of commercial games might support students’ learning of literacies in L1 classrooms. Key examples include students analysing and interpreting games as fic- tional texts (Berger & McDougal, 2013); students searching for, reading, and produc- ing various types of game-related paratexts, such as game journalism (Hanghøj et al., 2020) or imaginary expansion packs to existing games (Beavis, 2007); students de- signing their own game concepts by combining programming skills with narrative ideas (Burn, 2007); or students developing critical literacy by producing game- related Let’s Play videos on YouTube and gaining insight into the commercial aspects of the business models surrounding games (Burwell, 2017). As the variety of these examples suggests, digital games represent complex texts and flexible tools that can be linked to the curricular aims of L1 education in multiple ways. There exist several different ways of defining games. Abstract Abstract A fairly large body of research has documented how digital games can be used in L1 education. However, there is still a lack of detailed studies on how literacy teachers go about teaching with games as multi- modal texts in the classroom. Revisiting earlier empirical work on the use of the sandbox game Minecraft in primary school, the aim of this paper is to explore how a specific game challenge is enacted in practice as seen from a dialogic perspective. Drawing on theories on games and literacies, dialogic education, and teachers as professional practitioners, the paper presents the Game as Educational Challenge (GEC) model in order to understand how L1 teachers frame specific game challenges and facilitate dialogue with the students in relation to their game experiences. The model is used to reanalyse empirical examples of how teachers from three primary schools adopted a teaching unit with Minecraft through different pedagogi- cal approaches. The findings show not only how the teachers’ framing of the game challenges reflected their familiarity with the game, but also how they taught and related the game challenges to curricular aims in different ways. Moreover, it is found that the teachers negotiated authorial positions quite differ- ently when facilitating classroom discussions with students about their game experiences. Keywords: digital games, dialogic education, educational framing, teacher facilitation, Minecraft 2 T. HANGHØJ It is a well-known fact that children all over the world spend a considerable amount of time playing digital games outside school – often more than two hours each day (WHO, 2016). Moreover, it is often argued that the multimodal communication that takes place during videogame play also extends beyond the actual game activities (e.g., Enslinn et al., 2019; Gee, 2003). In this way, it is not surprising if teachers choose to use commercial digital games in L1 education to engage students in mean- ingful literacy activities, which build on and extend their out-of-school game experi- ences in relation to curricular aims. As the two reviews by Bacalja (2022) and by Hanghøj et al. (2022) in this special issue find, several case studies have shown how the use of commercial games might support students’ learning of literacies in L1 classrooms. Abstract However, Deterding (2015) argued that many definitions of games assume that "a game consists of one or more interconnected challenges a player is trying to overcome, which emerge from the player taking actions in pursuit of goals, and rules and objects/opponents that make attaining those goals challenging” (2015, p. 299; see also Plass et al., 2015). In this way, a game’s challenge, which could be a specific kind of difficulty or resistance requiring nontrivial effort and skill to overcome (e.g., solving puzzles in an adventure game), is at the heart of its game play experience. Building on this definition, I use digital game as an umbrella term for any type of game that is being played on an electronic device (computers, phones, consoles, and handheld devices), which in- cludes both videogames and computer games. Studies on digital games in L1 classrooms often focus on students’ experiences and learning processes. But how do L1 teachers go about using digital games in their classrooms to teach literacies? Drawing on literacy research as well as game re- search, Apperley and Beavis (2013) have argued convincingly that teachers using games need “gaming literacies,” which involve both “textual” literacies (e.g., when reading/playing or writing/producing multimodal texts in and around games) as well as literacies that are “specifically linked to the action-based processes of digital game play” (p. 2). In contrast to many other types of texts in the expanded text repertoire of the L1 curriculum, the authors emphasised how digital games cannot be under- stood simply on textual terms. This means that teachers using games in the class- room must pay attention to the action layer of games—how the specific games are TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT 3 3 played in the classroom and how that relates to students’ out-of-school gaming prac- tices. Apperley and Beavis described several important aspects of the games-as-text and the games-as-action layers in order to help “build classroom activities” that in- volve both curricular aspects and “young people’s out-of-school experiences of digi- tal games and gameplay” (p. 9.). Their theoretical work has inspired several other researchers working with games and literacies (e.g. Bacalja, 2017; Elliott, 2018; Mar- con & Faulkner, 2016), which makes it a highly valuable and influential contribution to the research on games and literacies in L1. Abstract However, even though Apperley and Beavis’s work has been used extensively to describe literacy teachers’ different ap- proaches towards games (e.g., Beavis et al., 2017), their theoretical framework pro- vides relatively limited analytical insight into how teachers frame and adopt digital games or facilitate dialogic interaction with students about their game experiences in classroom contexts. At the same time, several studies have pointed out that it is highly important to understand and provide more detail on how teachers try to cre- ate meaningful integration between facilitating games and achieving curricular aims (Arnseth et al., 2018; Bacalja, 2022). It is often unclear from the research how teach- ers manage (or fail) to link particular games with curricular aims and how they facil- itate games in the literacy classroom. In this way, there is a need for a more contex- tual understanding of game-based teaching in the L1 classroom, which maps how teachers create coherent connections between games as texts and as social actions when planning and facilitating specific game-related units. This paper explores a practice-oriented perspective on game-based teaching through a theoretical framework and an analytical model that focuses on under- standing how literacy teachers integrate games as a part of their teaching. In order to do so, I will pursue a dialogic perspective on how teachers frame, enact and facil- itate discussions on students’ game experiences in the classroom (Arnseth et al., 2018). Furthermore, I will argue that specific game challenges represent a key focal point when L1 teachers frame and enact games in their teaching, which involves cre- ating meaningful links between game goals and educational aims, as well as between game design (textual) aspects and social, action-oriented aspects of meaning. The empirical focus of my paper is based on a research project involving Mine- craft, which is a widely used and studied digital game within L1 education. The game is an open world (also known as a “sandbox” game) that offers the player a wide range of possibilities for exploring worlds, finding and crafting resources, and build- ing new constructions through interactions with other players. The game has sold more than 100 million copies and has a wide appeal among both girls and boys be- tween the ages of six and 12 (Mavoa et al., 2018). Abstract Given the huge popularity of the game and its flexible design space with almost unlimited possibilities for world-build- ing activities, it is not surprising that many L1 educators and researchers have taken a keen interest in the game. Moreover, there exists an educational version of the game specifically for schools, which has made the game more accessible to thou- T. HANGHØJ 4 sands of educators and students all over the world. In this way, Minecraft has be- come a game-changer, which demonstrates that it is possible for teachers who are not necessarily specialised “teacher-gamers” (Holan Lucci, 2018) to access and teach with complex commercial games in the classroom. p g The paper draws on my earlier empirical work on using Minecraft in the primary literacy classroom. Previously, I analysed how primary teachers positioned them- selves through different pedagogical approaches by executing, improvising, and transforming a game-based teaching unit (Hanghøj, 2017) and how the same group of teachers facilitated students’ writing of diaries in relation to their game experi- ences (Hanghøj et al., 2018). In this paper, I revisit and reinterpret the empirical data through a dialogic perspective by introducing a reworked version of the Game as Educational Challenge (GEC) model, which I have presented in an earlier version else- where (Hanghøj, 2017). In this way, I use the GEC model as an analytical tool in order to explore how the literacy teachers framed and enacted specific Minecraft game challenges in relation to game goals and curricular aims and how they assumed dif- ferent dialogic positions when facilitating the students’ game experiences in the classroom. The analysis is guided by the following research question: When teaching with Minecraft in an L1 classroom, how do literacy teachers frame and enact game challenges as well as facilitate dialogue with students about their game experiences? It is my hope that the theoretical and empirical insights that follow from reinterpret- ing the data through the GEC model will provide valuable perspectives on how teach- ers bring digital games into the L1 classroom. In this way, the paper aims to provide a more detailed understanding of how teachers do not simply use games as neutral tools, but adopt them by negotiating the meaning of specific game challenges and by orchestrating classroom discussions with students around their game experi- ences. The paper is structured as follows. 1. TEACHING MINECRAFT IN L1 CLASSROOMS There exists a growing body of literacy research on the use of Minecraft, which shows that the game can be used for teaching literacies in many different ways across pri- mary, lower-secondary, and upper-secondary education. Some studies focused on using Minecraft to increase student interest in working with literary texts, such as by letting upper-secondary students recreate and interpret Susan Hinton’s novel The Outsiders (1967) in the multimodal 3D game world (Marlatt, 2018) or by inviting lower-secondary students to read a book with a Minecraft theme in a reading club (Stufft, 2018). Other studies documented how primary students learn to write diaries or log entries by combining text and images in response to their game experiences in the Minecraft world (Dezuanni & Zagami, 2017; Hanghøj et al., 2020). In this way, the literacy focus is sometimes on creating multimodal designs in Minecraft inspired by existing literary texts and sometimes on letting students write their own texts based on their experiences in the game world. Several studies focused on how the game can be used to appeal to different groups of students who might not be engaged in L1 education. One study docu- mented how teaching with Minecraft can engage disadvantaged lower secondary students in the literacy classroom by drawing on their existing knowledge of the game outside school and positioning them as game experts (Elliot, 2016). Similarly, Marcon and Faulkner (2016) explored how Minecraft appealed to lower-secondary boys and girls, who worked strategically and collaboratively as they immersed them- selves in the game and engaged in problem-solving activities, as well as distributed forms of learning by linking their game constructions with social media. Other stud- ies have explored how children’s game play in an after-school setting with Minecraft employs a broad range of different modalities and collaborative performances (Bai- ley, 2016) or how the learning of literacies takes place when children play the game at home with their peers (Abrams, 2017; Dezuanni, 2018). Taken together, these findings indicate that Minecraft is well suited for engaging students across different age levels and for teaching many forms of literacies (e.g., reading, writing, or multi- modal production), whether the learning context is formal, informal, or a combina- tion thereof. Although there is rich potential for using Minecraft to learn literacies, several studies also mention that it can be quite demanding for teachers to facilitate such a complex game (Hanghøj, 2017; Prestridge, 2017). TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT 5 Abstract First, I review relevant research on using Mine- craft in L1, as well as research on how teachers teach with games. Next, I describe dialogic perspectives on game-based teaching, as well as how teachers frame games as a part of being reflective practitioners. Based on these theoretical perspectives, I then introduce the GEC model for conceptualising how game challenges relate to different aspects of game goals, curricular aims, games as designs (texts), and games as social actions. Third, I present an empirical case involving design interventions with a Minecraft unit in primary education. Using the categories of the GEC model, I then present two analytical themes that address teachers’ framing of game chal- lenges in Minecraft and their dialogic facilitation of classroom discussions in relation to the game. The paper concludes with a discussion on how Minecraft and other commercial digital games can be meaningfully related to the L1 curriculum, as seen from a dialogic perspective. 5 1. TEACHING MINECRAFT IN L1 CLASSROOMS In their recent cross-disciplinary review of research on Minecraft in education, Baek et al. (2020) pointed to several issues that teachers should consider when teaching with the game. First, teachers need support when learning how to play and teach with Minecraft, as the open- ended game world can be overwhelming at first. This might involve getting help from more capable colleagues or students. Moreover, students often will have different levels of expertise in relation to the game, with some students being novice players (“noobs”) and others being expert players. In this way, teachers must consider ped- agogical strategies for including students who are less familiar with the game to seek T. HANGHØJ 6 help from more capable peers. Consequently, teacher preparation is crucial when planning students’ learning activities and establishing meaningful links between the game and the curriculum. Baek et al. (2020) also emphasised that there might be curriculum inflexibility when teaching the game, as it is not necessarily obvious to teachers or students how the game aligns with particular standards. Moreover, there might be incompatibility between the knowledge practices of formal literacy educa- tion and children’s informal game practices, which involve different domain-specific norms and values on what counts and does not count as valid knowledge (Bacalja, 2020; Hanghøj, 2011). In general, research on teachers’ use of games documents the importance of teachers’ game literacy, as well as their pedagogical approaches to games. A survey conducted in the U.S. found that teachers who are not familiar with commercial games are far less likely to be using them for educational purposes (Takeuchi & Vaala, 2014). Likewise, a qualitative review of research on game-based teaching em- phasised the importance of teachers’ reflection on different pedagogical activities when using games, which involve planning game units, orienting students towards relevant aspects of the game, and facilitating game play and post-game reflection (Kangas et al., 2017). These examples illustrate how the discussion has shifted during the last two decades from trying to argue why it might be a good idea to use games for learning literacies (Gee, 2003) toward developing practice-oriented perspectives on how teachers could be teaching with specific games and in relation to what cur- ricular aims and topics (Arnseth et al., 2018). Consequently, there is a need for more studies that go into detail exploring and understanding the messy realities of how teachers adopt games in the literacy classroom. 2. THEORETICAL FRAMEWORK I will now present a theoretical framework for analysing how literacy teachers might use Minecraft and other digital games in the classroom. This involves bringing to- gether theories on dialogic education, as well as on teachers’ framing of games as a part of their professional practice. Building on earlier studies (Hanghøj, 2008, 2017), the framework will be linked to the GEC model, which is used to describe different aspects of how literacy teachers frame, enact, and facilitate Minecraft in the class- room. 2.1 A dialogic perspective on games in the classroom Digital games represent multimodal texts designed to offer specific affordances in terms of game aesthetics, narrative structures, and game mechanics that allow play- ers to navigate and interact with the game world (Apperley & Beavis, 2013; Burn, 2007; Toh & Lim, 2020). At the same time, it is problematic to assume that game designs, themselves, have direct effects on pedagogy and learning, as the potentials 7 TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT of games need to be facilitated and realised in educational practice (Clark et al., 2016). Following a dialogic perspective, I assume that digital games should be viewed as flexible designs or tools, which might take on myriad dialogic meanings to be ex- plored, negotiated, and legitimised when specific games are taught, played, and “talked into being” in the context of the classroom (Arnseth et al., 2018, p. 126). Research on dialogic education comprises a broad field that involves several theo- retical frameworks and research traditions (Mercer et al., 2019). For the purpose of this study, I am interested in how educational game experiences can be used to cre- ate dialogic spaces in and around the game being played (Arnseth et al., 2018; Hang- høj, 2008; Staaby, 2020). The notion of dialogic space refers to how talk is used in the classroom to create and explore difference as a shared meaning space “of un- certainty, multiplicity and potential” (Wegerif, 2010, p.346). The conceptualisation of games as enablers of dialogic spaces is highly inspired by the work of Bakhtin (1981, 1986). Bakhtin’s dialogic philosophy is grounded in the understanding that dialogue always is based on a “mutuality of differences” (Holquist, 2002, p. 41), which revolves around a preoccupation with how the juxta- position and “inter-animation of different voices” allow new meanings to emerge and develop (Bakhtin, 1981). Moreover, Bakhtin (1986) argued there can be no final word in dialogue, which makes dialogue inherently creative. This means that whilst each dialogic space is unique, what they all have in common is their potential for infinite meaning (Wegerif & Major, 2018). In this way, games can be used to create shared dialogic spaces that allow different voices among teachers and players to in- teract, where learning may occur. 2.1 A dialogic perspective on games in the classroom Similarly, when working with digital games in the classroom, teachers need to balance dialogically the authorial positions in relation not only to the students, but also to the authorial positions offered and denied by the specific game being taught. The focus here is not on teacher authority, understood as the isolated pedagogical practices of the individual teacher. Instead, I am interested in understanding autho- rial positions as a relational phenomenon among teacher, student, and game, to be challenged and maintained within particular classroom contexts. This means that dif- ferent authorial positions typically are present as a defining aspect of classroom dis- course. However, it is important to define empirically the purpose and nature of au- thorial positions and how they might serve valid educational ends. For the purpose of this paper, I am mainly interested in how L1 teachers facilitate dialogue with stu- dents’ about their game experiences in the classroom and how this involves negoti- ation of mutual dialogic positions between teacher, game, and students. This brings me to one last assumption of Bakhtin’s dialogic philosophy, which is highly relevant when describing teachers’ use of games in the L1 classroom. Accord- ing to Bakhtin (1981), all forms of communication and culture are subject to centrip- etal (or unifying) and centrifugal (or disunifying) forces. A centripetal force is the drive to impose one version of the truth, while a centrifugal force involves a range of possible truths and interpretations. Classroom dialogue is affected by both cen- tripetal and centrifugal forces in the ongoing negotiation of “truths” between teach- ers and students. In the words of Bakhtin, “Truth is not born nor is it to be found inside the head of an individual person, it is born between people collectively search- ing for truth, in the process of their dialogic interaction” (Bakhtin, 1984, p. 110). Dig- ital games represent a similar duality, as they involve centripetal forces, such as rules, constraints, and procedures designed to achieve order, as well as centrifugal forces, such as unexpected or disruptive events that might undermine order for no particular reason and allow for new meanings to emerge (Jensen, 2013, p. 75f). The dynamic between centripetal and centrifugal forces in a digital game opens up a pro- ductive dialogic possibility space, wherein players need to orient themselves toward the designed constraints of the game and take new possible actions, which might be more or less surprising. 2.1 A dialogic perspective on games in the classroom For Bakhtin, a voice does not necessarily imply the spoken or written words of a person but is a broader theoretical construct that refers to the position of the speaker: where the utterance is coming from and how it is responsive toward other voices. Bakhtin’s (1984) key example is an analysis of how Dostoevsky’s novels are multivoiced, as they are populated with many characters, each speaking in their own distinct voice, coming together through dialogic imagination to create a polyphonic work of art. Similarly, game designs might also appeal to the player through multiple voices attributed to specific game characters, game narratives, or intertextual refer- ences to other texts (Coelho, 2015). Educational researchers have extended Bakh- tin’s notions to school contexts in order to describe how classrooms are multivoiced in the sense that each student brings different voices with them, which might or might not be given attention and offered possibilities for articulation by the teacher (Dysthe, 1996). In this way, teaching with a game in a classroom involves not only the voices of the teacher and the students, but also the voices embedded in the game and the interanimation between these voices (de Sousa et al., 2018; Lacasa et al., 2008; Silseth, 2012; Staaby, 2020). In addition to identifying the interplay of voices as a key aspect of communica- tion, Bakhtin’s dialogic philosophy addresses different aspects of authority, which are also central to understanding how games can be used in L1 education. Bakhtin 8 T. HANGHØJ writes about the dialogical tensions between authoritative discourse, which he also describes as monologic, and what he terms internally persuasive discourse, which refers to language use directed towards mutual communication and the mutual con- struction of knowledge by revealing “ever new ways to mean” (Bakhtin, 1981, pp. 345-6). In this way, Bakhtin was interested in how humans author themselves and each other in the ongoing negotiation between dialogic and monologic meaning making. Similar to embracing the concept of voice, educational researchers have also adopted Bakhtin’s analytical focus on authorship within the context of classroom di- alogue (e.g., Matusov, 2011; Morson, 2004; Skaftun, 2019). The assumption is that teachers and students continually author each other through negotiation between many authorial positions, with the teacher having the overall authorial responsibil- ity. 2.2 Framing game challenges In order to provide a detailed understanding of literacy teachers’ dialogic approaches to games, I argue that teachers need to identify and frame game challenges in rela- tion to local educational aims. As mentioned, games open up possibility spaces that involve productive tension between centripetal and centrifugal forces. I further ar- gue that Bakhtin’s philosophical terms are particularly relevant when trying to un- derstand how the learning potentials of digital games revolve around specific game challenges. Game challenges carries three overall meanings, which all resonate with the dialogic perspectives presented above. Generally speaking, a challenge signifies a call or invitation to participate in a demanding situation. Consequently, digital games have been designed to offer challenges that involve a sense of agency (Deterding, 2015; Plass et al., 2015). This means that a player actively must choose to engage with particular aspects of a given game, which comes close to their voice and how they see themselves as a player. This also involves relations to other play- ers, as well as specific choices and consequences to be explored in the game. Simi- larly, teachers must familiarise themselves with the game in order to author and fa- cilitate specific educational challenges that students encounter within or in relation to a game. Finally, the word, challenge, also denotes different ways of disputing the truth or authorial position of something. Teachers and players might have different experiences and productive disagreements about the rules, strategies, or outcomes of a game. In this way, game challenges might serve as useful drivers for promoting dialogue and discussion in the classroom through the interanimation of different voices and experiences. In summary, the notion of a game challenge in an educa- tional context might refer to (1) the possible in-game challenges embedded in a spe- cific game design, (2) the players’ interpretation, acknowledgement, and exploration of specific game challenges, and (3) the teachers’ linking of specific game challenges to educational aims. If we take Minecraft as an example, the game offer players numerous in-game challenges that teachers need to identify and consider when bringing the game into the classroom. When teachers and students play the game in Survival Mode, the core game challenges concern being able to survive in the game when fighting monsters, getting enough food, and building a shelter to be safe at night. 2.1 A dialogic perspective on games in the classroom In this way, teachers need to facilitate games in the class- room by striking a balance between maintaining the centripetal, or rule-regulated 9 TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT aspects of digital games, and embracing the centrifugal, or open-endedness of unex- pected game events (Hanghøj, 2008). 2.2 Framing game challenges In Creative Mode, the in-game challenges relate less to surviving and more to selecting and using resources (e.g., mining iron and crafting tools) or creating constructions that have specific aes- thetic qualities. Based on the mode of play, the game challenges of Minecraft are offered to the player by the affordances of different game elements (e.g., game rules, navigation, and game mechanics) and textual elements (e.g., the meanings of the game narrative, the game aesthetics, the players’ avatar, and the cultural aspects 10 T. HANGHØJ of the game). However, in the L1 classroom, game challenges relate not only to in- teraction with game design features or interpreting textual aspects, but also to how they are enacted and negotiated through the participants’ social actions, which in- volve dialogic interaction between the players and the teacher in the classroom con- text. When L1 teachers use Minecraft in the classroom, game activities might involve students’/players’ knowledge-sharing or co-construction of buildings, often sup- ported by written (e.g., chat) or spoken forms of communication. Following the work of Schön (1983), I argue that educators adopting a game for their teaching need to frame the game in relation to specific game challenges. Ac- cording to Schön (1983), teachers act as reflective practitioners who continually an- alyse the interpretive frames of classroom interaction in order to name (identify) and frame pedagogical approaches that address unexpected situations or problems. Schön argued that teachers should not be seen as rational problem-solvers, as it is impossible to apply pre-defined knowledge that can cater to all the unexpected sit- uations that arise when teaching. Instead, teachers need to set the problems for which they try to design solutions: When we set the problem, we select what we will treat as the ‘things’ of the situation, we set the boundaries of our attention to it, and we impose upon it a coherence which allows us to say what is wrong and in what directions the situation needs to be changed. Problem setting is a process where we name the things to which we will attend and frame the context in which we will attend to them (Schön, 1983, p. 40). Moreover, Schön also stressed that teachers must be able to reframe problems or situations through reflection-in-action. 2.2 Framing game challenges Teachers’ ongoing processes of naming, framing, and reframing problems are very important when teaching with complex games such as Minecraft, as the interaction patterns of the social participants in a game world involve different interpretive framings than everyday classroom interac- tion (Goffman, 1974; Hanghøj, 2008). When playing Minecraft, students-as-players might die by falling off a cliff, and teachers might choose to teleport themselves into different positions on the virtual game map in order to visit and help students at their different locations. In this way, it is important for teachers to name the main game challenges in Minecraft and decide how to educationally frame the game chal- lenges in response to specific curricular aims that might be pre-planned or arise from unexpected situations. 3. THE GAME AS EDUCATIONAL CHALLENGE MODEL Drawing on the theoretical perspectives outlined above, I now present the Game As Educational Challenge (GEC) model that can be used to understand how digital games are enacted in the literacy classroom. Elsewhere, I have presented other ver- sions of the model in order to describe how teachers position themselves and choose different pedagogical approaches when facilitating games in the classroom, such as by executing, improvising, or transforming a game-related teaching unit with Mine- TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT 11 craft (Hanghøj, 2017). Although previous versions of the model were used for ana- lytical purposes, this work lacked a comprehensive theoretical foundation. Thus, in this article, I adopt the GEC model to exemplify how the model can be used as an analytical tool to explore how L1 teachers frame game challenges and facilitate stu- dents’ game experiences. The GEC model (see Figure 1) is based on the assumption that the enactment of games in the L1 classroom requires teachers to identify and to create meaningful links between selected game challenges and relevant educational challenges. Figure 1. The Game as Educational Challenge (GEC) model Figure 1. The Game as Educational Challenge (GEC) model Figure 1. The Game as Educational Challenge (GEC) model Moreover, it is assumed that teachers address specific game challenges through a dynamic interplay of two different dimensions of meaning making, which are shown as vertical and horizontal dimensions in the model. The vertical dimension illustrates how teachers orient students towards specific game challenges by balancing a con- tinuum between curricular aims and game goals. This distinction is based on an ear- lier study of digital games in L1, which showed how teachers and students experi- ence meaningful integration, as well as clashes of meaning and expectations, when faced with the mixed intentions of curricular aims and game goals in a classroom context (Hanghøj, 2011). Seen from a dialogic perspective, the vertical dimension can be used to analyse how teachers facilitate discussion on students’ game experi- ences that address different voices in the L1 classroom, such as voices that primarily relate to being a “student” or being a “player.” Informed by Schön (1983), the verti- cal dimension of the model also can be used to analyse how teachers frame specific in-game challenges by balancing game goals (e.g., survival in Minecraft) in relation to curricular aims (e.g., learning about the narrative structure of a survival story). T. HANGHØJ The horizontal dimension in the model refers to teachers’ facilitation of game challenges between focusing on the game as design (text) and the game as social action. As mentioned, digital games might offer numerous affordances for interac- tion in terms of game mechanics or game narratives (Toh & Lim, 2020). Seen from a literacy perspective, the multimodal design features of digital games can be concep- tualised and understood as textual aspects that relate to students’ existing knowledge of games, as well as broader game discourses and meaning making (Bea- vis & Apperley, 2013; Burn, 2007). This means that L1 teachers using games in their classroom must both address the affordances of specific games (e.g., the game struc- ture and mechanics of Minecraft’s open-ended game world) and how the game re- lates to relevant literacy concepts (e.g., establishing connections between the Mine- craft game structure and the generic narrative structure of a Robinsonade story). In this way, “game as design/text” might refer to the affordances of specific game ele- ments and/or textual aspects, depending on which aspects are emphasised in the classroom. This implies a situated understanding of how teachers and students ex- perience games as design and/or as text in the classroom, which differs from the more abstract theoretical distinction between games-as-text and games-as-action suggested by Beavis and Apperley (2013). Moreover, the horizontal dimension of the GEC model addresses how the use of specific digital games in educational contexts is enacted and explored as social ac- tion. This refers to the game play practices that emerge when teachers and students explore a given game world and how specific literacy activities are organised in rela- tion to the students’ game experiences. Here, it is useful to distinguish between game activities that take place within the game (e.g., when students communicate or build constructions in Minecraft) and literacy activities that take place around the game (e.g., when students’ game experiences in Minecraft are linked to writing dia- ries, Dezuanni & Zagami, 2017; Hanghøj et al., 2020). It is important to bear in mind how teachers’ and students’ game play practices are influenced by their interest in, knowledge of, and previous experiences with games outside school, which might be used to “bridge” literacies across school and non-school domains (Abrams & Gerber, 2014; Hanghøj et al., 2021). 3. THE GAME AS EDUCATIONAL CHALLENGE MODEL Moreover, it is assumed that teachers address specific game challenges through a dynamic interplay of two different dimensions of meaning making, which are shown as vertical and horizontal dimensions in the model. The vertical dimension illustrates how teachers orient students towards specific game challenges by balancing a con- tinuum between curricular aims and game goals. This distinction is based on an ear- lier study of digital games in L1, which showed how teachers and students experi- ence meaningful integration, as well as clashes of meaning and expectations, when faced with the mixed intentions of curricular aims and game goals in a classroom context (Hanghøj, 2011). Seen from a dialogic perspective, the vertical dimension can be used to analyse how teachers facilitate discussion on students’ game experi- ences that address different voices in the L1 classroom, such as voices that primarily relate to being a “student” or being a “player.” Informed by Schön (1983), the verti- cal dimension of the model also can be used to analyse how teachers frame specific in-game challenges by balancing game goals (e.g., survival in Minecraft) in relation to curricular aims (e.g., learning about the narrative structure of a survival story). 12 T. HANGHØJ This dual focus on viewing the use of games both as designs/texts and as social actions that relate to specific contextual framings (in-game and off-game) is inspired by Scollon (2001), who has insisted on the analytical importance of foregrounding social actions when conducting discourse analysis and not only focusing on the meaning of texts (or designs or discourses). This analytical distinction makes it pos- sible to account for how games are enacted not only as designs/texts, but also as local practices that are accomplished with teachers and students in relation to other pedagogical practices in the classroom context (Hanghøj, 2008). In line with the dia- logic perspective presented earlier, the horizontal dimension of the GEC model ad- dresses how teachers engage in dialogue with and facilitate students’ game experi- ences in relation to their understanding of game designs (e.g., specific game ele- ments or textual aspects) or the social actions that unfold inside or around the game. TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT 13 Similarly, the horizontal dimension of the model can also be used to analyse how teachers frame game challenges by orienting students towards game design aspects (e.g., specific game elements or literacy aspects in relation to Minecraft) or towards specific activities that take place inside or around the game. The relations between the two meaning-making dimensions when teachers facil- itate game challenges in the L1 classroom are illustrated in the GEC model shown above. There exist no sharp ontological distinctions between the two dimensions (horizontal and vertical) and four different aspects (literacy aspects, game elements, game activities, and literacy activities), as the different categories will overlap in ac- tual practice when teaching with games. Instead, the different dimensions and as- pects should serve to establish analytical focal points when trying to describe and to understand how teachers bring games to life in the classroom through dialogic inter- actions with games and students. The model does not assume specific pedagogic values or norms in relation to how teachers should introduce specific games as de- signs (texts), facilitate game/literacy activities, or establish and assess meaningful links between game goals, game challenges, and curricular aims. In this way, the GEC model is mainly intended as a descriptive tool for analysing and qualifying an under- standing of how teachers frame, enact, and facilitate game challenges in the literacy classroom. 4. ENTER: THE MYSTERIOUS ISLAND In order to provide deeper insight into how literacy teachers go about teaching with digital games, I will now revisit earlier work (Hanghøj, 2017) revolving around an em- pirical case that explored how primary school teachers teach with Minecraft in L1. The case is based on data collected in a research project funded by the Danish Min- istry of Education (2013–2015) as part of a larger research project on “ICT in the Innovative School.” The data were gathered at three different Danish schools from Grades 1 to 2 (age between six and seven years). Teachers from the three schools took part in design interventions with an open-world Minecraft game map entitled, “The Mysterious Island” (see Figure 2). The game map as well as a YouTube video introducing the island had been developed by researchers and game experts in order to create a game-related literacy unit in Danish (L1) that could support students’ abilities of collaboration, creativity, and communication. Using The Mysterious Is- land in combination with a fictive Robinsonade storyline (Bell & Harkness, 2013), the teachers used the unit to invite students to explore and imagine surviving on a de- serted island, with loose inspiration from Daniel Dafoe’s Robinson Crusoe narrative. In order to provide deeper insight into how literacy teachers go about teaching with digital games, I will now revisit earlier work (Hanghøj, 2017) revolving around an em- pirical case that explored how primary school teachers teach with Minecraft in L1. The case is based on data collected in a research project funded by the Danish Min- istry of Education (2013–2015) as part of a larger research project on “ICT in the Innovative School.” The data were gathered at three different Danish schools from Grades 1 to 2 (age between six and seven years). Teachers from the three schools took part in design interventions with an open-world Minecraft game map entitled, “The Mysterious Island” (see Figure 2). The game map as well as a YouTube video introducing the island had been developed by researchers and game experts in order to create a game-related literacy unit in Danish (L1) that could support students’ abilities of collaboration, creativity, and communication. 4. ENTER: THE MYSTERIOUS ISLAND The students then left the game and engaged in brain- storming and discussion activities on what constructions they needed in order to en- sure their survival on the island. During the unit, the teacher introduced the students to taking screenshots and keeping a diary of what they experienced on the island, to be presented at the end of the unit. In summary, the curricular aims of the unit fo- cused on: (1) learning about the Robinsonade as a literary genre; (2) learning to col- laborate and communicate with other students; (3) learning to argue for particular design choices for surviving on The Mysterious Island; and (4) learning to write dia- ries involving various multimodal literacies. The unit involved the following activities. First, the students were introduced to the defining features of a Robinsonade narrative and what it means to survive on a de- serted island. Next, the students created their own avatars in the game, which they characterised and related to the overall survival narrative. The teacher then organ- ised the students into small groups, which entered the island at various entry points, where they could explore, find hidden objects, and become familiar with the naviga- tion and the game mechanics. The students then left the game and engaged in brain- storming and discussion activities on what constructions they needed in order to en- sure their survival on the island. During the unit, the teacher introduced the students to taking screenshots and keeping a diary of what they experienced on the island, to be presented at the end of the unit. In summary, the curricular aims of the unit fo- cused on: (1) learning about the Robinsonade as a literary genre; (2) learning to col- laborate and communicate with other students; (3) learning to argue for particular design choices for surviving on The Mysterious Island; and (4) learning to write dia- ries involving various multimodal literacies. The methodological approach of the study was inspired by Design-Based Re- search (e.g., Squire & Barab, 2004), which stresses how context matters in educa- tional interventions. Consequently, the study was organised around an iterative se- ries of interventions with the game-related teaching unit in order to document and qualify educational design principles for teaching with games in L1 classrooms. More specifically, the design interventions aimed to generate knowledge and pedagogical principles on how L1 teachers in primary education could teach with Minecraft. 4. ENTER: THE MYSTERIOUS ISLAND Using The Mysterious Is- land in combination with a fictive Robinsonade storyline (Bell & Harkness, 2013), the teachers used the unit to invite students to explore and imagine surviving on a de- serted island, with loose inspiration from Daniel Dafoe’s Robinson Crusoe narrative. T. HANGHØJ 14 Figure 2. Screenshot from a YouTube video introducing The Mysterious Island. Figure 2. Screenshot from a YouTube video introducing The Mysterious Island. Figure 2. Screenshot from a YouTube video introducing The Mysterious Island. Figure 2. Screenshot from a YouTube video introducing The Mysterious Island. The unit involved the following activities. First, the students were introduced to the defining features of a Robinsonade narrative and what it means to survive on a de- serted island. Next, the students created their own avatars in the game, which they characterised and related to the overall survival narrative. The teacher then organ- ised the students into small groups, which entered the island at various entry points, where they could explore, find hidden objects, and become familiar with the naviga- tion and the game mechanics. The students then left the game and engaged in brain- storming and discussion activities on what constructions they needed in order to en- sure their survival on the island. During the unit, the teacher introduced the students to taking screenshots and keeping a diary of what they experienced on the island, to be presented at the end of the unit. In summary, the curricular aims of the unit fo- cused on: (1) learning about the Robinsonade as a literary genre; (2) learning to col- laborate and communicate with other students; (3) learning to argue for particular design choices for surviving on The Mysterious Island; and (4) learning to write dia- ries involving various multimodal literacies. The unit involved the following activities. First, the students were introduced to the defining features of a Robinsonade narrative and what it means to survive on a de- serted island. Next, the students created their own avatars in the game, which they characterised and related to the overall survival narrative. The teacher then organ- ised the students into small groups, which entered the island at various entry points, where they could explore, find hidden objects, and become familiar with the naviga- tion and the game mechanics. 4. ENTER: THE MYSTERIOUS ISLAND The project was part of a larger research project involving 35 schools, which meant that each of the three schools was recruited through contact with the school principals. This meant that none of the teachers had actively chosen to be part of the research project with the Minecraft teaching unit, and each consequently felt quite different TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT 15 senses of ownership toward the project. Moreover, all teachers were “non-gamer teachers” (Prestridge, 2017), as they had quite limited game experience. None of them had previous experience playing or teaching with Minecraft prior to the inter- vention. The project teachers took part in all-day workshops before and after the interventions, led by researchers and consultants from university colleges, wherein the teachers were introduced to and asked to reflect on Minecraft, as well as the teaching unit with The Mysterious Island. During the workshops, the teachers could explore the game map, ask questions, and receive advice on how to teach with the game. Moreover, they were encouraged to make changes to the teaching unit to meet local curricular needs and demands and ensure meaningful adaptation. The pedagogical choices involved in adapting the Minecraft unit to the local demands and interests at the three different schools were made collectively among the teach- ers at each school, who sometimes taught together in pairs, especially when intro- ducing the game unit to the students. The research team collected data by following the L1 teachers’ enactment of the teaching unit in three different primary schools. This involved group interviews (two or three participants) with the literacy teachers at each school before the interven- tion and individual teacher interviews after teaching the Minecraft unit. Moreover, the research team conducted classroom observations at each of the three schools. The observations followed the enactment of the unit at each school and involved observations of selected lessons in two second-grade classes (age eight) at School 1 over the course of three weeks, two weeks of full-day observations in three, first- grade classes (age seven) at School 2, and a three-week observation period of se- lected lessons in two, first-grade classes (age seven) at School 3. In addition to taking field notes and collecting various texts from the students, the team documented the observations by taking photos, as well as making video and sound recordings of se- lected lessons. 4. ENTER: THE MYSTERIOUS ISLAND Based on the collected data, studies have documented the teachers’ different pedagogical approaches (Hanghøj, 2017), as well as the students’ game-related lit- eracy practices (Hanghøj et al., 2018). The data were analysed through thematic analysis (Braun & Clarke, 2006), which focused on mapping and coding significant patterns and events in the teachers’ local practices and their facilitation of classroom dialogue when unfolding the teaching unit. In this way, the focus was less on docu- menting learning outcomes or the individual teachers’ experiences or values and more on understanding the relational aspects of their dialogic interaction with the students as the game activities unfolded. Next, key events from the observations were selected for further thematic coding and analysis, which indicated important differences in the teachers’ pedagogical approaches to adopting the game in the classroom. For the purpose of this study, I will use the GEC model (Figure 1) to revisit the analysis of the teachers’ pedagogical approaches to the game-based teaching unit (Hanghøj, 2017). In this way, I am interested in exploring how the teachers in the 16 T. HANGHØJ research project framed the Minecraft game challenges and facilitated classroom discussions in relation to students’ game experiences. 5.1 Framing and reframing of game challenges in relation to curricular aims This analytical theme involves two examples taken from two different schools in or- der to show how the project teachers chose quite different approaches to framing the game challenges in the Minecraft unit. 5. ANALYSIS The analysis will use the GEC model to explore two analytical themes in relation to the teachers’ pedagogical approaches to the Minecraft unit: (1) teachers’ framing and reframing of game challenges in relation to curricular aims, and (2) dialogic po- sitions when facilitating the students’ experiences of challenges in and around the game. Each theme focuses on classroom events involving dialogic perspectives on the teacher and students’ interaction in relation to the game as well as the teachers’ authorial positioning. TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT Teacher 1A: But that’s actually what’s good about being in this world… that you can’t actually, you can’t die Teacher 1A: But that’s actually what’s good about being in this world… that you can’t actually, you can’t die Boy: Aw! Boy: Ugh! Boy: Aw, that’s a downer! NOISE* Teacher 1A: So… What else could it be that you don’t like? Alma: * Teacher 1B: Alma is actually saying something. It’s a survival island we’re on… so we have to survive Anders: But, but… you can’t survive when you can’t die! You can’t! Boy: Then it’s easy to survive! Girl: Well, that’s not the purpose, is it, Anders?! Teacher 1B: Alma is actually saying something. It’s a survival island we’re on… so we have to survive Anders: But, but… you can’t survive when you can’t die! You can’t! Boy: Then it’s easy to survive! Girl: Well, that’s not the purpose, is it, Anders?! Anders: But, but… you can’t survive when you can’t die! You can’t! As the example shows, some of the students, particularly the boys, had very high expectations about playing Minecraft at school and spontaneously engaged in loud comments and discussions about the game with their classmates. They became quite disappointed when they found out that they were not able to “whack” anyone in the game, which is how the game is played in Survival Mode. Instead, the students had to imagine their “survival” was a part of the storyline narrative, which is different from actual in-game survival. In contrast, the teachers presented the lack of dying as a “good thing” about The Mysterious Island, as they wanted the island to be a safe place for all students, a premise that they shared with several of the girls but not the boys. The example illustrates how the students had quite different expectations of the game, presumably based on their different game experiences outside school. In this way, bringing the commercial game, Minecraft, into the classroom calls for the ne- gotiation and reframing of specific game challenges in order to make the game meaningful as a learning resource within the school domain. However, the two teachers were quite unfamiliar with the game, and their reluctant framing of the “survival challenge” did not make it clear to the students what actions they were expected to accomplish in the game world as a part of the Robinsonade storyline. 5.1.1 Example 1: You can’t survive when you can’t die! The first example is from School 1, where the two teachers just introduced the teach- ing unit to the students and showed the short YouTube video designed by the re- search team in order to engage teachers and students in the survival narrative (see Figure 2). The video follows a ship filled with children, which is lost in a storm and eventually winds up on the shores of The Mysterious Island with no adults in sight. After viewing the video, the teachers asked the students to close their eyes and im- agine that they were on the ship just about to stop at The Mysterious Island. The students, who were quite familiar with playing Minecraft outside school and eager to try the game, found it difficult to follow the teachers’ directives. One student blurted aloud: “I’m not on the ship!” Instead, the students commented on different phenomena in the game setting shown in the video that they found interesting, such as the lava on the island. Next, the teachers opened up a general classroom discus- sion on what the students thought about exploring Minecraft: Teacher 1A: Is there anything that isn’t nice or that you don’t like… [in Minecraft]? Girl: Getting hit by others in Minecraft Teacher 1B: That’s the thing… if that was possible Boy: Yes, getting whacked! Boy: Can we whack each other? NOISE* (…) Teacher 1A: Is there anything that isn’t nice or that you don’t like… [in Minecraft]? Girl: Getting hit by others in Minecraft Teacher 1B: That’s the thing… if that was possible Boy: Yes, getting whacked! Boy: Can we whack each other? NOISE* (…) NOISE*** (…) 17 TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT The lack of framing becomes even clearer when a student later on in the same lesson directly asked the teachers: “Why are we actually working with Minecraft?” One of the teachers responded rather bluntly: “Because we’re working with the Robin- sonade in Danish class.” This monologic response narrows the aims of the unit to understanding the Robinsonade as a well-established literary genre within the school subject, thereby reducing the game challenge to learning a narrative text. In relation to the GEC model, the teacher is mainly focused on linking the game challenge to the literacy aspects of the unit as shown in the left upper quadrant of the model (Figure 1). In this way, the teacher omits to acknowledge the other important literacy and 18 T. HANGHØJ game activities of the unit, which are linked to social actions inside and around the game. This included the students’ collaborative exploration of the game map (in game), brainstorming and planning ideas for survival (outside the game), building constructions (in game), using screenshots to document creations (in game), and writing about game experiences (outside the game). Based on their limited familiar- ity with Minecraft, the teachers presented the game as a resource for literacy activ- ities, but primarily framed the game world as a safe place, which implied a centripe- tal orientation (i.e., unifying focus) towards ensuring social order and following agreed-upon rules. 5.1.2 Example 2: Staging a “crisis” The next example comes from School 2, where the three teachers opted for a radi- cally different approach to adapting the teaching unit. In comparison to the teachers at School 1, who primarily executed the teaching unit through a reluctant and rela- tively passive approach to the game as a narrative text, the three teachers at School 2 decided to “go all in,” as one of them remarked in the pre-intervention interview. This meant that the teachers not only engaged with the demanding task of becoming familiar with the navigation, mechanics, and teacher interface in the Minecraft game design (cf. the left lower quadrant of the GEC model), but also further decided to redesign the use of the whole unit. Their transformed unit involved careful scaffold- ing of the students’ literacy activities (mainly diary writing), additional cross-curricu- lar activities that included physical education and music lessons, and a physical re- design of the classrooms and hallways in order to immerse the students in the blend- ing of the interpretive framing of the game design with the storyline narrative (see Figure 3). TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT 19 Figure 3. Photo of hallway outside the classroom redesigned by the teachers. Figure 3. Photo of hallway outside the classroom redesigned by the teachers. Moreover, the teachers’ transformative approach to the unit involved a clearer focus on linking the key game challenge to the educational aims, especially in relation to teaching narrative structures. The teachers decided that they wanted to use the teaching unit for educating students about the classical narrative structure of a story with a beginning, a middle with a crisis, and an ending. Because Minecraft is an open- ended sandbox game with no pre-set goal, the teachers lacked a clear “crisis” on The Mysterious Island for the students to overcome. Not wanting the students to get killed by each other in the Survival Mode of the game, they, instead, chose to engage with a team of experienced Minecraft players from Grade 8 who were interested in helping the teachers. Unknown to the Grade 1 students, the Grade 8 students logged into the game from computers at another location and suddenly appeared as strangers (“cannibals”) on The Mysterious Island, where they had been given per- mission by the teachers to use TNT blocks to blow up some of the Year 1 students’ in-game constructions. 5.1.2 Example 2: Staging a “crisis” This unexpected visit caused considerable chaos and frustra- tion among the Grade 1 students, and the event was later labelled “the crisis” when the teachers evaluated the survival narrative with the students (see Figure 4). Moreover, the teachers’ transformative approach to the unit involved a clearer focus on linking the key game challenge to the educational aims, especially in relation to teaching narrative structures. The teachers decided that they wanted to use the teaching unit for educating students about the classical narrative structure of a story with a beginning, a middle with a crisis, and an ending. Because Minecraft is an open- ended sandbox game with no pre-set goal, the teachers lacked a clear “crisis” on The Mysterious Island for the students to overcome. Not wanting the students to get killed by each other in the Survival Mode of the game, they, instead, chose to engage with a team of experienced Minecraft players from Grade 8 who were interested in helping the teachers. Unknown to the Grade 1 students, the Grade 8 students logged into the game from computers at another location and suddenly appeared as strangers (“cannibals”) on The Mysterious Island, where they had been given per- mission by the teachers to use TNT blocks to blow up some of the Year 1 students’ in-game constructions. This unexpected visit caused considerable chaos and frustra- tion among the Grade 1 students, and the event was later labelled “the crisis” when the teachers evaluated the survival narrative with the students (see Figure 4). Moreover, the teachers’ transformative approach to the unit involved a clearer focus on linking the key game challenge to the educational aims, especially in relation to teaching narrative structures. The teachers decided that they wanted to use the teaching unit for educating students about the classical narrative structure of a story with a beginning, a middle with a crisis, and an ending. Because Minecraft is an open- ended sandbox game with no pre-set goal, the teachers lacked a clear “crisis” on The Mysterious Island for the students to overcome. Not wanting the students to get killed by each other in the Survival Mode of the game, they, instead, chose to engage with a team of experienced Minecraft players from Grade 8 who were interested in helping the teachers. 5.1.2 Example 2: Staging a “crisis” Unknown to the Grade 1 students, the Grade 8 students logged into the game from computers at another location and suddenly appeared as strangers (“cannibals”) on The Mysterious Island, where they had been given per- mission by the teachers to use TNT blocks to blow up some of the Year 1 students’ in-game constructions. This unexpected visit caused considerable chaos and frustra- tion among the Grade 1 students, and the event was later labelled “the crisis” when the teachers evaluated the survival narrative with the students (see Figure 4). T. HANGHØJ 20 Figure 4. ‘Oh no, we’ve got enemies’ Figure 4. ‘Oh no, we’ve got enemies’ Figure 4. ‘Oh no, we’ve got enemies’ Note: The image is an excerpt of poster made by the teachers, which illustrates “the crisis.” The screen- shot shows an example of one of the student’s construction sites after a TNT explosion. 5.2 Dialogic positions when facilitating students’ experiences of game challenges This second analytical theme focuses on how the teachers assumed different autho- rial positions when orchestrating discussions with the students about their game ex- periences. In this way, the focus is on how the teachers facilitated dialogic spaces that enabled the students’ different experiences of the game challenges to emerge, which involved shared negotiation of meaning and an interplay of different voices in the classroom. Note: The image is an excerpt of poster made by the teachers, which illustrates “the crisis.” The screen- shot shows an example of one of the student’s construction sites after a TNT explosion. Having caused havoc and destruction, the cannibals disappeared, but the Grade 8 students then returned to the game map as helpers who offered the younger stu- dents assistance with reconstructing their buildings. The rebuilding process was or- ganised through online communication in the in-game chat, with which the Grade 1 students had only limited experience prior to the event. An excerpt of the chat com- munication between helpers and students is shown below, which illustrates how the teacher stepped back and supported the students’ engagement in authentic com- munication with the helpers: <Hole> do you want roof on Noah? <noah> yes <Teacher 2A> It is nice that you help us Hole <Hole> no problem <Frederik> aw whatabout 2 fish then <xpfreva> what else <Hole> what about light noah? <noah> yes <isabella> hole help us <lival> hi Hole will hailp [help] lival <Hole> mr.bean helps you lival:-) <MrBean> What should I help you with? <MrBean> Can I help you Lival? :D 21 TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT <Frederik> ghost you can buy things by xpfreva [player’s screenname] <Frederik> ghost you can buy things by xpfreva [player’s screenname] <Frederik> ghost you can buy things by xpfreva [player’s screenname] <Hole> should I help Robina? <Robina> yes <Frederik> ghost you can buy t <Hole> should I help Robina? <Robina> yes <Hole> should I help Robina? <Robina> yes These data excerpts show how the teachers managed to organise game activities that created a meaningful crisis, which combined in-game actions (e.g., TNT explo- sions and rebuilding activities) with the imagined survival in the overall storyline nar- rative. Even though there were considerable differences between the language skills of the grade 1 and 8 students, both groups found the communication meaningful. In summary, the teachers at School 2 actively staged and redesigned the teaching unit by not only identifying, but also reframing the key game challenge in order to fit their local needs and aims. In contrast to the teachers at School 1, who did not engage with the in-game challenges and mainly focused on presenting the game as a safe and orderly experience, the teachers at School 2 introduced a new threat (the “can- nibals”), which enabled the students to experience a crisis in order to learn about narrative structures. In this way, the three teachers actively promoted a disruptive form of game play with a strong centrifugal or open-ended orientation, which simul- taneously served a clear educational purpose. Viewed through the analytical lens of the GEC model (Figure 1), the teachers reinterpreted and reframed the overall game challenge of the unit in a way that both created more meaningful links to the game goals, game elements and game activities (cf. the lower half of the model), as well as to their planned curricular aims, literacy aspects and literacy activities (cf. the upper half of the model). 5.2 Dialogic positions when facilitating students’ experiences of game challenges 5.2.1 Example 3: Teachers as learners Student 2: You use a potato, so you do it on a female or a male, and then when the male meets a female, they get married Teacher 3A: Could you just explain the thing with the potato, Lucas? Teacher 3B: I would like that, too Teacher 3A: I was lost there Teacher 3B: I was as well. What is it with the potato? Is it inside the game? Student 1: Then how do we get the animals to mate? Student 2: You use a potato, so you do it on a female or a male, and then when the male meets a female, they get married Teacher 3A: Could you just explain the thing with the potato, Lucas? Teacher 3B: I would like that, too Teacher 3A: I was lost there Teacher 3B: I was as well. What is it with the potato? Is it inside the game? Student 2: You use a potato, so you do it on a female or a male, and then when the male meets a female, they get married Teacher 3B: I was as well. What is it with the potato? Is it inside the game? The example shows how the teachers invited the students to share their knowledge as game experts. In the discussion that ensued, Lucas explained how to get the cows to mate, and other students supported his explanation while negotiating different meanings of the specific game mechanic. The discussion ended with the teachers’ recognition of the students’ knowledge: Teacher 3A: I am very glad for the explanation. I did not know any of it Teacher 3B: Me neither As the example shows, the two teachers at School 3 were quite curious and support- ive of the students’ exploration of the game world and the fairy-tale narrative, which they co-constructed with the students. In this way, the teachers clearly tried to facil- itate classroom discussions in order to create a collaborative learning experience that allowed different student voices to emerge. At the same time, it is clear from the interviews and the classroom dialogue that the teachers had rather limited knowledge of the different game elements in Minecraft (cf. the left lower quadrant of the GEC model). 5.2.1 Example 3: Teachers as learners The example is taken from School 3, where the two teachers chose an improvisa- tional approach to the teaching unit in the sense that they did not know much about Minecraft but were interested in learning about the game and reacting to it as the game play unfolded. In this way, they positioned themselves by adopting the voice of a learner in relation to the students-as-players’ knowledge about the game. As one of the teachers said in the interview before the intervention: 22 T. HANGHØJ Then I can just tell the students, “But, hey, I’m quite simply no good at Minecraft, it’s not me…When I was little, I used to play with.…” In that way, I can pull myself out and then kind of pass it on to them [the students], so that they find solutions on their own. In order to achieve deeper insight into and support for using the game, the teachers teamed up with a student from the Grade 5, who showed the teachers how to build in the game and how to make cows mate and produce offspring. The teachers used this knowledge as inspiration for an unfolding fairy-tale storyline, which they decided to develop around the game activities on The Mysterious Island together with the students. In order to achieve deeper insight into and support for using the game, the teachers teamed up with a student from the Grade 5, who showed the teachers how to build in the game and how to make cows mate and produce offspring. The teachers used this knowledge as inspiration for an unfolding fairy-tale storyline, which they decided to develop around the game activities on The Mysterious Island together with the students. As the unit progressed, the teachers facilitated classroom discussions with the students about their constructions in Minecraft, which created dialogic spaces for letting different student voices and experiences emerge. In the dialogue excerpt shown below, the students talked about how to get the cows in the game to mate in order to increase their number, which could provide food for their hometown: Student 1: Then how do we get the animals to mate? Student 1: Then how do we get the animals to mate? 5.2.1 Example 3: Teachers as learners By following an improvisational approach and going along with the students, the teachers provided the students with limited knowledge of how their in-game actions should be linked to the educational aims, literacy aspects or literacy activities of the unit (cf. the upper half of the GEC model). When I inter- TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT 23 viewed the teachers after the intervention, I noticed that they were slightly disap- pointed with the students’ learning outcomes of the somewhat unpredictable unit and doubted “whether it was sound to mix up Minecraft with the fairy tale genre.” Having assumed the authorial position primarily as learners of the game, the teach- ers found themselves up in a position where they felt unable to legitimise the stu- dents’ game experiences in relation to the curricular aims of L1 education. 5.2.2 Example 4: Challenging students’ game decisions The final example is chosen to illustrate how other teachers facilitated dialogic spaces with students around their game experiences, which both acknowledged as well as challenged students’ game decisions. Moreover, the example shows how in- game challenges can be contextualised into educational challenges outside the game. The example is taken from a grade 1 classroom at School 2, where the teach- ers had extended the teaching unit to include aims for citizenship education through group-work activities and democratic decision-making on what constructions to build. In the excerpt that follows, the students just finished their idea development, which involved brainstorming, intense negotiations, and democratic voting on what they should build in order to survive on The Mysterious Island. Several of the groups had decided to build police stations in order to secure their imagined life on the is- land. At the prospect of creating a new society on The Mysterious Island based mostly on police stations, the teacher decides to challenge her students’ decisions while still playing along with the imaginary outcomes of the survival narrative: Teacher 3A: Why do you think it’s so important to have a police station? Frederik: If there’s a lot of people making trouble Teacher 3A: Well, you’re the ones who are making the trouble because you’re the only ones living there…What could you be up to? Frederik: We might end up hitting someone because we had too much alcohol or what- ever Teacher: Yes, and where would you get that alcohol from, Frederik? Frederik: From a store or whatever Teacher: Where would you get the store from, Frederik? Frederik: I would get it from a company Teacher: Where would you get the company from, Frederik? Frederik: Er... I would build it (laughs) Teacher: Okay, what are you going to build it with, Frederik? Frederik: Wood Teacher: Well, what’s in the company, and where would you get it from, Frederik? Frederik: Er… electricity? Teacher 3A: Why do you think it’s so important to have a police station? Frederik: If there’s a lot of people making trouble Teacher 3A: Well, you’re the ones who are making the trouble because you’re the only ones living there…What could you be up to? Frederik: We might end up hitting someone because we had too much alcohol or what- ever Teacher: Yes, and where would you get that alcohol from, Frederik? Teacher: Where will you get electricity from, Frederik? Teacher: Where will you get electricity from, Frederik? Teacher: Sometimes you all tend to forget what this is, and I can understand why. But you have to remember that you’re sitting on a deserted island with nothing on it. Can you remember that we talked about what you were going to build? And you said, “You know, we need a fire station, and if something happens, we just call 911.” But what are you going to call with? “Well, the phone.” And I said, where will the phone come from? “We’re just going to build that.” With what? “Er… with iron.” And I said: But are phones only made of iron? “No, we also need some electricity in it.” But where do you get the electricity from?! Teacher: So, you see, it’s not that simple… that you just grab your iPad or phone and then you say: “Hello hello, can you come over, because there’s a fire here!” Eva: Well, it’s enough with just one police station for the whole island. There also only needs to be one hospital Through continual questioning of Frederik’s design choice and his underlying rea- sons, the teacher tries to help him and the rest of the class reflect on the complex relations between needs, causes, and effects for different phenomena in a modern world when surviving on a deserted island. The teacher’s persistent interrogation is held in a humorous tone, as she is deliberately asking an almost endless series of questions that verge on the absurd until Frederik and the class finally break down in laughter. What is important here is that the teacher is not so much seeking a prede- termined answer as she is trying to open up a playful dialogic space for reflective thinking with the students while playing along in the co-construction of different so- lutions to the imaginary problem of surviving on The Mysterious Island. After the teacher wraps up the discussion, Eva’s final comment indicates how some of the stu- dents have started reflecting on and reconsidering their choices by prioritising a broader set of needs and constructions to ensure their survival. The example shows how the teacher dialogically played with the narrative fram- ings of survival, both in relation to the possible actions in the Minecraft game world and the imaginary scenario of surviving on a deserted island in the real world. 5.2.2 Example 4: Challenging students’ game decisions Frederik: From a store or whatever Teacher: Where would you get the store from, Frederik? Frederik: I would get it from a company Teacher: Where would you get the company from, Frederik? Frederik: Er... I would build it (laughs) Teacher: Okay, what are you going to build it with, Frederik? Frederik: Wood Teacher: Well, what’s in the company, and where would you get it from, Frederik? Frederik: Er… electricity? Teacher 3A: Well, you’re the ones who are making the trouble because you’re the only ones living there…What could you be up to? Frederik: We might end up hitting someone because we had too much alcohol or what- ever 24 T. HANGHØJ 6. DISCUSSION The analysis of the two themes—(1) teachers’ framing and reframing of game chal- lenges in relation to curricular aims, and (2) dialogic positions when facilitating the students’ experiences of challenges in and around the game—points to important differences in how the L1 teachers from the three schools addressed the Minecraft game in their classrooms. There was striking variation in the teachers’ understanding of Minecraft as a game design and as a narrative text, their understanding of how the game challenge could be related to curricular aims, and how the game unit could create a possibility space for emerging social actions (activities) in and around the game. Moreover, it is a key finding that the teachers’ ability to name and frame the game challenge of the Minecraft unit was highly influenced by their experience and familiarity with the game. In this way, the teachers at School 1 and 3 remained “non- gamer teachers” (Prestridge, 2017), whereas the teachers at School 2 who went “all in” managed to develop expertise with Minecraft and The Mysterious Island and even reframed the game unit to fit with their local aims. Following the methodolog- ical approach of Design-Based Research (Barab & Squire, 2004), this key finding can be phrased as an educational design principle that might qualify teachers’ use of dig- ital games in L1 – namely, that literacy teachers need sufficient experience with the specific game being taught in order to understand the game as not only a text, but also a design with specific game elements (e.g., game mechanics, narrative structure) that involve specific social actions (e.g., collaboration, construction) related to spe- cific game challenges (e.g., what it means to survive on The Mysterious Island). In this way, the teachers needed sufficient knowledge and understanding of specific game challenges to transform them into meaningful educational challenges that re- lated to local curricular aims. However, the differences in the teachers’ dialogic facilitation of classroom dis- cussions across the three schools cannot be reduced to a question of familiarity with linking Minecraft challenges and curricular aims. Through their facilitation, the teachers dialogically took up different authorial positions on a continuum between enacting centrifugal forces through playful events (e.g., the ‘crisis’ that opened up new meanings of the narrative in the unit) and orienting themselves towards cen- tripetal or ludic forces (e.g., ensuring rules for safe play), which strive for order and unity of meaning. Teacher: Where will you get electricity from, Frederik? This last aspect also includes voices that relate to the students’ everyday understanding, such as the need to feel safe from troublemakers by being able to catch them or the need for electricity in order to communicate. In this way, the teachers’ dialogic facil- itation implied authorial positions that both acknowledged the students’ game de- sign choices and challenged them by asking reasons for their choices in order to pro- mote a wider variety of relevant ideas and constructions that fit with the survival narrative. Translated to the GEC model (Figure 1), the teacher is familiar with the limitations and possibilities when building constructions in Minecraft that might en- sure survival (cf. the lower half of the model). However, instead of just acknowledg- ing the students’ game design choices, she adopts a playful dialogic position, where 25 TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT she tries to broaden the students’ game experiences in relation to the overall edu- cational challenge of understanding what it means to reason and what it takes to survive in a modern world (cf. the upper half of the model). 6. DISCUSSION In this way, the teachers from the three schools chose quite dif- ferent dialogic approaches to enact authorial positions and thereby legitimising the students’ game experiences (Hanghøj, 2008). This finding points to another design principle for teaching digital games in L1: Teachers might benefit from facilitating classroom dialogue around games, which strikes an authorial balance between play- ing along with and playing against the contingent possibility spaces of the challenges 26 T. HANGHØJ that can emerge from a specific game world. Teachers should be able to accept the playful challenges offered by games but also be able and willing to facilitate the in- teranimation of voices from the students and the game in tune with their voices as teachers, which are shaped by their pedagogical values and professional expertise as reflective practitioners. The use of Minecraft or other digital games in the literacy classroom is not simply a question of embracing game worlds or categorizing them as texts according to different literacy aspects. Rather, the enactment of games in the literacy classroom requires teachers to engage in dialogue with students’ expe- riences of different game challenges to create meaningful educational connections. The analysis has shown how the dimensions and aspects of the GEC model can be adopted to describe and understand teachers’ enactment of digital games in the L1 classroom through framing of game challenges and dialogic facilitation of stu- dents’ game experiences. However, the GEC model can also be used as a teacher tool for identifying and providing an overview of how a specific digital game relates to curricular aims, literacy aspects, and literacy activities. In Figure 5, I have used the GEC model to interpret the intended game challenge, as well as the game and liter- acy aspects of the unit with The Mysterious Island. Figure 5. Exemplified GEC model: The Mysterious Island unit Figure 5. Exemplified GEC model: The Mysterious Island unit Figure 5. Exemplified GEC model: The Mysterious Island unit As the exemplified model shows, the Mysterious Island teaching unit involved the key challenge of surviving on the Mysterious Island. This challenge mainly referred to the imagined Robinsonade storyline, as the students played in the Creative Mode, which did not allow the players to die, in contrast to playing the game in Survival Mode. 6. DISCUSSION Moreover, the teaching unit involved learning about narrative aspects of the As the exemplified model shows, the Mysterious Island teaching unit involved the key challenge of surviving on the Mysterious Island. This challenge mainly referred to the imagined Robinsonade storyline, as the students played in the Creative Mode, which did not allow the players to die, in contrast to playing the game in Survival Mode. Moreover, the teaching unit involved learning about narrative aspects of the As the exemplified model shows, the Mysterious Island teaching unit involved the key challenge of surviving on the Mysterious Island. This challenge mainly referred to the imagined Robinsonade storyline, as the students played in the Creative Mode, which did not allow the players to die, in contrast to playing the game in Survival Mode. Moreover, the teaching unit involved learning about narrative aspects of the TEACHERS’ FRAMING AND DIALOGIC FACILITATION OF MINECRAFT 27 Robinsonade and writing diaries, as well as various phases of multimodal design pro- cesses. Similarly, the game aspects of the teaching unit required the students to ex- plore The Mysterious Island, learn how to take screenshots, and collaboratively build new constructions as a part of the storyline survival narrative. In this way, the GEC can serve as a tool or semiotic resource for helping teachers to create a metalan- guage (Toh & Lim, 2020) that can establish conceptual links between digital games (as designs/texts and social actions) and curricular aims by relating them to specific educational challenges. Finally, it should be mentioned that this empirical study has obvious limitations in terms of generalisability, as none of the teachers initially signed up for the re- search project but were pulled into it by their local management. The teachers’ dif- fering senses of ownership of the project meant that only the teachers from School 2 went “all in” when teaching with the game unit, whereas the teachers from the two other schools operated at the edge of their comfort zone. As mentioned by Baek et al. (2020), the Minecraft game world is quite complex and can be experienced as an overwhelming possibility space. Moreover, the teachers were given a rather lim- ited introduction to the game unit during the project workshops, and not one of them was instructed in dialogic approaches to facilitating the game unit. 6. DISCUSSION In this way, the teachers had to dedicate considerable time on their own or together with col- leagues, local ICT supporters, and/or expert student gamers in order to become fa- miliar with the different game elements. This involved dealing with various practical and technical obstacles in relation to installing and running the game from dedicated servers, which required extra preparation time. The design of the teaching unit also might be criticised for creating insufficient links between the game challenges and the Robinsonade narrative as a literary genre, which sometimes led to confusion of narrative frames, such as the different meanings of surviving in the game versus in the storyline narrative. Arguably, the teaching unit could have benefitted from fo- cusing more on the existing genres, paratexts, and literacy practices of the Minecraft ecosystem (cf. Bailey, 2016; Dezuanni, 2018), which the majority of the students were familiar with from playing the game outside school. In spite of these limitations, the dialogic perspective on how teachers go about framing and facilitating the Minecraft unit presented here should have broader rel- evance when trying to understand how teachers use the game (and other digital games) in L1 classrooms. Even though the data were collected back in 2014, and dig- ital games tend to come and go easily, Minecraft is still a popular game both inside and outside educational contexts (Baek et al., 2020). As the theoretical framework and GEC model suggest, it is important to generate more knowledge on how teach- ers enact Minecraft and other digital games in L1, which involves possible tensions between curricular aims and game goals, as well as different orientations towards games as designs/texts and games as social action. It is important to understand the meaning of these different dimensions and aspects, and how they relate in situ when teachers bring digital games and specific game challenges alive in the literacy class- 28 T. HANGHØJ room. By mapping teachers’ pedagogical practices and dialogic approaches to teach- ing with games, it becomes possible to unpack the black box of game-based teaching and provide more detailed answers to the question of what role digital games can or should play as a valuable, but also somewhat unpredictable, possibility space in L1 education. 6. DISCUSSION Seen from a dialogic perspective, digital games might contribute to the multivoiced classroom in ways that could, on the one hand, create more complexity and challenge teachers’ authorial positions, but, on the other hand, enable mean- ingful dialogic spaces that allow different voices and new questions to emerge. In this way, teaching with digital games can open up engaging and even transforma- tional learning experiences, which might provide teachers and students with new perspectives and understandings of themselves as well as of the L1 subject. 7. CONCLUSION Though there exists an impressive body of research on the use of digital games in L1 education, there has been a lack of detailed studies on how teachers integrate games in the literacy classroom. In response to this gap, this study has presented theoretical perspectives and an empirical analysis in order to map how primary school L1 teach- ers approach Minecraft as a part of a game-related unit on narratives and multi- modal production. The findings show how the teachers chose approaches to framing game challenges and facilitating students’ game experiences, which differed in their centripetal (monologic) as well as centrifugal (open-ended) orientations toward game goals and educational aims. This suggests that teachers must be able to ad- dress games not only as texts or as narratives. Games always involve specific chal- lenges that open up possibility spaces to be enacted through situated social actions in and around games, which means that teachers must relate to selected literacy aspects and activities through their dialogic facilitation. In this way, the study points to the need for further research on how L1 teachers might benefit from being famil- iar with the game elements of the games they teach, how they frame specific game challenges in relation to literacy aims, how they combine game activities with literacy activities, and how they assume different authorial (dialogic) positions when facili- tating classroom discussions around games. This could help literacy teachers to un- derstand how digital games represent not only a specific type of text, but also a val- uable resource for developing a broad range of literacies and offering meaningful forms of participation in the multivoiced classroom. Abrams, S. S. (2017). 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https://openalex.org/W1867484126	http://ojs.ecologiaaustral.com.ar/index.php/Ecologia_Austral/article/download/25/28	Portuguese	null	Invertebrados aquáticos associados a serapilheira de Salix humboldtiana em um riacho subtropical	Ecología austral/Ecología austral	2,014	cc-by	7,738	Invertebrados aquáticos associados a serapilheira de Salix humboldtiana em um riacho subtropical 1. Programa de Pós-Graduação em Biologia de Ambientes Aquáticos Continentais, Instituto de Ciências Biológicas, Laboratório de Limnologia, Universidade Federal do Rio Grande, Rio Grande, Brasil. 2. Programa de Pós-Graduação em Ecologia. Universidade Regional Integrada do Alto Uruguai e das Missões, Laboratório de Biomonitoramento, Erechim, Brasil. 3. Endereço atual: COMUSA - Serviços de Água e Esgoto de Novo Hamburgo, Novo Hamburgo, Brasil. RESUMO. O material alóctone da mata ripária é fonte importante para a cadeia alimentar detritívora em riachos ﬂ orestados. Este estudo teve como objetivos i) estimar a perda de massa foliar de Salix humboldtiana Willd., ii) analisar as alterações na composição química dos detritos durante o período de perda de massa, e iii) avaliar a assembleia de invertebrados colonizadoresem um córrego arenoso na planície costeira sul do Rio Grande do Sul (Brasil), com a hipótese de que os detritos de S. humboldtiana constituem habitat favorável a colonização por invertebrados, e que estes contribuem para sua degradação. Foram incubadas 28 bolsas de folhiço na superfície do sedimento do córrego, com retiradas em sete datas amostrais. Mudanças na composição química e a abundância, riqueza e grupos tróﬁ cos funcionais de invertebrados durante a perda de peso foram analisadas. Aos 184 dias foi registrada a perda de 70% da biomassa dos detritos (k=0.0066 dia-1). Foram registrados 14197 organismos, distribuídos em 36 táxons. Oligochaeta (43.6%), Hydrobiidae (16.6%), Chironominae (11.3%) e Hydracarina (6.6%) foram os mais representativos. O desenvolvimento do bioﬁ lme, observado por meio do incremento das concentrações de nitrogênio e fósforo totais nos detritos, pode ter favorecido a colonização por raspadores na fase ﬁ nal do experimento. A hipótese inicial foi corroborada em parte, pois considerando a baixa abundância de potenciais fragmentadores, a contribuição da comunidade de invertebrados para a degradação dos detritos é reduzida, sendo os coletores - catadores o grupo tróﬁ co funcional mais abundante da assembleia. [Palavras-chave: decomposição, riacho arenoso, detritívoros, grupos tróficos funcionais] ABSTRACT. Aquatic invertebrates associated with Salix humboldtiana litter in a subtropical stream: Allochthonous material from riparian forest is an important source for detritivore food chain in streams. This study aimed to: i) estimate the leaf mass loss of Salix humboldtiana Willd., ii) analyze the changes in the detritus chemical composition during mass loss, and iii) analyze the invertebrate colonizers assemblage of a sandy stream in south coastal plain of the Rio Grande do Sul state (Brazil). We hypothesized that S. 220 Ecología Austral 24:220-228. Agosto 2014 Asociación Argentina de Ecología 220 Ecología Austral 24:220-228. Agosto 2014 Asociación Argentina de Ecología Invertebrados aquáticos associados a serapilheira de Salix humboldtiana em um riacho subtropical humboldtiana debris constitute favorable habitat for colonization by invertebrates, which in turn contribute to debris degradation. Twenty eight bags were incubated on the sediment surface of the stream in seven sampling dates. During the decomposition process we analyzed changes of detritus chemical composition and the abundance, richness and functional trophic groups of invertebrates. After 184 days we recorded an average loss of 70% of debris (k=0.0066 day-1) and counted a total of 14197 invertebrate individuals, distributed in 36 taxa. Oligochaeta (43.6%), Hydrobiidae (16.6%), Chironominae (11.3%) and Hydracarina (6.6%) were the most representative. The bioﬁ lm development, observed through increased concentrations of total phosphorus and nitrogen in the debris, may have favored colonization by scrapers at the end of the decomposition period. The initial hypothesis was partially corroborated as the abundance of potential shredders was low, the contribution of invertebrates assemblage to litter decomposition was low, and the gathering-collectors were the most abundant group. [Keywords: decomposition, sandy stream, detritivores, functional trophic groups] Recibido: 26 de agosto de 2013; Fin de arbitraje: 5 de noviembre; Última versión: 9 de diciembre; Aceptado: 30 de enero de 2014. Editor asociado: Fernando Unrein INTRODUÇÃO Minshall & Rugenski 2006). Em linhas gerais, o processamento do detrito orgânico passa por uma fase de lixiviação dos compostos solúveis, condicionamento microbiano e fragmentação por invertebrados aquáticos (Webster & Benfield 1986). O aporte de matéria orgânica alóctone constitui fontede energia e nutrientes para a biota em ecossistemas lóticos sombreados (Vanotte et al. 1980; Webster & Benfield 1986). Nestes ambientes, a cadeia de detritos representa a principal via de circulação de matéria e energia (Begon et al. 2007). A entrada de matéria orgânica alóctone, a retenção e a decomposição dos detritos constituem processos fundamentais para o metabolismo de riachos de pequeno porte (Abelho 2001; A comunidade de invertebrados aquáticos desempenha importante papel nos ecossistemas de água doce, participando das teias alimentares como presas para peixes, anfíbios e aves (Albertoni & Palma-Silva 2010). Editor asociado: Fernando Unrein * dmbefa@furg.br 221 INVERTEBRADOS AQUÁTICOS EM SERAPILHEIRA Agosto de 2014 A espécie arbórea Salix humboldtiana Willd. (Salicaceae) desenvolve-se nas margens de cursos d’água e em ambientes com água subterrânea em abundância, nas Américas do Sul e Central (Backes & Irgang 2002; Carvalho 2003). Esta espécie é constituinte comum da vegetação ripária de lagos e riachos arenosos típicos da planície costeira do Rio Grande do Sul (Batista et al. 2007; Trindade et al. 2010). Salix humboldtiana apresenta elevada produção de serapilheira foliar, sendo responsável por importante aporte de matéria orgânica e nutrientes aos ambientes adjacentes onde se desenvolve (Aceñolaza et al. 2010). Estudos com decomposição de S. humboldtiana são escassos destacando-se Leguizamon et al. (1992), Capello et al. (2004) e Poi de Neiff et al. (2006), desenvolvidos no sistema hidrológico do rio Paraná, Argentina, e Telöken et al. (2011) em um lago raso no sul do Brasil. Para a planície costeira sul do Brasil, a literatura sobre o processo de decomposição desta espécie em riachos é inexistente, apesar de sua abundância e ampla distribuição como constituinte da vegetação ripária nos ambientes aquáticos. Considerando a abundância de S. humboldtiana junto aos riachos arenosos da planície costeira do extremo Sul do Brasil, este estudo tem como objetivos i) estimar a perda de massa foliar de S. humboldtiana em um riacho arenoso, ii) analisar as alterações na composição química durante o período de perda de massa e iii) estimar a densidade, riqueza e composição funcional da assembleia de invertebrados colonizadores ao longo do tempo. A hipótese central do estudo é de que os detritos de S. INTRODUÇÃO humboldtiana constituem habitat favorável a colonização por invertebrados aquáticos, e que estes contribuem através dos fragmentadores para a degradação foliar desta espécie. Em adição, a associação dos invertebrados aquáticos aos detritos orgânicos e o seu processamento tem sido reportados por estudos em diversos ecossistemas (ver revisão de Graça 2001). Embora esta associação ainda não seja completamente clara, sabe-se que os detritos podem prover alimento, abrigo ou ambos para estes organismos (Dudgeon & Wu 1999; Graça 2001; Janke & Trivinho-Strixino 2007). Assim, os invertebrados aquáticos atuam sobre o detrito orgânico auxiliando na transformação da matéria orgânica particulada grossa (MOPG) em matéria orgânica particulada fina (MOPF) (Webster & Benfield 1986). Os estudos sobre o papel dos invertebrados no processamento dos detritos em ambientes subtropicais ainda são insipientes. Ainda assim, alguns estudos reportam que uma abordagem interessante no estudo da associação de invertebrados em detritos vegetais é a análise funcional de sua alimentação baseada nos mecanismos morfocomportamentais de aquisição e consumo de alimentos (Cummins et al. 2005; Silva et al. 2010; Telöken et al. 2011, entre outros). Entre os recursos utilizados, a MOPG e MOPF constituem fontes alimentares para os fragmentadores e coletores que colonizam os detritos vegetais em decomposição (Capello et al. 2004; Graça & Cressa 2010; Biasi et al. 2013). Ainda podem se associar aos detritos, organismos raspadores, utilizando-se do perifíton aderido, e os predadores que predam sobre os outros grupos (Cummins et al. 2005; Merritt & Cummins 2006). Embora a planície costeira sul do Brasil apresente cerca de 40% de sua superfície com ambientes aquáticos, estudos sobre os processos ecossistêmicos nestes ainda são escassos. A região costeira do extremo sul do Brasil apresenta uma ampla área de terras baixas, sendo os ambientes lagunar-lacustre os mais predominantes (Vieira & Rangel 1988), em sua maior parte ocupada pelo sistema lagunar Patos-Mirim (Villwock & Tomazelli 1995). A unidade mórfica básica é a restinga, originada a partir de acumulações sedimentares continentais e marinhas tendo como constituição básica as areias (Vieira & Rangel 1988). Como consequência das baixas declividades, os riachos apresentam reduzida correnteza. Em períodos com baixa precipitação pluviométrica, ocorre perda significativa da força hidráulica acarretando em um intenso processo de acumulação de sedimentos no leito do canal, originando bancos arenosos (Vieira & Rangel 1988). Experimento de campo e procedimentos em laboratório Ao final do período estudado foi determinada a taxa de perda foliar (k), utilizando o coeficiente exponencial negativo proposto por Webster & Benfield (1986) respectivamente com as equações %R=(Wt/W0)*100 (para porcentagem de peso remanescente) e k=-ln(%R/100)/t, onde Wt é o peso seco no tempo t (em dias) e W0 é o peso seco inicial. Foram avaliadas as diferenças entre as concentrações dos compostos químicos do detrito ao longo do tempo por meio de análise de variância (ANOVA-one way) seguida por pós-teste de Tukey (HSD), considerando significativo P<0.05. Os valores de riqueza e densidade dos grupos taxonômicos ao longo do tempo foram analisados por meio de análise de variância (ANOVA-one way) seguida por pós-teste de Tukey (HSD), considerando significativo P<0.05. O experimento foi realizado entre agosto/2009 e fevereiro/2010. Foram utilizadas folhas senescentes de S. humboldtiana coletadas às margens do riacho estudado. No laboratório, as folhas foram secas a temperatura ambiente (20±2 ºC) durante duas semanas. Foram confeccionadas 28 bolsas de folhiço com telas de nylon e polietileno, de dimensões 20x30 cm e aberturas de malha de 0.1 cm2 na face inferior (em contato com o sedimento) e 1.0 cm2 na face oposta (adaptado de Bedford 2004). Em cada bolsa foi adicionado 6.0±0.1 g de folhas. As bolsas foram dispostas no fundo do riacho presas com fios de nylon entre si e a tijolos para que ficassem submersas. Após 1, 7, 20, 50, 90, 126 e 184 dias de incubação, quatro bolsas de detritos foram retiradas aleatoriamente do riacho. Os detritos remanescentes foram lavados em água corrente, sobre peneira de 250 µm, sendo posteriormente secos em estufa (60 °C/48 h) para determinação do peso seco remanescente. Área de estudo (2005) e Merritt et al. (2008). Os invertebrados foram depositados na Coleção de Invertebrados Límnicos Subtropicais do Laboratório de Limnologia da Universidade Federal do Rio Grande. Perda de massa foliar Após lavagem e remoção dos organismos, os detritos secos foram triturados e submetidos a análises químicas quanto às concentrações de nitrogênio total (NT; Kjeldahl; Allen et al. 1974), fósforo total (PT) (Fassbender 1973; Baumgarten & Rocha 1996), matéria orgânica (incineração 550 °C/ 4 h) (Wetzel & Likens 1991) e polifenóis (adaptado de Bärlocher & Graça 2005). As concentrações de carbono foram estimadas multiplicando os valores de matéria orgânica por 0.465 (Westlake 1965). As concentrações químicas foram expressas em porcentagem por peso seco de detrito (%PS), com exceção de polifenóis (U.D.O./g PS). Ao final do período estudado foi determinado o peso remanescente de 69.48±3.81% representando um coeficiente de perda de massa de k=0.0066±0.0007 dia-1 (Figura 1). Em 24 h foram lixiviados 32.16±2.86% do peso inicial dos detritos, representando k=0.3908±0.0437 dia-1 (Figura 1). Área de estudo O estudo foi desenvolvido em um riacho localizado na região costeira do Rio Grande do Sul (32º09'27'' S e 52º11'18'' O). O clima é descrito como Cfa (subtropical úmido) de acordo com a classificação de Köppen. A região encontra-se em uma faixa de transição entre condições tropicais e temperadas com temperaturas médias anuais entre 15 e 18 oC e precipitação pluviométrica anual variando de 1200 a 1500 mm (SEPLAG 2012). A área de drenagem do riacho é composta por campos, banhados e mata ripária composta basicamente pelas espécies arbóreas Salix humboldtiana Willd., Erythrina crista-galli L., Ficus organensis Miq., Sebastiania commersoniana (Baillon) L. B. Smith & R. J. Downs, Erytroxylum argentinum O. E. Schulz, entre outras (Batista et al. 2007). 222 F TELÖKEN ET AL. Ecología Austral 24:220-228 O arroio Bolaxa é semelhante a maioria dos arroios da planície costeira sul do Brasil, com baixa declividade e movimento lento da água (Vieira & Rangel 1988). O substrato do riacho estudado é basicamente composto por areia e eventualmente ocorre o desenvolvimento de estandes de macrófitas aquáticas, entre elas Potamogeton pectinatus L., Salvinia auriculata Aubl., Myriophyllum brasiliensis Camb. (Batista et al. 2007). Durante o período de realização do experimento o pH da água permaneceu próximo à neutralidade (6.70±0.38), enquanto que a condutividade elétrica e alcalinidade total foram elevadas (245.5±22.7 µS/ cm e 1373.1±135.1 meq/L CO2, respectivamente). Os teores de oxigênio dissolvido variaram de 1.48 a 7.38 mg/L, porém, esta oscilação é normal para os riachos da região em épocas de estiagem (Tagliani et al. 1992). As concentrações de nutrientes e clorofila- a mantiveram-se baixas durante todo o período (Nitrogênio total: 0.85±0.41 mg/L; Fósforo total: 0.04±0.03 mg/L; Clorofila-a: 1.74±1.90 µg/L). Buckup 1999; Merritt et al. 2008; Mugnai et al. 2010). Foi determinada a riqueza e abundância de táxons, considerando-se como táxon o grupo identificado, conforme corrente uso na literatura com abordagem de avaliação da comunidade de invertebrados associada a detritos (Yamamuro & Lamberti 2007; Resende et al. 2010; Moulton et al. 2010, entre outros). A densidade de organismos foi expressa como número de indivíduos por grama de peso seco de detrito (indivíduos/g PS). Para determinar os padrões de sucessão de organismos em relação ao uso dos recursos, os invertebrados foram classificados em grupos tróficos funcionais (GTF) desde que bem estabelecido em literatura para o nível taxonômico utilizado no presente estudo, de acordo com classificação proposta por Callisto et al. (2001), Cummins et al. Análises químicas dos detritos leaf litter (percentage of remaining weight) in the period between August/2009 and February/2010 in a sandy stream, coastal plain of Rio Grande do Sul (Brazil). apresentaram incremento significativo ao longo do tempo, enquanto as concentrações de fósforo total não apresentaram variações ao longo do experimento. Coletor Raspador Predador Coletor Raspador Predador Figura 2. Distribuição em porcentagem dos grupos tróficos funcionais dos invertebrados associados a serapilheira de Salix humboldtiana Willd., entre agosto/2009 e fevereiro/ 2010, em um córrego arenoso da planície costeira do Rio Grande do Sul, excluindo Chironomidae, e considerado Hyallelidae como coletor. Figura 2. Distribuição em porcentagem dos grupos tróficos funcionais dos invertebrados associados a serapilheira de Salix humboldtiana Willd., entre agosto/2009 e fevereiro/ 2010, em um córrego arenoso da planície costeira do Rio Grande do Sul, excluindo Chironomidae, e considerado Hyallelidae como coletor. Análises químicas dos detritos Tempo (dias) %NT %PT %MO %C Polifenóis (U.D.O./g PS) N : P C : N C : P 0 0.98 ± 0.04 a 0.08 ± 0.00 94.96 ± 0.22 ac 44.63 ± 0.10 ac 13.53 ± 0.41 a 12 : 1 46 : 1 558 : 1 1 1.65 ± 0.38 bc 0.09 ± 0.05 96.29 ± 0.14 b 45.26 ± 0.06 b 10.72 ± 0.45 a 18 : 1 27 : 1 503 : 1 7 1.40 ± 0.06 ab 0.11 ± 0.00 95.29 ± 0.11 a 44.79 ± 0.05 a 4.27 ± 0.24 b 13 : 1 32 : 1 407 : 1 20 1.69 ± 0.07 bc 0.17 ± 0.01 94.18 ± 0.11 cd 44.27 ± 0.05 cd 1.76 ± 0.03 c 10 : 1 26 : 1 260 : 1 50 1.93 ± 0.09 bc 0.11 ± 0.00 94.21 ± 0.20 cd 44.28 ± 0.09 cd 1.53 ± 0.07 df 18 : 1 23 : 1 403 : 1 90 2.37 ± 0.08 c 0.11 ± 0.01 94.72 ± 0.27 ac 44.52 ± 0.13 ac 2.83 ± 0.07 d 22 : 1 19 : 1 405 : 1 126 2.19 ± 0.05 c 0.10 ± 0.01 93.85 ± 0.08 d 44.11 ± 0.04 d 1.86 ± 0.06 e 22 : 1 20 : 1 441 : 1 184 2.19 ±0.04 c 0.13 ± 0.02 90.92 ± 0.03 e 42.73 ± 0.02 e 1.97 ± 0.07 f 17 : 1 20 : 1 329 : 1 0 20 40 60 80 100 0 1 7 20 50 90 126 184 % Remanescente de folhas Dias 0 1 7 20 50 90 126 184 Días % Remanescente de folhas % Remanescente de folhas % Remanescente de folhas No primeiro dia de colonização foram registrados 16 táxons colonizando os detritos (Tabela 2). No 7º dia foi registrada a maior riqueza (29 táxons) e posterior redução até o final do experimento (184º dia com 15 táxons). 1 7 20 50 90 126 184 Dias de decomposição 100% 75% 50% 25% 0% Dias Días Figura 1. Perda de massa da serapilheira de Salix humboldtiana Willd. (em porcentagem de peso remanescente) no período entre agosto/2009 e fevereiro/ 2010 em um córrego arenoso, planície costeira do Rio Grande do Sul (Brasil). Figure 1. Mass loss of Salix humboldtiana Willd. Análises químicas dos detritos Variação na composição química da serapilheira de Salix humboldtiana Willd., entre agosto/2009 e fevereiro/ 2010, em um córrego arenoso da planície costeira do Rio Grande do Sul (Brasil). Tempo zero=folhas senescentes não- incubadas. Médias±desvios-padrões de nitrogênio total (%NT), fósforo total (%PT), matéria orgânica (%MO), carbono (%C) e concentrações de polifenóis (U.D.O./g PS). N:P, relação nitrogênio:fósforo, C:N, relação carbono:nitrogênio, C: P, relação carbono fósforo. Letra sdiferentes (a-f) representam diferenças significativas (P<0.05). Table 1. Changes in the chemical composition of Salix humboldtiana Willd. leaf litter, between august 2009 and February 2010, in a sandy stream of coastal plain of Rio Grande do Sul (Brazil). Time-0=senescent leaves not incubated. Mean±SD of total nitrogen (%NT), total phosphorus (%PT), organic matter (%MO), carbon (%C) and polyphenol concentration (U.D.O./g PS). N:P, nitrogen:phosphorus relationship; C:N, carbon:nitrogen relationship; C:P, carbon:phosphorus relationship. Different letters (a-f) represent significant difference (P<0.05). Coletor 1 7 20 50 90 126 184 Dias de decomposição Raspador Predador 0 1 7 20 50 90 126 184 Días % Remanescente de folhas 100% 75% 50% 25% 0% Table 1. Changes in the chemical composition of Salix humboldtiana Willd. leaf litter, between august 2009 and February 2010, in a sandy stream of coastal plain of Rio Grande do Sul (Brazil). Time-0=senescent leaves not incubated. Mean±SD of total nitrogen (%NT), total phosphorus (%PT), organic matter (%MO), carbon (%C) and polyphenol concentration (U.D.O./g PS). N:P, nitrogen:phosphorus relationship; C:N, carbon:nitrogen relationship; C:P, carbon:phosphorus relationship. Different letters (a-f) represent significant difference (P<0.05). Análises químicas dos detritos As concentrações de matéria orgânica dos detritos mantiveram-se sempre superiores a 90% durante todo o período de estudo, porém com variações significativas ao longo do tempo (Tabela 1). As concentrações de polifenóis apresentaram rápida redução até o 20º dia. As concentrações de nitrogênio total Os invertebrados retidos na peneira (malha de 250 µm) foram fixados em etanol 80% e identificados em um estereomicroscópio até o menor nível taxonômico possível utilizando chaves taxonômicas específicas (Thorp & Covich 1991; Elmoor-Loureiro 1997; Buckup & Bond- 223 INVERTEBRADOS AQUÁTICOS EM SERAPILHEIRA Agosto de 2014 Tabela 1. Variação na composição química da serapilheira de Salix humboldtiana Willd., entre agosto/2009 e fevereiro/ 2010, em um córrego arenoso da planície costeira do Rio Grande do Sul (Brasil). Tempo zero=folhas senescentes não- incubadas. Médias±desvios-padrões de nitrogênio total (%NT), fósforo total (%PT), matéria orgânica (%MO), carbono (%C) e concentrações de polifenóis (U.D.O./g PS). N:P, relação nitrogênio:fósforo, C:N, relação carbono:nitrogênio, C: P, relação carbono fósforo. Letra sdiferentes (a-f) representam diferenças significativas (P<0.05). Tabela 1. Variação na composição química da serapilheira de Salix humboldtiana Willd., entre agosto/2009 e fevereiro/ 2010, em um córrego arenoso da planície costeira do Rio Grande do Sul (Brasil). Tempo zero=folhas senescentes não- incubadas. Médias±desvios-padrões de nitrogênio total (%NT), fósforo total (%PT), matéria orgânica (%MO), carbono (%C) e concentrações de polifenóis (U.D.O./g PS). N:P, relação nitrogênio:fósforo, C:N, relação carbono:nitrogênio, C: P, relação carbono fósforo. Letra sdiferentes (a-f) representam diferenças significativas (P<0.05). apresentaram incremento significativo ao longo do tempo, enquanto as concentrações de fósforo total não apresentaram variações ao longo do experimento. Invertebrados associados Foram contabilizados 14197 organismos distribuídos em 36 grupos taxonômicos (Tabela 2). Os mais representativos foram Oligochaeta (43.60%), Hydrobiidae (16.63%), Chironominae (11.26%), Hydracarina (6.61%), Ostracoda (4.80%) e Orthocladiinae (4.69%), totalizando 87.60% da assembleia. Em 24 horas, a densidade média de invertebrados foi 11.41±1.58 individuos/g PS (Tabela 2). Foi observado aumento da densidade até o 20º dia (524.16±83.61 indivíduos/g PS), com posterior diminuição destes valores, chegando a 40.99±17.13 indivíduos/g PS no 126º dia. No primeiro dia de colonização foram registrados 16 táxons colonizando os detritos (Tabela 2). No 7º dia foi registrada a maior riqueza (29 táxons) e posterior redução até o final do experimento (184º dia com 15 táxons). 0 20 40 60 80 100 0 1 7 20 50 90 126 184 % Remanescente de folhas Dias Figura 1. Análises químicas dos detritos Perda de massa da serapilheira de Salix humboldtiana Willd. (em porcentagem de peso remanescente) no período entre agosto/2009 e fevereiro/ 2010 em um córrego arenoso, planície costeira do Rio Grande do Sul (Brasil). Figure 1. Mass loss of Salix humboldtiana Willd. leaf litter (percentage of remaining weight) in the period between August/2009 and February/2010 in a sandy stream, coastal plain of Rio Grande do Sul (Brazil). Figura 2. Distribuição em porcentagem dos grupos tróficos funcionais dos invertebrados associados a serapilheira de Salix humboldtiana Willd., entre agosto/2009 e fevereiro/ 2010, em um córrego arenoso da planície costeira do Rio Grande do Sul, excluindo Chironomidae, e considerado Hyallelidae como coletor. Figure 2. Percentage distribution of functional feeding groups associated with Salix humboldtiana Willd. leaf litter, between august 2009 and February 2010, in a sandy stream, coastal plain of Rio Grande do Sul (Brazil), excluding Chironomidae, Hyallelidae as colector. Tempo (dias) %NT %PT %MO %C Polifenóis (U.D.O./g PS) N : P C : N C : P 0 0.98 ± 0.04 a 0.08 ± 0.00 94.96 ± 0.22 ac 44.63 ± 0.10 ac 13.53 ± 0.41 a 12 : 1 46 : 1 558 : 1 1 1.65 ± 0.38 bc 0.09 ± 0.05 96.29 ± 0.14 b 45.26 ± 0.06 b 10.72 ± 0.45 a 18 : 1 27 : 1 503 : 1 7 1.40 ± 0.06 ab 0.11 ± 0.00 95.29 ± 0.11 a 44.79 ± 0.05 a 4.27 ± 0.24 b 13 : 1 32 : 1 407 : 1 20 1.69 ± 0.07 bc 0.17 ± 0.01 94.18 ± 0.11 cd 44.27 ± 0.05 cd 1.76 ± 0.03 c 10 : 1 26 : 1 260 : 1 50 1.93 ± 0.09 bc 0.11 ± 0.00 94.21 ± 0.20 cd 44.28 ± 0.09 cd 1.53 ± 0.07 df 18 : 1 23 : 1 403 : 1 90 2.37 ± 0.08 c 0.11 ± 0.01 94.72 ± 0.27 ac 44.52 ± 0.13 ac 2.83 ± 0.07 d 22 : 1 19 : 1 405 : 1 126 2.19 ± 0.05 c 0.10 ± 0.01 93.85 ± 0.08 d 44.11 ± 0.04 d 1.86 ± 0.06 e 22 : 1 20 : 1 441 : 1 184 2.19 ±0.04 c 0.13 ± 0.02 90.92 ± 0.03 e 42.73 ± 0.02 e 1.97 ± 0.07 f 17 : 1 20 : 1 329 : 1 Tabela 1. Invertebrados associados Foram contabilizados 14197 organismos distribuídos em 36 grupos taxonômicos (Tabela 2). Os mais representativos foram Oligochaeta (43.60%), Hydrobiidae (16.63%), Chironominae (11.26%), Hydracarina (6.61%), Ostracoda (4.80%) e Orthocladiinae (4.69%), totalizando 87.60% da assembleia. Em 24 Figure 2. Percentage distribution of functional feeding groups associated with Salix humboldtiana Willd. leaf litter, between august 2009 and February 2010, in a sandy stream, coastal plain of Rio Grande do Sul (Brazil), excluding Chironomidae, Hyallelidae as colector. Figure 2. Percentage distribution of functional feeding groups associated with Salix humboldtiana Willd. leaf litter, between august 2009 and February 2010, in a sandy stream, coastal plain of Rio Grande do Sul (Brazil), excluding Chironomidae, Hyallelidae as colector. 224 Ecología Austral 24:220-228 F TELÖKEN ET AL. Ecología Austral 24:220-228 Tabela 2. Invertebrados associados a serapilheira de Salix humboldtiana Willd., entre agosto/2009 e fevereiro/2010, em um córrego arenoso da planície costeira do Rio Grande do Sul (Brasil). Densidades médias (indivíduos/g PS), abundâncias (%), riqueza (estimada para os táxons identificados) e grupos tróficos funcionais (GTF) dos táxons em cada período de amostragem. Pr: predador, Co-Fi: coletor - filtrador, Co-Ca: coletor-catador, Ra: raspador, Fr: fragmentador. Letras diferentes representam diferenças significativas (a - c) (P< 0.05). Table 2. Invertebrates associated with Salix humboldtiana Willd. leaf litter, between August/2009 and February/2010 in a sandy stream of coastal plain of Rio Grande do Sul (Brazil). Mean densities (individuos/g DW) abundance (%), richness (calculated for all taxa) and functional trophic groups (FTG) of taxain each sampling period. Pr: predators, Co-Fi: filtering-collectors, Co-Ca: gathering-collectors, Ra: scrapers, Fr: shredders. Different letters represent significant differences (a-c) (P< 0.05). Invertebrados associados Taxa GTF Densidades Médias (ind/g PS) % 1 7 20 50 90 126 184 Rhizopoda Pr 0 7.49 154.52 0 0 0 1799.27 0.76 Porifera Co-Fi 0 0 0 0 0 15.02 0 0.01 Tricladida Co-Ca / Pr 0 14.32 0 0 8.33 0 0 0.02 Nemertea Pr 0 0 0 8.80 0 0 0 0.01 Nematoda Co-Ca 0 157.51 149.73 530.86 293.87 290.51 743.83 1.42 Hirudinea Pr 0 7.27 53.96 297.84 66.67 60.33 0.38 Oligochaeta Co-Ca 33.69 4224.37 34182.02 8797.37 1294.42 808.84 1205.19 43.60 Hydracarina Pr 345.06 3063.49 1604.85 1117.92 427.50 150.97 1353.37 6.61 Cladocera Chydoridae Co-Ca 106.68 990.85 581.73 17.24 0 0 28.88 1.66 Daphniidae Co-Fi 0 40.98 0 8.62 0 0 0 0.05 Macrothricidae Co-Ca 0 6.83 0 0 0 0 0 0.01 Amphipoda Hyalellidae Co-Ca 43.73 190.18 342.59 141.11 8.33 15.02 0 0.67 Isopoda Sphaeromatidae Co-Ca 0 7.00 0 0 0 0 0 0.01 Cyclopoida Co-Ca / Pr 0 779.69 306.32 17.61 0 0 0 1.07 Harpacticoida Co-Ca / Pr 0 14.53 31.15 8.62 0 0 0 0.05 Ostracoda Co-Ca 6.81 905.57 1268.08 163.67 58.58 15.02 7288.32 4.80 Collembola Entomobryidae Co-Ca 6.74 36.51 32.53 18.87 82.64 0 57.76 0.17 Ephemeroptera Baetidae Co-Ca / Ra 6.43 0 30.44 132.04 8.33 45.05 0 0.17 Caenidae Co-Ca / Ra 34.19 815.38 479.61 155.90 125.13 75.21 34.72 1.55 Odonata Coenagrionidae Pr 6.81 34.33 8.31 0 0 0 0 0.05 Coleoptera Elmidae Co-Ca / Ra 0 0 7.62 0 33.46 136.36 0 0.09 Staphylinidae Pr 13.55 21.01 0 0 0 75.76 34.72 0.08 Hydrophilidae Co-Ca / Pr 0 51.96 156.34 35.21 0 0 0 0.22 Chironomidae Tanypodinae Co-Ca / Pr 6.81 35.46 53.55 8.47 0 45.05 0 0.12 Orthocladiinae Co-Ca / Fr / Pr / Ra 143.55 779.45 3201.26 1042.18 12.56 45.05 0 4.69 Chironominae Co-Ca / Co-Fi / Fr / Pr / Ra 231.70 803.52 5064.96 5628.82 864.98 1273.82 185.13 11.26 Pupas 29.79 78.32 425.50 466.19 45.90 15.02 0 0.90 Ceratopogonidae Co-Ca / Pr / Ra 0 7.27 22.82 17.42 0 0 0 0.04 Simuliidae Co-Fi 0 0 32.17 431.20 0 45.05 0 0.39 Sciomyzidae Pr 0 7.27 0 0 0 0 0 0.01 Muscidae Pr 0 0 0 0 8.33 0 0 0.01 Trichoptera Hidroptilidae Co-Ca / Ra 0 21.01 0 34.55 31.23 0 28.57 0.08 Gastropoda Ampullaridae Ra 0 0 16.27 27.67 0 0 0 0.04 Ancylidae Ra 44.78 646.66 559.93 650.58 16.67 120.26 666.57 2.01 Hydrobiidae Ra 80.54 1930.27 3501.72 1526.06 850.70 851.74 23385.46 16.63 Planorbidae Ra 0 63.91 148.05 51.58 33.46 15.02 84.03 0.30 Bivalvia Co-Fi 0 0 0 0 0 0 180.91 0.07 Densidade total (ind/g PS) 11.41 a 157.42 bc 524.16 c 213.54 abc 42.71 ab 40.99 ab 370.77 bc Riqueza 16 ab 29ab 26 a 27 ab 19 b 19 ab 15 ab p g ( ) ( ) 225 INVERTEBRADOS AQUÁTICOS EM SERAPILHEIRA Agosto de 2014 decomposição tenha se tornado um fator facilitador para a colonização microbiana e por invertebrados. DISCUSSÃO O coeficiente de perda de massa estimado no presente trabalho foi menor do que os valores obtidos em estudos com Salix humboldtiana em diferentes ambientes no sistema hidrológico do Rio Paraná (Argentina). Os coeficientes de decomposição de S. humboldtiana foram maiores em um pântano (k=0.019/dia) (Poi de Neiff et al. 2006), e em um canal da planície aluvial (k=0.0101/dia) (Capello et al. 2004) e em um rio (k=0.0119/dia) (Leguizamon et al. 1992). As taxas de degradação variam de acordo com a composição química da água, qualidade do detrito, ação do fluxo da correnteza e da ação de invertebrados, em especial fragmentadores. No entanto, acredita-se que a perda de massa mais lenta em relação aos estudos citados tenha ocorrido por efeitos da dinâmica do sedimento do riacho. O fundo arenoso é instável a mudanças no fluxo, o que acarreta em suspensão do mesmo e posterior deposição sobre o detrito, retardando o processo de perda de massa e dificultando a colonização por invertebrados. Detritos enterrados apresentam menores taxas de degradação do que os dispostos na superfície do sedimento, sendo isto causado principalmente pela diminuição da abrasão física com a água em movimento, baixas concentrações de oxigênio dissolvido e limitação na ação de invertebrados (Herbst 1980; Smock et al. 1989; Metzler & Smock 1990). Aliado ao aumento na concentração de nitrogênio, a diminuição nas concentrações de polifenóis favorece também a colonização por invertebrados, pois proporcionam um incremento na qualidade do detrito. Recentes estudos sugerem que altas concentrações de compostos secundários nos detritos (polifenóis) inibem a colonização de invertebrados retardando o processo de fragmentação (Wantzen et al. 2008; Trevisan & Hepp 2007; Hepp et al. 2009; Biasi et al. 2013). No presente estudo, foi observada a redução da relação C:N e C:P em cerca de 50% e do conteúdo de polifenóis em cerca de 70% durante o período de degradação, o que refletiu na melhoria da qualidade do detrito e, consequentemente, na densidade de invertebrados colonizadores. Os coletores catadores representaramos organismos mais abundantes na assembleia de invertebrados, e aos 20 dias foram dominados por Oligochaeta, que chegou a 65% de abundância na assembleia. A dominância de coletores-catadores em detritos é associada à acumulação de MOPF originado do próprio detrito, ou de partículas orgânicas em suspensão na coluna d’água que acumulam na superfície dos substratos (Richardson 1992; Grubbs et al. 1995; Serra et al. 2013). Invertebrados associados Os coletores-catadores representaram a maior parcela da assembleia (52.8%), sendo 70% destes Oligochaeta. Os raspadores representaram 30%, sendo 80% destes Hydrobiidae. Os predadores representaram 11.6%, potenciais fragmentadores 3% e coletores-filtradores 2.6%. Excluindo os potenciais fragmentadores (Chironomidae), considerando Hyallelidae como coletor, e agrupando coletores catadores e filtradores como um grupo, a representação dos GTF ao longo do processo de degradação é mostrada na Figura 2. O condicionamento microbiano é importante para o processo de aumento da palatabilidade do detrito, o que facilita a colonização do mesmo por parte dos invertebrados detritívoros (Graça 2001; Graça et al. 2001; Wantzen et al. 2008). A variação na concentração de NT e PT dos detritos ao longo do tempo reflete o condicionamento microbiano do detrito, o qual diminui as relações C:N e C:P refletindo o aumento do valor nutricional dos detritos (Graça & Cressa 2010). Neste estudo, o incremento de nutrientes, em especial nitrogênio, é um forte indicador do condicionamento microbiano do detrito (Lester et al. 1994) pois é reconhecido que o aumento de nitrogênio com o tempo de degradação vegetal favorece a atuação dos decompositores (fungos e bactérias) (Brum & Esteves 2001). DISCUSSÃO A abundância de raspadores no final do período do experimento, representados principalmente por Hidrobiidae, pode estar relacionada com o incremento nas concentrações de NT e PT no detrito com o decorrer da decomposição, o que A perda de massa neste estudo foi rápida durante as primeiras horas devido à lixiviação de compostos solúveis. O material remanescente, composto por polissacarídeos estruturais é mais resistente à degradação, sendo sua perda de peso mais lenta (Bianchini Jr. 1999). Polifenóis também são compostos facilmente lixiviados do detrito (Hepp et al. 2009). Provavelmente a rápida lixiviação dos polifenóis nos primeiros 20 dias de 226 F TELÖKEN ET AL. Ecología Austral 24:220-228 provavelmente favoreceu o estabelecimento do perifíton associado à MOPF. et al. 2013). Irons et al. (1994) propõem que a importância de fragmentadores para o processo de decomposição é maior em ambientes temperados, enquanto que nos ambientes tropicais e subtropicais os microrganismos possuem maior importância. Os resultados referentes a assembléia de invertebrados e sua categorização funcional no riacho estudado ainda são insipientes, e ressaltam a importância de estudos mais detalhados sobre a categorização funcional dos organismos encontrados. Futuros experimentos devem ser conduzidos para definir principalmente a real participação de fragmentadores no processo de degradação foliar, de forma a aplicar ou modificar as classificações propostas em outros trabalhos em diferentes ecossistemas nas várias regiões geográficas. Embora o nível de identificação taxonômica não permita categorizar funcionalmente com precisão alguns grupos (e.g., Chironomidae), para a grande maioria a classificação trófica funcional é bem estabelecida em literatura. Os organismos de Amphipoda apresentaram abundância muito baixa durante o processo de degradação (menos de 1%), e foram todos pertencentes a família Hyallelidae (Hyallela curvispina), reportada por Capello et al. (2004) e Wantzen & Wagner (2006) como coletores, embora Cummins et al. (2005) e Zilli et al. (2008) categorizem esta família como fragmentadora. Neste trabalho adotamos a classificação dos dois primeiros autores. Outros potenciais fragmentadores seriam alguns gêneros de Chironomidae, não identificados no presente estudo. Cummins et al. (2005) categorizam a família Chironomidae como coletores-catadores em vários riachos de Mata Atlântica no Brasil. Conforme colocam Wantzen & Wagner (2006), em geral as espécies descritas como fragmentadores para a região neotropical apresentam baixa biomassa, e provavelmente têm menor influência sobre a degradação dos detritos comparada a regiões temperadas. Biasi et al. DISCUSSÃO (2013), em recente estudo sobre Chironomidae associados a decomposição foliar em riachos subtropicais reportam que apenas 2.2% dos gêneros foram classificados como fragmentadores, Zilli et al. (2008) reportam dois gêneros em diferentes mesohabitats aquáticos da planície de inundação do rio Paraná (Argentina) pertencentes a este grupo trófico e Silva et al. (2010) reportam apenas um gênero de Chironomidae fragmentador em um lago próximo ao riacho estudado. Considerando a composição química dos detritos de S. humboldtiana, a redução da concentração de polifenóis e das proporções C:N e C:P ao longo do tempo, sugere-se que estes detritos melhoraram a qualidade ao longo do processo de degradação, favorecendo a colonização por invertebrados e corroborando parte da hipótese inicial de nosso estudo. Por outro lado, mesmo com a categorização taxonômica utilizada, constatou- se que o número de potenciais fragmentares foi reduzido ao longo do processo de perda de massa foliar, refutando parte de nossa hipótese de contribuição efetiva destes para a velocidade de degradação de S. humboldtiana. Embora a identificação taxonômica não tenha possibilitado identificar gêneros de Chironomidae potenciais fragmentadores, a participação em termos de abundância destes organismos ao longo do período experimental sempre foi inferior aos principais grupos tróficos (predomínio de coletores nas fases iniciais e raspadores na fase final), o que ressalta a importância da MOPF como recurso alimentar para estes organismos. O papel da MOPF no estabelecimento de comunidades de invertebrados em riachos foi recentemente demonstrado por Callisto & Graça (2013), onde concluem que Chironomidae foi fortemente relacionado com as zonas deposicionais onde é maior a quantidade de MOPF. Por outro lado, a dinâmica do sedimento dos riachos arenosos subtropicais, condicionado às variações climáticas sazonais, pode atuar como fator regulador do processamento da matéria orgânica alóctone. Assim, com base na baixa abundância de potenciais fragmentadores registrada durante a degradação de S. humboldtiana, pode ser considerado que este grupo funcional não contribuiu de forma significativa para a velocidade de degradação dos detritos. Os fragmentadores consistem no principal GTF que contribui para a degradação da matéria orgânica particulada grossa em regiões temperadas (Wallace & Webster 1996; Graça et al. 2001; Serra et al. 2013). No entanto, estudos em regiões tropicais e subtropicais têm registrado baixas abundâncias destes organismos colonizando os detritos (Dobson et al. 2002; Stripari & Henry 2002; Gonçalves et al. 2006, 2007; Carvalho &Uieda 2009; Poi de Neiff et al. 2009; Telöken et al. REFERÊNCIAS Food quality, feeding preferences, survival and growth of shredders from temperate and tropical streams. Freshw. Biol., 46:947-957. BEGON, M; CR TOWNSEND & JL HARPER. 2006. Ecologia: de indivíduos a ecossistemas.4a ed. Porto Alegre, Artmed. Pp. 752. 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https://openalex.org/W4385408528	https://ejurnal.bunghatta.ac.id/index.php/JMN/article/download/23257/18913	English	null	Omni-Channel Capability, Online Experience & Value Co-Creation and Their Impact to Customer Satisfaction	Jurnal Manajemen Universitas Bung Hatta	2,023	cc-by-sa	7,037	Keywords: Omni-Channel Capability Online Experience Value Co-Creatio Keywords: Omni-Channel Capability Online Experience Value Co-Creatio Received: Revised: Corresponding author: syafrizal. ajosyafrizal@gmail.com Accepted: ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) Omni-Channel Capability, Online Experience & Value Co-Creation and Their Impact to Customer Satisfaction Arbi Abdullah Saleh1, Syafrizal2, Ali Vafaei Zadeh3 1,2Department of Management, Faculty of Economics and Business Universitas Andalas 3Graduate School of Business (GSB), Universiti Sains Malaysia, Penang, Malaysia Management Department, Faculty of Economics and Business, Universitas Bung Hatta Introduction 266 Management Department, Faculty of Economics and Business, Universitas Bung Hatta ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) Management Department, Faculty of Economics and Business, Universitas Bung Hatta Customer Value Co-Creation The complexities of customer needs, along with the ever-changing nature of the business environment, has driven organizations to include all stakeholders in knowledge development via an open system. Customers have been given the ability to play major roles in the generation of value by sharing operand and operant resources (Preikschas et al., 2017). (Vargo & Lusch, 2014) stated that the consumer is an active participant in the exchange process rather than a passive actor, and that value is always co-created with the customer. Firms interact in order to meet the particular demands of their customers, and in doing so, they were able to generate new ideas. Co-creation is based on the interchange of information and knowledge between actors, in which the consumer is a vital player and actively participates in the sharing of knowledge and information. It is possible to infer that the client is an operant resource, sharing information and skills with the company (Vargo & Lusch, 2016). According to (Preikschas et al., 2017), the company is employing customers as a source of expertise and thereby putting them to work. ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) Customer Satisfaction enhances comprehension of the distinctions between offline and online experiences. Customer satisfaction is one of the most extensively studied concepts in marketing literature. It is significant in a competitive economy since of its capacity to hold existing customers while also recruiting new ones . define satisfaction as an emotion by an individual of delight or dissatisfaction as a result of a comparison of goods and service performance and anticipation. (Oliver, 2010), defined customer satisfaction as the customer's satisfying reaction. It’s a choice regarding a characteristic of a product or service, or when the goods or service has provided (or continues to provide) a satisfying degree of consumption satisfaction. According to and (Giese et al., 2009) customer satisfaction consists of three important parts: a response (cognitive or emotional) linked to specific emphasis (product, consuming experience, expectations, etc.) identified in a specific moment; a response (cognitive or emotional) linked to specific emphasis product, consuming experience, expectations, etc.) identified at some specific moment; and a response (emotional or cognitive) linked to (after consumption, after choice, based on accumulated experience, etc.). Based on the most recent study in online experience literature, there is presently no consensus on the concept of online experience. The literature uses a variety of terms to describe online experience, including online customer experience , website experience , and online purchase experience . Management Department, Faculty of Economics and Business, Universitas Bung Hatta Omni-Channel Capability defines omni-channel capabilities as organizational practices that fulfill major organizational objectives in the providing of omni-channel services, in line with the resource- based definition of organizational capabilities. Using recent industry research and practices, omni- channel capabilities reveals customer views of omni-channel strategies (Galipoğlu et al., 2018) The goal of omni-channel capabilities is frequently said to be customer acquisition and retention. However, businesses that just attract users are frequently unable to retain them when other, more convenient services emerge and steal them away. As a result, it is vital to assess not only the quality of a service but also, one step further, its potential to gain consumer pleasure. Customers will be less inclined to switch to other providers if customer loyalty is increased. As a result, the actual goal of omni-channel capabilities is to increase client loyalty . Omni-channel retailing necessitates specific characteristics (both conventional and dynamic), typified by fully integrated retail software systems that combine data on items, customers, shopping, and communication, to be available across online and physical channels 267 Management Department, Faculty of Economics and Business, Universitas Bung Hatta 3. Customer The notion of customer experience has been investigated in a wide variety of business contexts, including tourism, service delivery, consumer marketing, and commerce, but currently it is also in the online setting (Novak et al., 2000). Online shopping a variety of product categories, online services such as travel, financial services, and theater bookings, accessibility to latest information, and social network for leisure or business reasons are now all part of the online experience. Given the rising performance of online sales, online experience has become a significant concept for retailer responsibility for the online Business-to-Customer (B2C) environment, especially regarding to online shopping. from that in omni-channel context, customer experience congruence has a positive effec on all of the variables tested in the research. support all hypotheses confirm the positive relationships between all customer experience components in omni-channel context. , also stated that positive a significant effect on where customer experience is the mediating variable, while the omni-channel is an independent variable. Based on the results of research, the first hypothesis t can be : p y g g pp g he by . that the results of a positive online experiences in online shopping are customer satisfaction. In line with also stated that online shopping experience affect marketing outcome variables especially satisfaction. In addition, the online experience is used as mediating variable in several studies as positive and significant towards customer satisfaction which resulted in full mediation. These studies are from , (Chen et al., 2021), (Kumar et al., 2022), and . Based on the results of research, the hypothesis t can be : H1: 1. Customer define omni-channel capabilit as organizational practices fulfill organization to provid omni-channel services, in line with the resource-based definition of organizational capabilities. Using recent industry research and practices, omni-channel capabilities reveals customer views of omni-channel strategies . The goal of omni-channel capabilit is frequently said to be customer acquisition and retention. H2: Research Hypothesis that there are several sources of value co- creation that be omni-channel. Based on the results of research, the hypothesis t can be : 2. Omni-channel retailing necessitates specific characteristics (both conventional and dynamic), typified by fully integrated retail software systems that combine data on items, customers, shopping, and communication available online and physical channels (Solem et al., 2023). (Cui et al., 2022) stated that online and offline omni channel interactivity and value co- creation positive relationship. (Telli, 2020) identifie the importance of omni-channel shopping services and value co-creation processes, where value co-creation is the mediating variable. It is also stated by H3: H3: Management Department, Faculty of Economics and Business, Universitas Bung Hatta Customer Online Experience The notion of customer experience has been investigated in a wide variety of business contexts, including tourism, service delivery, consumer marketing, and commerce, but currently it is also in the online setting (Novak et al., 2000). Online shopping across a wide variety of product categories, online services such as travel, financial services, and theater bookings, accessibility to latest information, and social network for leisure or business reasons are now all part of the online experience. Given the rising performance of online sales, online experience has become a significant concept for retailer responsibility for the online Business-to-Customer (B2C) environment, especially regarding to online shopping. Value co-creation refers to the joint development and delivery of services by both customers and enterprises . The value co-creation paradigm, in contrast to the traditional business model in which corporations control the service development and delivery process, stresses consumer engagement and contribution. Customers and enterprises engage in a dynamic development process that benefits both sides to produce product value based on mutual understanding Even though there is the existence of two retail settings today, namely face-to-face (offline) and Internet-based (online), it is necessary to compare the two. (Rose et al., 2011) stated that clarity about the distinctions between them 268 Management Department, Faculty of Economics and Business, Universitas Bung Hatta ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) Methods H4: 4. 4. Recent research on consumer engagement in value co-creation has that co-creation is a 269 Management Department, Faculty of Economics and Business, Universitas Bung Hatta Management Department, Faculty of Economics and Business, Universitas Bung Hatta ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) H5: direct predictor of customer satisfaction and loyalty . (Preikschas et al., 2017) discovered proof that sharing customer expertise develop solutions and satisfy the customer. According to (Opata et al., 2020), customer engagement in value co-creation has a large and beneficial effect on customer satisfaction. According to ,value co- creation a larger effect o satisfaction for both B2B and B2C clients. In addition, val is used as mediating variable in several studies as positive and significant towards customer satisfaction which resulted in full mediation. These studies are from , (Liu & Jo, 2020), and & . Based on the results of research, the hypothesis t can be H5 Based on the literature review, research framework can be proposed as in Figure 1. Figure 1. Theoretical Research Framework Omni- Channel Capability (OCC) Customer Satisfaction (CS) Online Experience (OX) Value Co- Creation (VCC) H1 H2 H3 H4 Online Experience (OX) Omni- Channel Capability (OCC) Customer Satisfaction (CS) H2 Value Co- Creation (VCC) Figure 1. Theoretical Research Framework Management Department, Faculty of Economics and Business, Universitas Bung Hatta Research Results nd Discussion VCC4 0.731 VCC5 0.853 VCC6 0.836 VCC7 0.859 VCC8 0.784 VCC9 0.809 VCC10 0.752 VCC11 0.828 VCC12 0.765 VCC13 0.766 VCC14 0.756 Source: Output data from SmartPLS 4.0 (2022) 5. The complexit of customer needs, along with the nature of business environment , has driven organizations to include all stakeholders in knowledge development via an open system. Customers have been given the ability to play major roles in the generation of value by sharing operant resources stated that the consumer is an active participant in the exchange process rather than a passive actor, and value always co-created with the customer. argue that when customers get purchasing goal-congruent promotions rather than purchasing goal- incongruent promotions, they are more likely to be happy with their omni-channel buying experience. The findings of indicated that customers are more satisfied when an organization provides a barrier-free buying experience, and omni-channel is one of them. stated that omni-channel gets strengthened because of customer satisfaction. Based on the results of research, the hypothesis t can be . 270 Management Department, Faculty of Economics and Business, Universitas Bung Hatta ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 Management Department, Faculty of Economics and Business, Universitas Bung Hatta Hypothesis Testing Tabel 5 Structural Model Analysis Regression Path Original sampl (O) Sample me n (M) Standard d viation (STDEV) T tatistics (\|O/STDEV\|) P alues Omni- Channel Capability -> Online Experience 0.742 0.742 0.046 15.954 0.001 Omni- Channel Capability -> Value Co- Creation 0.696 0.698 0.054 12.886 0.002 Online Experience - > Customer Satisfaction 0.213 0.21 0.12 1.783 0.075 Value Co- Creation -> Customer Satisfaction 0.37 0.367 0.124 2.974 0.003 Omni- Channel Capability -> Customer Satisfaction 0.181 0.187 0.098 1.841 0.066 Source: Output data from SmartPLS 4.0 (2022) Tabel 5 Structural Model Analysis Tabel 5 Tabel 5 Structural Model Analysis Structural Model Analysis Based on the table 3, the value of AVE root in the variable column with the variable row is higher than the value for the variable column with other variable rows. This that AVE root value in the latent correlation variable has met the requirements. Meanwhile, the value of AVE root in the variable column with the variable row of nline xperience 271 ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) and alue o- reation is higher than the value for the variable column with other variable rows. This that AVE root value in the latent correlation variable has met the requirements. Based on able 4 , it can be seen that R-Square value of ustomer satisfaction variable is 0.486, which means that 48.6% of the ustomer Satisfaction variable is influenced by the mni- hannel apability, nline xperience, and alue o- reation variables, then the remaining 51.4% is influenced by other factors outside of this research. Table 3 Final Fornell’s Lacker Criterion Variable Custo- mer Satis- factio n Omni- Channe l Capabi lity Online Experience Value Co- Creatio n Customer Satisfaction 0.847 Omni-Channel Capability 0.597 0.801 Online Experience 0.629 0.742 0.796 Value Co- Creation 0.658 0.696 0.759 0.772 Source: Output data from SmartPLS 4.0 (2022) Table 3 Final Fornell’s Lacker Criterion R-Square value of nline xperience variable is 0.55, it can be that nline xperience variable is influenced by the mni- hannel apability as much as 55%, then the remaining 45% is influenced by other factors outside the variables of this research. The R-Square value of alue o- reation variable is 0.485, it can be said that the alue o- reation variable is influenced by the mni- hannel apability variables is 48.5%, then the remaining 51.5% is influenced by other factors outside the variables of this research. This section contains a description of the obtained data. Data descriptions can be presented in descriptive statistics, frequency distributions accompanied by graphs, mean values, explanation of the results of the research instrument testing. Also, a brief and concise explanation of the development, testing, and results of hypotheses is presented in this section. The explanation is limited to the interpretation of data processing results on statistical figures and the meaning of these results. The discussion section describes whether the hypothesis is supported or rejected by scientific theory and evidence accompanied by logical reasoning. measurement model include construct validity tests (convergent validity and discriminant validity) and composite reliability analysis. The purpose of convergent validity tes is to the validity of relationship between each indicator used in the study and its latent. the table that outer loading value of variable indicator ha met the requirements or is above 0.50, so it can be that each indicator of variable valid. There are two types validity test, namely by at the value of outer loading and the value of Average Variance Extracted (AVE). uter loading value the correlation between indicator and its construct and a low outer loading value indicates that the indicator measurement model does not work. T expected outer loading value is greater than 0.50 and the AVE value must be greater than 0.5. The detail value of outer loading for can be seen in table 1 Table 2 Final Average Variance Extracted Table 2 Final Average Variance Extracted Variable (rho_a) (rho_ ) Customer Satisfacti on 0.803 0.813 0.884 0.717 Omni- Channel Capability 0.721 0.731 0.843 0.642 Online Experienc e 0.917 0.919 0.933 0.634 Value Co- Creation 0.924 0.925 0.936 0.597 Source: Output data from SmartPLS 4.0 (2022) Tabl Final Outer Loadin Indicators Squared Loading CS1 0.892 CS2 0.902 CS3 0.805 OCC1 0.753 OCC2 0.748 OCC3 0.779 OCC4 0.765 OCC5 0.766 OCC6 0.79 OCC7 0.746 OX1 0.822 OX2 0.83 OX3 0.754 OX4 0.797 OX5 0.86 OX6 0.856 OX7 0.857 OX8 0.841 OX9 0.843 VCC1 0.8 VCC2 0.808 VCC3 0.793 Tabl Final Outer Loadin Source: Output data from SmartPLS 4.0 (2022) Based on the table 2 it can be that the inal AVE values mni- hannel apability, ustomer atisfaction, nline xperience, and alue o- reation variable have met the requirements or the AVE value is above 0.50 so it can be said that convergent validity all indicators are valid. After the convergent validity test, then proceed to the next stage which is discriminant validity. Discriminant validity testing can be done by AVE root value on latent variable correlation and cross loading value. Based on the table 2, the value of AVE root in variable column with the variable row of ustomer atisfaction and mni- hannel apability is higher than the value for the variable column with other variable rows. Structural Model Analysis Inner Model testing was conducted to see the relationship between the construct, significance value and R-Square of the research model. Table 4 R-Square Variable R-square R-square adjusted Customer Satisfaction 0.486 0.476 Online Experience 0.55 0.547 Value Co-Creation 0.485 0.482 Source: Output data from SmartPLS 4.0 (2022) H1: 272 Management Department, Faculty of Economics and Business, Universitas Bung Hatta ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) able , by the values in original sample, it shows that there is a positive between omni-channel capability online experience. T-Statistics value is 15.954 and the beta value is (β= 0.742) which means that there a significant effect of omni-channel capability online experience. In addition, the P value of the path coefficient table is 0 which meets the criteria P Value must also be less than 0.05 to accept the hypothesis. the hypothesis is accepted. shows that there is a positiv effect between value co-creation customer satisfaction. The T- Statistics value is 2.974 and the beta value is (β= 0.367) which means that there is a significant and positive effect of value co- creation customer satisfaction. In addition, P value of path coefficient table is 0.003 which meets the criteria that the P Value must also be less than 0.05 to accept the hypothesis. Thus, the hypothesis is accepted. H5: H2: the tructural odel nalysis able by the values in original sample, it that there is a positive effect between omni-channel capability customer satisfaction. The T-Statistics value is 1.841 and the beta value is (β= 0.187) which means that there is an insignificant and positive effect of omni-channel capability customer satisfaction. In addition, the P value of the path coefficient table is 0.066 which the criteria that the P Value must also be less than 0.05 to accept the hypothesis. Thus, the hypothesis is rejected. tructural odel nalysis able , by the values in original sample, it that there is a positive effect between omni-channel capability value co-creation. T- Statistics value is 12.886 and the beta value is (β= 0.742) which means that there is a significant effect of omni-channel capability value co-creation. In addition, P value of the path coefficient table is 0 which meets the criteria P Value must also be less than 0.05 to accept the hypothesis. Thus, the hypothesis is accepted. Management Department, Faculty of Economics and Business, Universitas Bung Hatta Omni-Channel Capability and Customer Online Experience H3: This study found that omn -channe capabilit ha positive and significant effect on online experience. Th result in line with previous research. (Gao et al., 2021) that in an omni-channel context, customer experience congruence has a positive effect on all of variables tested in the research. (Tyrväinen et al., 2020) findings support all the hypotheses confirm the positive relationships between all customer experience components in omni-channel context. , (Yulisa & Permana, 2020) also stated that positive a significant effect on tructural odel nalysis able, by the values in original sample, it shows that there is a positive effect between online experience customer satisfaction. T-Statistics value is 1.783 and the beta value is (β= 0.21) which means that there is an insignificant and positive effect of online experience customer satisfaction. In addition, P value of path coefficient table is 0.075 which meet the criteria P Value must also be less than 0.05 to accept the hypothesis. Thus, the hypothesis is rejected. H4: This result is in accordance with the descriptive analysis of mni-channel apability variable, where customers of ACE Hardware Indonesia are able to use the online channel the tructural odel nalysis able , by values in original sample, it 273 Management Department, Faculty of Economics and Business, Universitas Bung Hatta ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) pleasantly. characteristics of respondents, most respondents are the younger generation who tend to use online shopping and are already used online shopping in their daily activities. They are educated and have no doubt to purchase their goods online through Omni-channel. is that most Indonesians tend to be lazy to understand about procedural things. Customer needs to get taught by other people so they can do and purchase in an online channel. This is also related to the lack of Indonesian people who have a high interest in reading. Omni-channel capability and value co-creation depend on each other so it is important for both variables to have positive and significant relationships. Another explanation of reason why there is a positive and significant relationship between omni-channel capability and online experience here is because there is a need for the online channels to omni-channel. Omni-channel cannot stand alone without online. Omni-Channel Capability and Customer Online Experience Also with omni- channel capability, customers will be able to feel a different experience. Where with a website or mobile application, customers will be able to experience purchasing goods with proper capability in an online channel. Customer Online Experience and Customer Satisfaction This study found that online experience doesn’t have significant effect on customer satisfaction. Despite the difference in conclusion between the hypothesis mentioned and this research finding, there is a number of studies that are in line with this research finding. For example, in Indonesia's context, (Mustikasari & Astuti, 2021) found that the shopping process experience in the context of online channel to customer satisfaction is not significant. stated that the effect of website appearance (one of the online experience items) on customer satisfaction is not significant. Another finding from Toon et al. (2013) that consumer online shopping experience does not have a significant effect on the relationship between customer satisfaction. (Jin & Park, 2006) mentioned that the source of consumer satisfaction remains the same regardless of the purchase experience. Management Department, Faculty of Economics and Business, Universitas Bung Hatta Omni-Channel Capability and Value Co- Creation So when the retailer decided to create this, the impact on customer satisfaction will become not as high as another variable, due to this is not what customers are concerned about the most. Most likely customers are looking for the best price or the best quality of the product, whether it is placed on the online channel or offline channel. contribute to ACE Hardware Indonesia. n the characteristics of respondents, most respondents are female who purchases goods for their home in ACE Hardware Indonesia. Whether it is the offline or online channel, customers still are able to give feedback to the retailer so that customers will be satisfied with the service provided. Another explanation why there is a positive and significant relationship between value co-creation and customer satisfaction here is that most people tend to want to teach other customers because they have a lot of free time to use. This is also related to the characteristics of customers who use the application less than three times. This means that there are still many who do not know and understand this. In addition, Indonesians tend to like to explain how to use websites and applications of omni-channel for those who understand. By maximizing value co- creation, customer satisfaction will increase and this is also important to further improve the quality of the omni-channel capability of ACE Hardware Indonesia. Another explanation is that customer was not really valuing delivery speed for the products they purchase, especially for home living and hobby goods, where Indonesians not valuing productivity and experience as much as people in western countries value them. These two products are the goods customer purchased only several times a year. The experience is not high (mostly 1-3 times in a year for online purchases), and customer needs to feel and touch the direct experience of product quality with their five sense. There is a need for product physical involvement in a retail business. In other words, the result is not increasing significantly, even though the experience is fulfilled. Customers already trust ACE Hardware Indonesia and don't really matter if it is purchased from an online channel or offline channel. Omni-Channel Capability and Value Co- Creation This study found that omni-channel capability has positive and significant effect on value co-creation. The result of this study in line with previous research. stated that online and offline omni channel interactivity and value co-creation positive relationship. (Telli, 2020) identifie the importance of omni- channel shopping services and value co-creation processes. It is also stated by that there are several sources of value co- creation that be omni-channel. This result is in accordance with the descriptive analysis of mni- hannel apability variable, where customers have no hesitation in giving feedback and collaborating with the retailer in order to make ACE Hardware and the online channel better. n the characteristics of respondents, most respondents are young females who tend to critique the retailer and also give an honest review, so that the service will become better. They mostly purchase the home living category in ACE Hardware Indonesia's online channel. This result is in accordance with the descriptive analysis of the nline xperience variable, where customers are most likely not satisfied maximally with the interface and overall experience of the online channel in ACE Hardware Indonesia. On the characteristics of respondents, most respondents use offline channels more than online channels. This might happen due to a lack of high satisfaction with the online channel experience from the customers. One of the explanations regarding the online experience variable is that here, the online experience was created by the retailer only to broaden the variation for customers to experience shopping in the traditional offline experience and Another explanation of reason why there is a positive and significant relationship between omni-channel capability and value co-creation here 274 Management Department, Faculty of Economics and Business, Universitas Bung Hatta ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) an online experience that is newly adopted by ACE Hardware Indonesia. So when the retailer decided to create this, the impact on customer satisfaction will become not as high as another variable, due to this is not what customers are concerned about the most. Most likely customers are looking for the best price or the best quality of the product, whether it is placed on the online channel or offline channel. an online experience that is newly adopted by ACE Hardware Indonesia. Omni-Channel Capability and Customer Satisfaction f This study found that omni-channel capability doesn’t have significant effect on customer satisfaction. However, despite the difference in conclusion between the hypothesis mentioned and this research finding, there is a number of studies that are in line with this research finding. (Sorkun et al., 2020) stated that the direct effect of mni- hannel apability on customer satisfaction is insignificant. Meanwhile, the findings of the research by indicate that Management Department, Faculty of Economics and Business, Universitas Bung Hatta Value Co-Creation and Customer Satisfaction f This study found that value co-creation ha positive and significant effect on customer satisfact . The result in line with previous research findings. Recent research on consumer engagement in value co-creation has that co- creation is a direct predictor of customer satisfaction and loyalty . discovered proof that sharing customer expertise develop solutions and satisfy the customer. According to (Opata et al., 2020), customer engagement in value co-creation has a large and beneficial effect on customer satisfaction. According to , value co- creation a larger effect o satisfaction for both B2B and B2C clients. This result is in accordance with the descriptive analysis of alue o- reation variable, where customers are able to comment, help and 275 Management Department, Faculty of Economics and Business, Universitas Bung Hatta ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) This result is in accordance with the descriptive analysis of the Omni-Channel Capability variable, where customers are most likely not satisfied maximally due to their experience in same-day delivery from online to a physical store and not really able to satisfy customers with their own requirements in ACE Hardware Indonesia. Regarding the characteristics of respondents, most respondents are female and mostly use offline channels more than online channels. This might happen due to a lack of high satisfaction with the same-day delivery experience from the customers. References Aditya Wicaksono Irawan; Aan Yusufianto; Dwi Agustina; Reagan Dean. (2020). Laporan Survei Internet APJII 2019 – 2020. In Asosiasi Penyelenggara Jasa Internet Indonesia (Vol. 2020, pp. 1–146). https://apjii.or.id/survei Research Implications One of the explanations why omni-channel capability is not significant to satisfaction is because their perceived omni-channel capability has exceeded what they expected so that the increase in omni-channel capability at ACE Hardware is no longer able to significantly increase their satisfaction. In other words, respondents' expectations for omni-channel capability are not too high and have been fulfilled by the current performance of omni-channel capability. Based on the research results that have been obtained, there are several implications obtained for in order to Another explanation is that the customer satisfaction of respondents in this study was not directly influenced by online channel capability (online delivery). This means that online delivery attributes such as goods can be ordered online, fast delivery times are not important in determining respondent satisfaction. This may have something to do with the profile of respondents who mostly order home living and hobby items, where the purchases are well planned and usually ordered in advance so that they are not items that are needed urgently. Thus, the speed of delivery does not have a significant impact on their satisfaction. In addition, the quality of ACE Hardware's offline delivery, which has been good so far, may also be one of the reasons why respondents' expectations are not too high on online delivery performance. Management Department, Faculty of Economics and Business, Universitas Bung Hatta Conclusions Based on the results and discussion of this study, the conclusion can be Blom, A., Lange, F., & Hess, R. L. (2021). Omnichannel promotions and their effect on Blom, A., Lange, F., & Hess, R. L. (2021). Omnichannel promotions and their effect on ISSN : 2615-537 (online) Jurnal Manajemen Universitas Bung Hatta Vol.18, No. 02, July 2023 ISSN : 1907-6576 (print) ISSN : 2615-5370 (online) https://doi.org/10.1016/j.tourman.2012.11.00 9 customer satisfaction. European Journal of Marketing, 55(13), 177–201. https://doi.org/10.1108/EJM-12-2018-0866 Galipoğlu, E., Kotzab, H., Teller, C., Yumurtaci, I. O., & Poeppelbuss, J. (2018). Omni-Channel Retailing Research – State of the art and intellectual foundation. International Journal of Physical Distribution & Logistics Management, 48. https://doi.org/10.1108/IJPDLM-10-2016- 0292 Brunero, S., Lamont, S., & Fairbrother, G. (2009). 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https://openalex.org/W3017148605	https://eventos.antac.org.br/index.php/sbtic/article/download/157/152	Portuguese	null	Concepção de projetos hidrossanitários com tecnologia BIM: estudo comparativo ao método conduzido em plataforma CAD	Simpósio Brasileiro de Tecnologia da Informação e Comunicação na Construção	2,019	cc-by	2,797	ABSTRACT The BIM (Building Information Modeling) concept is the digital representation of all elements, containing all the physical and functional characteristics and the graphical and non-graphic representations of a building. On this platform, it is possible to obtain all the information about a building life cycle in a project. The present work aims to analyze the use of BIM technology in the elaboration of projects applied to the hydrosanitary system. One case was analyzed: the transfer of a multi-storey building hydrosanitary project, made in CAD (Computer Aided Design), to the BIM platform. In the end, it was possible to make a comparison between the use of CAD and BIM, propose compatibilization solutions and evaluate the possibilities of this technology application in the elaboration of hydrosanitary projects. Keywords: BIM; CAD; Project; Hydrosanitary; Compatibilization. CONCEPÇÃO DE PROJETOS HIDROSSANITÁRIOS COM TECNOLOGIA BIM: ESTUDO COMPARATIVO AO MÉTODO CONDUZIDO EM PLATAFORMA CAD Design of hydro-sanitary projects using BIM technology: comparative study to the method conducted on CAD platform Matheus Alves Dariva Universidade Federal de Uberlândia \| Uberlândia, MG \| matheusalvesdariva@gmail.com André Luís de Araújo Universidade Federal de Uberlândia \| Uberlândia, MG \| andre.araujo@ufu.br André Luís de Araújo Universidade Federal de Uberlândia \| Uberlândia, MG \| andre.araujo@ufu.br RESUMO O conceito BIM (Building Information Modeling) é a representação digital de todos os elementos, contendo todas as características físicas e funcionais e as representações gráficas e não gráficas de um edifício. Nesta plataforma, é possível obter todas as informações sobre o ciclo de vida de um edifício em um projeto. O presente trabalho tem como objetivo analisar o uso da tecnologia BIM na elaboração de projetos aplicados ao sistema hidrossanitário. Um caso foi analisado: a transferência de um projeto hidrossanitário de um prédio de múltiplos pavimentos, feito em CAD (Computer Aided Design), para a plataforma BIM. No final, foi possível fazer uma comparação entre o uso do CAD e do BIM, propor soluções de compatibilização e avaliar as possibilidades de aplicação desta tecnologia na elaboração de projetos hidrossanitários. Palavras-chave: BIM; CAD; Projeto; Hidrosanitário; Compatibilização. Palavras-chave: BIM; CAD; Projeto; Hidrosanitário; Compatibilização. Palavras-chave: BIM; CAD; Projeto; Hidrosanitário; Compatibilização. 2 METODOLOGIA Primeiramente, efetuou-se uma pesquisa em escritórios de engenharia na cidade de Uberlândia - MG em busca de projetos conduzidos em plataforma CAD. Contatando algumas dessas empresas, foi possível adquirir os projetos arquitetônico e hidrossanitário de um edifício de múltiplos pavimentos realizados no software AutoCAD. Com isso, para o desenvolvimento do presente estudo de caso, foram pré-definidos quatro passos: Estudo do projeto arquitetônico feito em CAD; Remodelagem do projeto arquitetônico na plataforma BIM; Estudo do projeto hidrossanitário feito em CAD; Remodelagem do projeto hidrossanitário na plataforma BIM; 1 INTRODUÇÃO Atualmente, pesquisam-se estratégias de concepções construtivas que apresentem uma aproximação com a realidade dos canteiros de obra, a fim de se evitar problemas construtivos. A metodologia BIM se enquadra nesse cenário como uma possibilidade de inovação tecnológica para projetos de construção civil. De acordo com Eastman (2008), este conceito representa uma filosofia de trabalho que integra arquitetos, engenheiros e construtores (AEC) na elaboração de um modelo virtual preciso, que gera uma base de dados integrando tanto informações topológicas como os subsídios necessários para orçamento, cálculo energético, previsão de insumos e ações em todas as fases da construção. Desenvolvido pela empresa Autodesk, Inc., o Revit é um dos softwares com tecnologia BIM disponível no mercado e inclui recursos para projeto arquitetônico, engenharia de sistemas mecânicos, elétricos e hidráulicos, engenharia estrutural e construção. Ele ainda oferece suporte a um processo de projeto colaborativo e multidisciplinar, permitindo que todos os envolvidos se mantenham atualizados em relação as etapas de um empreendimento (AUTODESK, 2019). DARIVA, M. A.; ARAUJO, A. L. Concepção de projetos hidrossanitários com tecnologia BIM: estudo comparativo ao método conduzido em plataforma CAD. In: SIMPÓSIO BRASILEIRO DE TECNOLOGIA DA INFORMAÇÃO E COMUNICAÇÃO NA CONSTRUÇÃO, 2., 2019, Campinas. Anais [...]. Porto Alegre: ANTAC, 2019. Disponível em: https://www.antaceventos.net.br/index.php/sbtic/sbtic2019/paper/view/131 O projeto hidrossanitário visa planejar a instalação do conjunto de canalizações, aparelhos, peças e acessórios que formam o sistema hidráulico e sanitário de uma edificação. Um projeto elaborado de acordo com a normas técnicas brasileiras garante o funcionamento destas instalações, evitando problemas como falta de pressão, de vazão ou manifestação de mal cheiro proveniente de ralos e afins (ABNT, 1999). Segundo Pacheco (2018), do portal AltoQI, a elaboração de um projeto hidrossanitário pode representar uma redução de 20% do custo total da obra, trazendo também uma economia a longo prazo, pois, pode-se precaver imprevistos gerados pela falta de planejamento. De acordo com um estudo realizado em 2006 pelo Corpo de Bombeiros do estado de Goiás, cerca de 75% das patologias da construção são provenientes de problemas relacionados as instalações de água e esgoto. A aplicação da tecnologia BIM em projetos hidrossanitario ainda não é explorada de forma eficaz no Brasil, mas representa uma alternativa de estudo, viabilidade e planejamento a fim de se garantir a execução das instalações dentro das normas de forma compatível com os demais projetos complementares de uma edificação, evitando os problemas citados (PACHECO, 2018). 2.1 Estudo do projeto arquitetônico Trata-se de uma edificação de doze pavimentos para uso residencial com as seguintes características: Trata-se de uma edificação de doze pavimentos para uso residencial com as seguintes características: Tabela 1: Características da edificação PLANTA ÁREA (M²) Terreno 1.040,45 Térreo Garagem 736,05 945,45 Circulação Vertical + Recepção 73 Estacionamento descoberto 136,4 Pavimento 1 Garagem 314,08 861,41 Circulação + Banhos + DML 79,64 Vagas descobertas 467,69 Pavimentos tipos 2 e 3 767,64 Pavimentos tipos 4 ao 11 3.070,56 Pavimento 12 383,82 Total geral 6.028,88 Total coberto 5.319,79 Total descoberto 709,09 Padrão da obra conforme NBR 12.721/06 - R16 – N Fonte: Autor. Tabela 1: Características da edificação PLANTA ÁREA (M²) Terreno 1.040,45 Térreo Garagem 736,05 945,45 Circulação Vertical + Recepção 73 Estacionamento descoberto 136,4 Pavimento 1 Garagem 314,08 861,41 Circulação + Banhos + DML 79,64 Vagas descobertas 467,69 Pavimentos tipos 2 e 3 767,64 Pavimentos tipos 4 ao 11 3.070,56 Pavimento 12 383,82 Total geral 6.028,88 Total coberto 5.319,79 Total descoberto 709,09 Padrão da obra conforme NBR 12.721/06 - R16 – N Fonte: Autor. 2 2 Figura 1: Vista frontal - Planta baixa de um pavimento tipo - Cobertura Figura 1: Vista frontal - Planta baixa de um pavimento tipo - Cobertura Fonte: Autor. 2.2 Remodelagem do projeto arquitetônico em BIM Primeiramente configurou se as distâncias entre pavimentos nas vistas do Revit tomando como base as Fonte: Autor. Fonte: Autor. Fonte: Autor. 2.2 Remodelagem do projeto arquitetônico em BIM Primeiramente, configurou-se as distâncias entre pavimentos nas vistas do Revit, tomando como base as dimensões do projeto em CAD. Criou-se os níveis de cada planta, transferindo-as do AutoCAD para o Revit e relacionando-as em suas respectivas cotas, servindo assim de base para a remodelagem da arquitetura (paredes, pisos, esquadrias, telhados, etc.). Desse modo, e analisando os cortes, quadros de esquadrias e dimensões do projeto arquitetônico, foi possível construir o modelo virtual do edifício na plataforma BIM. Figura 2: Etapas da remodelagem do projeto arquitetônico no Revit Fonte: Autor. Figura 2: Etapas da remodelagem do projeto arquitetônico no Revit Fonte: Autor. 3 2.3 Estudo do projeto hidrossanitário O projeto hidrossanitário apresenta um conjunto de tubulações, conexões, acessórios e peças que compõem o projeto de água fria, águas pluviais e esgoto, com informações de diâmetro dos tubos, nomenclaturas, singularidades e localização dos elementos hidráulicos. Figura 3: Térreo e Pavimento Tipo 4 ao 11 (Esgoto e Pluvial) – Cobertura e Isométricos (Água Fria) Fonte: Autor. Figura 3: Térreo e Pavimento Tipo 4 ao 11 (Esgoto e Pluvial) – Cobertura e Isométricos (Água Fria) Fonte: Autor. 2.4 Modelagem do projeto hidrossanitário em BIM Lançou-se, primeiramente, as peças hidrossanitárias, em seguida, modelou-se as tubulações de água fria, começando pelo pavimento térreo com a alimentação e subindo para a cobertura. Para o lançamento dos tubos, adotou-se a estratégia de partir dos pontos de utilização com os sub-ramais, liga-los aos ramais, conecta-los as colunas de distribuição e adicionar todos os registros. Com todo o sistema lançado em um pavimento tipo, foi possível copia-lo e cola-lo alinhado aos demais pavimentos tipos, finalizando com a modelagem dos barriletes e do reservatório superior. Atribuiu-se cores para cada tipo de sistema: azul para água fria doméstica, verde para esgoto, cinza para ventilação e magenta para pluvial. Figura 4: Cores atribuídas a cada tipo de sistema e legenda do projeto hidrossanitário Fonte: Autor. Figura 5: Planta baixa do pavimento 03 (banheiros) e cobertura com o lançamento das tubulações de água fria Figura 5: Planta baixa do pavimento 03 (banheiros) e cobertura com o lançamento das tubulações de água fria Fonte: Autor. Fonte: Autor. Após o lançamento do sistema de água fria, iniciou-se o lançamento do sistema de esgoto e ventilação. A concepção partiu-se dos pontos de utilização, modelou-se os ramais de descarga e ramais de esgoto, ligando-os aos tubos de queda, foram lançados os tubos de ventilação primária próximo a cada caixa sifonada de cada banheiro. Posteriormente, todo sistema foi copiado e colado aos demais pavimentos tipos. O lançamento final deu-se no pavimento térreo, com a locação das caixas de inspeção, caixas de gordura e com o lançamento dos subcoletores e do coletor predial. Figura 6: Detalhes da remodelagem do sistema de esgoto Fonte: Autor. Figura 6: Detalhes da remodelagem do sistema de esgoto Fonte: Autor. 5 3 RESULTADOS 3.1 Analise de interferências entre projetos A medida que o projeto hidrossanitário era modelado no Revit, as interferências se tornavam evidentes. Foi possível observar alguns casos de sobreposição dos tubos de esgoto e ventilação com os tubos de água fria nos banheiros. Como no projeto realizado em AutoCAD as disciplinas de água fria e esgoto/pluvial são elaboradas separadamente, há grandes chances de existir incompatibilização entre o sistema. A figura a seguir destaca um dos casos de incompatibilidade encontrados no projeto. Figura 7: Planta baixa de um banheiro: Água Fria (AutoCAD) – Esgoto/Pluvial (AutoCAD) – Sistema completo/interferências (Revit) Fonte: Autor. 3.2 Soluções de compatibilização Utilizando a ferramenta BIM foi possível estudar as interferências para propor as melhores soluções para o sistema. Trabalhando com todo sistema unificado (água fria, ventilação, esgoto e pluvial) em diferentes vistas, foi possível criar uma nova concepção para os casos de sobreposição encontrados no projeto hidrossanitário, chegando a um modelo completo e compatibilizado. o: Água Fria (AutoCAD) – Esgoto/Pluvial (AutoCAD) – Sistema completo/interferências (Revit) uvial (AutoCAD) – Sistema completo/interferências (Revit) g 3.2 Soluções de compatibilização Utilizando a ferramenta BIM foi possível estudar as interferências para propor as melhores soluções para o sistema. Trabalhando com todo sistema unificado (água fria, ventilação, esgoto e pluvial) em diferentes vistas, foi possível criar uma nova concepção para os casos de sobreposição encontrados no projeto hidrossanitário, chegando a um modelo completo e compatibilizado. Utilizando a ferramenta BIM foi possível estudar as interferências para propor as melhores soluções para o sistema. Trabalhando com todo sistema unificado (água fria, ventilação, esgoto e pluvial) em diferentes vistas, foi possível criar uma nova concepção para os casos de sobreposição encontrados no projeto hidrossanitário, chegando a um modelo completo e compatibilizado. Figura 8: Solução para sobreposição de tubos nos banheiros Fonte: Autor. Figura 8: Solução para sobreposição de tubos nos banheiros Fonte: Autor. 6 6 Figura 9: Representação isométricas do sistema hidrossanitário da edificação Fonte: Autor. Figura 9: Representação isométricas do sistema hidrossanitário da edificação Fonte: Autor. 4.1 Concepção de projeto Ao remodelar o projeto hidrossanitário, observou-se a presteza em se desenvolver uma concepção para cada sistema (água fria, esgoto, ventilação e pluvial) de forma unificada dentro da plataforma BIM, essa facilidade se relaciona com o fato de podermos, a qualquer momento, analisar as várias vistas de qualquer área da edificação. Podemos criar cortes, analisar o modelo em 3 dimensões e até estudar uma vista interna da edificação de forma imediata, podendo evitar interferências do projeto à medida em que ele é realizado. Figura 10: Isométrico de todo sistema hidrossanitário de um banheiro Fonte: Autor. Figura 10: Isométrico de todo sistema hidrossanitário de um banheiro Fonte: Autor. Fonte: Autor. 7 Figura 11: Comparação de concepção em CAD (esquerda) e em BIM (direita) Fonte: Autor. Figura 12: Vista interna de um banheiro no Revit Fonte: Autor. análise da representação de um banheiro, enquanto que no CAD temos apenas representações gráficas elementos do projeto, o BIM abrange, além das representações gráficas, as relações espaciais, análises strutivas, geometria dos elementos, informações de materiais e fabricantes. A plataforma ainda esenta a possibilidade de aderir custos aos componentes e planejar cronogramas de obra por meio do delo virtual. Figura 11: Comparação de concepção em CAD (esquerda) e em BIM (direita) Fonte: Autor. Figura 11: Comparação de concepção em CAD (esquerda) e em BIM (direita) Figura 12: Vista interna de um banheiro no Revit Fonte: Autor. Figura 12: Vista interna de um banheiro no Revit Fonte: Autor. Fonte: Autor. Na análise da representação de um banheiro, enquanto que no CAD temos apenas representações gráficas dos elementos do projeto, o BIM abrange, além das representações gráficas, as relações espaciais, análises construtivas, geometria dos elementos, informações de materiais e fabricantes. A plataforma ainda apresenta a possibilidade de aderir custos aos componentes e planejar cronogramas de obra por meio do modelo virtual. 8 8 Figura 13: Representação de um banheiro em CAD (superior) e BIM (inferior) Fonte: Autor. Figura 13: Representação de um banheiro em CAD (superior) e BIM (inferior) Fonte: Autor. 4.3 Integração de todas informações de projeto Como a aplicação do BIM envolve a integração de todas as informações do projeto, qualquer alteração que seja feita em qualquer elemento construtivo, atualiza automaticamente os dados relacionados a ele. Se houver desejo de remodelar algum componente do projeto, isso pode ser feito de forma funcional e prática, podendo todas informações serem analisadas ou modificadas a qualquer momento. Ou seja, se for necessário, por exemplo, realizar uma alteração em uma caixa sifonada (excluir ou trocar por outro modelo), isso também será alterado nas tabelas de informações e em todas as vistas do projeto. Figura 15: Integração de informações de uma caixa sifonada Fonte: Autor. Figura 15: Integração de informações de uma caixa sifonada Figura 15: Integração de in Fonte: Autor. 4.2 Quantitativos de materiais Outro ponto a se destacar são os quantitativos de materiais que são gerados em tempo real, à medida que o projeto é realizado. Essa quantidade é exata e se aproxima do que realmente será utilizado na obra, podendo evitar desperdícios e retrabalhos. Além do quantitativo, existe a possibilidade de atribuir várias informações associadas aos elementos do projeto, como os fabricantes, links de onde adquirir o produto, preço, material, dimensões, catálogos técnicos, manuais técnicos, guias de execução, etc. Figura 14: Comparação entre tabelas de quantitativos em CAD (esquerda) e em BIM (direita) Fonte: Autor Figura 14: Comparação entre tabelas de quantitativos em CAD (esquerda) e em BIM (direita) Fonte: Autor. 9 5 CONCLUSÃO A conclusão do estudo comparativo do presente trabalho foi reproduzida por meio da Tabela 2. A conclusão do estudo comparativo do presente trabalho foi reproduzida por meio da Tabela 2. p p p p Tabela 2: Conclusão do estudo comparativo entre as plataformas CAD e BIM para a concepção de projetos hidrossanitários CRITÉRIOS\PLATAFORMAS CAD BIM Concepção de projeto Unifilar. Tridimensional. Nível de complexibilidade Baixo. Alto. Tratamento de informações Manual. Automático (desde que sejam criados parâmetros para os elementos). Possibilidades Representação gráfica; Integração com diversas plataformas. Representação gráfica; Relações espaciais; Análises construtivas; Informações de materiais; Fabricantes; Custos; Planejamento e cronogramas de obra; Geração de manuais de manutenção e segurança. Elementos construtivos Representados por elementos gráficos: Linhas, arcos, polígonos, círculos, etc. Representados por componentes paramétricos: Paredes, portas, tubos, conexões, peças hidrossanitárias, etc. Fluxo de trabalho São criados e editados desenhos individuais de forma independente, ou seja, cada elemento é único. Qualquer alteração reflete apenas no elemento modificado. Os elementos do projeto são unificados e integrados em todo modelo virtual, ou seja, as alterações são refletidas em todas as vistas. Os itens conectados e tabelas são atualizadas de forma automática. Flexibilidade Alta flexibilidade: Os comandos podem ser executados de forma superficial a qualquer momento. Baixa flexibilidade: Os elementos devem respeitar os parâmetros estabelecidos para eles, sendo impedidos de serem concebidos de forma incorreta. Fonte: Autor. 10 REFERÊNCIAS ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS-ABNT. NBR 10844: Instalações prediais de águas pluviais. Rio de Janeiro, 1989. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS-ABNT. NBR 5626: Instalação predial de água fria. Rio de Janeiro, 1999. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS-ABNT. NBR 8160: Sistemas prediais de esgoto sanitário- Projeto e execução. Rio de Janeiro, 1999. AUTODESK. Revit - Desenvolvido para Modelagem de Informação da Construção. [S. l.], 2019. Disponível em: <https://www.autodesk.com.br/products/revit/overview>. Acesso em: 1 fev. 2019. EASTMAN, C.; TEICHOLZ, P.; SACKS, R.; LISTON, K. Manual de Bim: Um guia de modelagem da informação da construção para arquitetos, engenheiros, gerentes, construtores e incorporadores. Porto Alegre: Bookman, 2014, 483 p. PACHECO, Adriano. 5 motivos que tornam o projeto hidráulico indispensável. 2018. Disponível em: <http://maisengenharia.altoqi.com.br/hidrossanitario/5-motivos-que-tornam-o-projeto-hidraulico-indispensavel/>. Acesso em: 25 jan. 2019.. 11 11
https://openalex.org/W3047852347	https://escholarship.org/content/qt1j0760d6/qt1j0760d6.pdf?t=rq4dfr	English	null	Open questions on the chemical composition of airborne particles	Communications chemistry	2,020	cc-by	4,166	UC Irvine Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ UC Irvine UC Irvine Previously Published Works Title Open questions on the chemical composition of airborne particles Permalink https://escholarship.org/uc/item/1j0760d6 Journal Communications Chemistry, 3(1) ISSN 2399-3669 Authors Finlayson-Pitts, Barbara J Wingen, Lisa M Perraud, Véronique et al. Publication Date 2020 DOI 10.1038/s42004-020-00347-4 Copyright Information This work is made available under the terms of a Creative Commons Attribution License availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Open questions on the chemical composition of airborne particles Permalink https://escholarship.org/uc/item/1j0760d6 Journal Communications Chemistry, 3(1) ISSN 2399-3669 Authors Finlayson-Pitts, Barbara J Wingen, Lisa M Perraud, Véronique et al. Publication Date 2020 DOI 10.1038/s42004-020-00347-4 Copyright Information This work is made available under the terms of a Creative Commons Attribution License availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Open questions on the chemical composition of airborne particles Permalink https://escholarship.org/uc/item/1j0760d6 Journal Communications Chemistry, 3(1) ISSN 2399-3669 Authors Finlayson-Pitts, Barbara J Wingen, Lisa M Perraud, Véronique et al. Publication Date 2020 DOI 10.1038/s42004-020-00347-4 Copyright Information This work is made available under the terms of a Creative Commo availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Open questions on the chemical composition of airborne particles Barbara J. Finlayson-Pitts 1✉, Lisa M. Wingen 1, Véronique Perraud 1 & Michael J Ezell 1 Barbara J. Finlayson-Pitts 1✉, Lisa M. Wingen 1, Véronique Perraud 1 & Michael J. Ezell 1 Michael J. Ezell 1 Airborne particles have signiﬁcant impacts on health, visibility, and climate. Here, an overview of what is known about particle chemical composition is presented, along with open questions and challenges that are central to relating composition to life cycles and impacts. Airborne particles have been known for at least 800 years to negatively impact visibility and health. For example, Moses Ben Maimonides (1135–1204) described air in Cairo as “stagnant, turbid, thick, misty, and foggy”, and attributed “dullness of understanding, failure of intelligence, and defect of memory” to “spoilage of the air”. A recent estimate is that exposure to outdoor particulate matter (PM) currently leads to 3.3 million premature deaths per year worldwide, and this could double by the year 20501. It is known that these particles impact climate as well2. The chemical composition of the particles is an important determinant of all of these atmospheric impacts. Hence, understanding the composition and processes affecting composition is critical for developing cost-effective control strategies. p g g A ﬁrst step is to distinguish “primary particles”, those that are directly emitted from natural and anthropogenic sources such as sea spray, transportation, industry, forest ﬁres, etc. from “secondary particles”, those formed from chemical reactions of gaseous precursors in air. The latter include both inorganics such as sulfuric acid/sulfate from SO2 oxidation and nitrate from NOx oxidation, as well as a host of volatile organics, which make up a major fraction of the particles around the world3. Because there are thousands of potential organic precursors in air, and a number of different oxidation reactions involving O3 and OH, Cl, and NO3 radicals (Fig. 1), the organic component of particles becomes very complex4. Further adding to the complexity of this secondary organic aerosol (SOA) are reactions in the condensed phase that can also form new products after the particle has formed5. Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org COMMUNICATIONS CHEMISTRY \| (2020)3:108 \| https://doi.org/10.1038/s42004-020-00347-4 \| www.nature.com/commschem 1 Department of Chemistry, University of California, Irvine, CA 92697-2025, USA. ✉email: bjﬁnlay@uci.edu Composition, size, and phase p p A critical property of atmospheric particles is their size. Particles must grow from sub-nm seeds formed by the gas phase chemistry to ~100 nm in order to scatter light efﬁciently. This reduces visibility and affects climate both directly through light scattering and indirectly through their impacts on clouds. This ~100 nm size also efﬁciently reaches the alveolar region of the lung. partment of Chemistry, University of California, Irvine, CA 92697-2025, USA. ✉email: bjﬁnlay@uci.edu 1 COMMENT COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-020-00347-4 Fig. 1 Schematic of the life cycle of secondary organic aerosol particles and their impacts. Volatile organic precursors are oxidized in air by O3, OH, NO3, or Cl, and the resulting highly oxygenated, low-volatility products formed from these reactions nucleate new particles, and/or condense on pre-existing ones to grow them. Different species participate in particle growth at different stages of the life cycle, with less oxygenated, more volatile species condensing on the larger particles. Chemical composition of these SOA particles will depend on the environmental conditions surrounding the particles (relative humidity, temperature, gas phase composition …etc.), as well as their physical properties (i.e., viscosity) governing the uptake of trace gases. Once they reach ~50–100 nm, these particles are able to impact climate directly by interacting with incoming solar radiation but also indirectly via cloud processing. The chemical composition and size of these particles will also determine their impact on health. Fig. 1 Schematic of the life cycle of secondary organic aerosol particles and their impacts. Volatile organic precursors are oxidized in air by O3, OH, NO3, or Cl, and the resulting highly oxygenated, low-volatility products formed from these reactions nucleate new particles, and/or condense on pre-existing ones to grow them. Different species participate in particle growth at different stages of the life cycle, with less oxygenated, more volatile species condensing on the larger particles. Chemical composition of these SOA particles will depend on the environmental conditions surrounding the particles (relative humidity, temperature, gas phase composition …etc.), as well as their physical properties (i.e., viscosity) governing the uptake of trace gases. Once they reach ~50–100 nm, these particles are able to impact climate directly by interacting with incoming solar radiation but also indirectly via cloud processing. The chemical composition and size of these particles will also determine their impact on health. that a complete molecular identiﬁcation of all components has not been achieved. Composition, size, and phase There is no one technique that provides a comprehensive analysis of all inorganic and organic species, and most approaches have been targeted to subsets of the components in limited size ranges. Elemental analysis shows C, H, O, N, S as well as trace metals are very common components, and it is known that sulfate, nitrate, and ammonium ions as well as organics are ubiquitous in particles3. The organic component of primary particles tends to be less oxidized (e.g., polycyclic aro- matic hydrocarbons in soot) and can contain unique compounds that serve as tracers of speciﬁc sources. The organic component of SOA is even more complex, including many different oxygenated species (e.g., acids, alcohols, aldehydes and ketones, esters, per- oxides, aldols, hemiacetals, and acetals) and nitrogen- and sulfur- containing organics (e.g., alkyl- or heterocyclic amines such as imidazoles, organonitrates, organosulfate esters). A growing number of measurements of SOA show that highly oxidized and high molecular weight oligomeric compounds with larger O:C ratios are relatively more important in smaller particles, whereas growth to larger diameters involves less oxidized, lower molecular weight species4. Most importantly, the chemical composition of particles changes during the life cycle of particle formation and growth, reﬂecting changes in the processes that occur at each stage6. For example, the O:C ratio of particles increases as reactions with atmospheric oxidants such as OH and O3 occur4. The composition also changes due to photolysis, hydrolysis and condensed-phase reactions that can, for example, form new light-absorbing spe- cies such as brown carbon7,8. Measuring particle composition from the nm to micron scale is thus important for understanding particle nucleation, growth, physicochemical transformations and impacts (Fig. 1). It had been assumed for many years that secondary organic particles were low-viscosity liquids, so that exchange with the gas phase and diffusion inside the particles (which facilitates gas uptake and in-particle chemistry) was relatively rapid. However, over the past decade, it has been demonstrated that under many conditions, particles are high viscosity solids or semi-solids9. In that case, uptake from the gas phase by diffusion into the particle following adsorption at the surface is slow, changing the mechanism of gas uptake that grows the particle and decreasing opportunity for in-particle chemistry10. In addition, the relative importance of uptake of gases into the particles may be different for high viscosity versus liquid particles, impacting the particle chemical composition. Composition, size, and phase Both the phase/viscosity of particles and the chemical composition are very sensitive to the environmental conditions, including temperature, relative humidity, and the gas phase composition during particle formation, particularly the concentrations of NOx. Approaches to determine the chemical composition of particles Historically, and continuing to this day, particles have been collected using ﬁlters or impactors and the bulk samples col- lected in this manner have typically been extracted into a sol- vent and analyzed using chromatographic, spectroscopic, and mass spectrometric methods11,12. While very useful, the results are dependent on a number of factors such as the nature of the extraction solvent. Those components that are most soluble in the chosen solvent will be over-represented in the analysis and Overview of chemical composition of particles Given the plethora of different compounds in particles, and particularly the organic component, it is perhaps not surprising COMMUNICATIONS CHEMISTRY \| (2020)3:108 \| https://doi.org/10.1038/s42004-020-00347-4 \| www.nature.com/commschem Overview of chemical composition of particles Given the plethora of different compounds in particles, and particularly the organic component, it is perhaps not surprising MMUNICATIONS CHEMISTRY \| (2020)3:108 \| https://doi.org/10.1038/s42004-020-00347-4 \| www.nature.com/commschem 2 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-020-00347-4 COMMENT Fig. 2 Probing the chemical composition of particles as a function of diameter. a Size-resolved bulk chemical composition of nanoparticles formed in a ﬂow reactor from the acid:base initiated reaction between gas phase methanesulfonic acid (MSA; 2.3 × 1011 molecules cm−3) and methylamine (MA; 1.4 × 1011 molecules cm−3) under dry conditions. Particles were sampled at 0.6 s and 9 s residence time, respectively, using a thermal desorption chemical ionization mass spectrometer13; b size distributions of monodisperse 220 nm glutaric acid particles coated with malonic acid with coating thickness shown on top axis and c ratio of malonic acid (coating) signal to glutaric acid (core) signal detected for the particles in b using sonic spray ionization mass spectrometry16. Fig. 2 Probing the chemical composition of particles as a function of diameter. a Size-resolved bulk chemical composition of nanoparticles formed in a ﬂow reactor from the acid:base initiated reaction between gas phase methanesulfonic acid (MSA; 2.3 × 1011 molecules cm−3) and methylamine (MA; 1.4 × 1011 molecules cm−3) under dry conditions. Particles were sampled at 0.6 s and 9 s residence time, respectively, using a thermal desorption chemical ionization mass spectrometer13; b size distributions of monodisperse 220 nm glutaric acid particles coated with malonic acid with coating thickness shown on top axis and c ratio of malonic acid (coating) signal to glutaric acid (core) signal detected for the particles in b using sonic spray ionization mass spectrometry16. be very challenging. In this regard, chemical ionization approa- ches have proven powerful for sensitive and molecular detection of speciﬁc classes of compounds7,12. Commonly used ionization reagents include protons in the positive ion mode and O2−, iodide, nitrate, and CF3O−ions in the negative ion mode. Some more exotic reagents such as N-methyl-2-pyrrolidone have been recently used for measurement of species that can be difﬁcult to detect such as ammonia in nanoparticles13. The use of elemental analysis with high-resolution techniques and factor analysis of mass spectra has notably increased our understanding of particle sources, level of oxidation, and the ubiquity of organic aerosol in air3. for some solvents such as methanol, there is a possibility of reactions (e.g., esteriﬁcation of acids) occurring during extrac- tion. Overview of chemical composition of particles Filtration methods tend to be biased toward larger parti- cles that dominate the sample by mass. There is also the possibility of reactions of the particles on the ﬁlter during collection, particularly if oxidants are not removed from the air prior to ﬁltration. p This led to the development of a number of on-the-ﬂy mass spectrometric approaches for analysis of bulk composition of airborne particles or single particle analysis without the need for prior collection7,8,12. Mass spectrometry (MS) is a very powerful technique for providing both elemental information and in the case of high resolution MS, molecular formulae of complex, higher molecular mass organics. All of the on-the-ﬂy approaches involve a number of steps: (1) sampling the particles in such a way that there is sufﬁcient mass for detection; (2) transferring the particle components from the condensed into the gas phase; (3) ionizing the particle components; and (4) characterizing the ions using MS. The ﬁrst step, concentrating the particles from air into a beam, is commonly accomplished using aerodynamic lenses. The second step typically involves either laser ablation or heating. In the case of laser ablation, lasers in the UV have most com- monly been used, which efﬁciently vaporize the particles, including any metallic components and simultaneously causes ionization of the vaporized components, providing elemental composition. Alternatively, infrared lasers can be used for vaporization, followed by UV radiation for ionization. The high energies involved with UV laser ionization tend to cause extensive fragmentation of organics, with a few exceptions such as the polycyclic aromatic hydrocarbons. On the other hand, vaporiza- tion by heating is not effective for most metals but does preserve more of the molecular structures of organics with the exception of decomposition of thermally sensitive molecules. A relatively new approach uses ambient ionization, which combines the transfer to the gas phase and ionization by inter- secting a particle ﬂow with a stream of charged droplets or a metastable plasma12,14. The former can be generated using dif- ferent techniques such as electrospray or sonic spray. These techniques have the advantages of providing molecular infor- mation for organics, but sensitivity for the small mass of ambient levels of SOA is a challenge15,16. COMMUNICATIONS CHEMISTRY \| (2020)3:108 \| https://doi.org/10.1038/s42004-020-00347-4 \| www.nature.com/commschem References Mass spectrometry of atmospheric aerosols - recent developments and applications. Part II: on-line mass spectrometry techniques. Mass Spectrom. Rev. 31, 17–48 (2012). q p 9. Reid, J. P. et al. The viscosity of atmospherically relevant organic particles. Nat. Commun. 9, 956 (2018). 10. Shiraiwa, M., Zuend, A., Bertram, A. K. & Seinfeld, J. H. Gas-particle partitioning of atmospheric aerosols: interplay of physical state, non-ideal mixing and morphology. Phys. Chem. Chem. Phys. 15, 11441–11453 (2013). 11. Heard, D. Analytical Techniques for Atmospheric Measurements. (Wiley- Blackwell, 2006). 12. Johnston, M. V. & Kerecman, D. E. Molecular characterization of atmospheric organic aerosol by mass spectrometry. Annu. Rev. Anal. Chem. 12, 247–274 (2019). 13. Perraud, V., Li, X., Jiang, J., Finlayson-Pitts, B. J. & Smith, J. N. Size-resolved chemical composition of sub-20 nm particles from methanesulfonic acid reactions with methylamine and ammonia. ACS Earth Space Chem. https:// doi.org/10.1021/acsearthspacechem.0c00120 (2020). doi.org/10.1021/acsearthspacechem.0c00120 (2020). 14. Domin, M. & Cody, R. Ambient Ionization Mass Spectrometry. (Royal Society of Chemistry, 2015). MS techniques that can examine the molecular surface com- position of such particles versus their bulk composition are on the rise and will help us to understand differences in composition between high viscosity and liquid particles. An example of one approach is shown in Fig. 2b, c, where laboratory-generated model glutaric acid particles were coated with increasing thick- nesses of malonic acid and the ratio of coating to core signals measured using sonic spray ionization MS. As the coating thickness increases, less of the core is detected relative to the coating, with the probe depth estimated to be 2–4 nm16. 15. Kumbhani, S. et al. New mechanism of extractive electrospray ionization mass spectrometry for heterogeneous solid particles. Anal. Chem. 90, 2055–2062 (2018). 16. Wingen, L. M. & Finlayson-Pitts, B. J. Probing surfaces of atmospherically relevant organic particles by easy ambient sonic-spray ionization mass spectrometry (EASI-MS). Chem. Sci. 10, 884–897 (2019). 17. Thompson, J. E. Airborne particulate matter human exposure and health effects. J. Occup. Environ. Med. 60, 392–423 (2018). 18. Bzdek, B. R. & Johnston, M. V. New particle formation and growth in the troposphere. Anal. Chem. 82, 7871–7878 (2010). 19. Riemer, N., Ault, A. P., West, M., Craig, R. L. & Curtis, J. H. Aerosol mixing state: measurements, modeling, and impacts. Rev. Geophys. 57, 187–249 (2019). Just as major progress has been made over the last decades we can expect signiﬁcant progress in the coming years. References 1. Lelieveld, J., Evans, J. S., Fnais, M., Giannadaki, D. & Pozzer, A. The contribution of outdoor air pollution sources to premature mortality on a global scale. Nature 525, 367–374 (2015). g 2. IPCC. Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. 151 (Geneva, Switzerland, 2014). 3. Jimenez, J. L. et al. Evolution of organic aerosols in the atmosphere. Science 326, 1525–1529 (2009). 4. Akimoto, H. & Hirokawa, J. Atmospheric Multiphase Chemistry: Fundamentals of Secondary Aerosol Formation. (Wiley-Blackwell, 202 4. Akimoto, H. & Hirokawa, J. Atmospheric Multiphase Chemistry: Fundamentals of Secondary Aerosol Formation (Wiley Blackwell 5. McNeill, V. F. Aqueous organic chemistry in the atmosphere: Sources and chemical processing of organic aerosols. Environ. Sci. Technol. 49, 1237–1244 (2015). y y g g A third challenge is analyzing particles not only as a function of size, but also as a function of their 3-D structure. If they are liquid and homogeneous, rapid diffusion results in well-mixed particles. However, this is not always likely to be the case. For example, liquid-liquid phase separation has been documented in both laboratory studies and ﬁeld samples19 where aqueous and organic phases are immiscible. In addition, the mechanisms by which high viscosity solid and semi-solid particles grow will not be primarily by quasi-equilibrium uptake from the gas phase as is the case for liquid particles where solubility is a determining factor. In the high viscosity case, growth will depend on the interaction of the gas with the surface of the particle, and whether the attractive forces are strong enough to bind the gas to the surface for sufﬁcient time to become incorporated into the par- ticle20. The factors determining growth in this case are different from those for liquid particles, with the nature and composition of the particle surface driving uptake. This may lead to a com- position that differs from that expected based on solubility con- siderations alone, but little is known regarding such potential differences. 6. Zhang, R. Y., Khalizov, A., Wang, L., Hu, M. & Xu, W. Nucleation and growth of nanoparticles in the atmosphere. Chem. Rev. 112, 1957–2011 (2012). 7. Laskin, J., Laskin, A. & Nizkorodov, S. A. Mass spectrometry analysis in atmospheric chemistry. Anal. Chem. 90, 166–189 (2018). 8. Pratt, K. A. & Prather, K. A. References As has often been the case, the development of new analytical techniques that are sufﬁciently gentle and provide accurate molecular informa- tion will largely drive new understanding. In this vein, having techniques that can peel particles layer by layer “like an onion” could be quite valuable. It is sometimes the case that doing the measurement actually changes the sample during analysis, a sort of “atmospheric uncertainty principle.” Techniques that do not alter the sample composition during analysis will thus be important. Minimally destructive techniques will also help in tracing which products are formed by chemistry within the par- ticle versus uptake from the gas phase. Finally, development and application of quantitative methods that cover the entire periodic table in an integrated approach over a wide size range is the 20. Vander Wall, A. C., Perraud, V., Wingen, L. M. & Finlayson-Pitts, B. J. Evidence for a kinetically controlled burying mechanism for growth of high viscosity secondary organic aerosol. Environ. Sci. Process. Impacts 22, 66–83 (2020). Key open questions and challenges Received: 18 June 2020; Accepted: 2 July 2020; Received: 18 June 2020; Accepted: 2 July 2020; Received: 18 June 2020; Accepted: 2 July 2020; Another challenge is being able to measure all particle com- ponents, both inorganic (including metals) and organic simulta- neously, with identiﬁcation of speciﬁc organic molecules. The SOA portion in particular is known to contain many compounds that are fragile with respect to the vaporization and ionization processes used to detect them. Although elemental information such as O:C and H:C ratios can be obtained using a number of techniques, identifying speciﬁc compounds or even all of the functional groups remains difﬁcult. For example, peroxides and hydroperoxides, as well as higher molecular mass compounds representing the reaction products of the backbones of two or more of the parent species (often referred to as dimers or oli- gomers) are commonly found in SOA7,12. Transferring such compounds into the gas phase without causing decomposition or secondary chemistry is a signiﬁcant challenge. Acknowledgements g We are grateful to the National Science Foundation (Grants #1647386, 1916993, 1928252, and 1707883) and the Army Research Ofﬁce (Grant # W911NF1710105) for support of our work. We are grateful to the National Science Foundation (Grants #1647386, 1916993, 1928252, and 1707883) and the Army Research Ofﬁce (Grant # W911NF1710105) for support of our work. Key open questions and challenges Despite the great strides we have made in elucidating key species responsible for particle formation and growth over the past sev- eral decades, there are many open questions and challenges remaining regarding the inorganic and the molecular organic composition of particles as a function of the nature of precursors and of the formation, growth, and aging processes that occur in air. These are driven by the need to address the impacts that particles have on health, visibility, and climate. For example, there is increasing evidence that ultraﬁne particles <100 nm in size may have disproportionate health effects17 that are not captured in their contribution to the total mass. The mass of such small particles is exceptionally low, making detection a major challenge, especially for measurements made on-the-ﬂy. At present, air quality standards are currently set for the mass of particles <2.5 μm in diameter (PM2.5) and those up to 10 μm (PM10). In addition to laser ionization, there are a number of less- energetic approaches to ionization. Traditional electron impact ionization has the advantage that there is a wealth of data on the fragmentation patterns of many different potential particle components, and it can be less fragmenting/damaging than some UV ionization techniques. With the complex mixtures found in SOA there are so many different contributions to the mass spectrum that identifying parent compounds (those from the sampled particles that are ionized but have not fragmented) can However, measuring the composition of these smaller (<100 nm) but more numerous particles remains largely an open issue6,18. Thus, although it is known that acid-base reactions play a signiﬁcant role in new particle formation, this may not occur in a simple 1:1 acid:base stoichiometric fashion. For example, Fig. 2a COMMUNICATIONS CHEMISTRY \| (2020)3:108 \| https://doi.org/10.1038/s42004-020-00347-4 \| www.nature.com/commschem 3 COMMENT COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-020-00347-4 ultimate goal that provides an enormous challenge for the coming decade. However, it is the essential basis of quantitative models that can provide a predictive capability for assessing particle impacts and developing effective control strategies. shows the acid:base molar ratio for nanoparticles formed from the gas phase reaction of methanesulfonic acid with methylamine, where the smallest particles (<9 nm) contain more acid than base13. The capability of measuring chemical composition down to ~1 nm is crucial for understanding how stable clusters form and subsequently grow to detectable particles. p g The authors declare no competing interests. p g The authors declare no competing interests. COMMENT COMMENT COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-020-00347-4 © The Author(s) 2020 Author contributions B.J.F.P., L.M.W., V.P., and M.J.E. all contributed to the preparation of this manuscript. COMMUNICATIONS CHEMISTRY \| (2020)3:108 \| https://doi.org/10.1038/s42004-020-00347-4 \| www.nature.com/commschem 4 Competing interests 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/. Additional information Correspondence and requests for materials should be addressed to B.J.F.-P. Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. COMMUNICATIONS CHEMISTRY \| (2020)3:108 \| https://doi.org/10.1038/s42004-020-00347-4 \| www.nature.com/commschem 5 Reproduced with permission of copyright owner. Further reproduction prohibited without permission. Reproduced with permission of copyright owner. Further reproduction prohibited without permission. Reproduced with permission of copyright owner. Further reproduction prohibited without permission.
https://openalex.org/W2125318483	https://bmcprimcare.biomedcentral.com/counter/pdf/10.1186/1471-2296-13-42	English	null	Quality of life among caregivers of patients with schizophrenia: a cross-cultural comparison of Chilean and French families	BMC family practice	2,012	cc-by	4,986	* Correspondence: laurent.boyer@ap-hm.fr 1Aix-Marseille University, EA 3279 Research Unit, 27 Boulevard Jean Moulin, Marseille 13284, France Full list of author information is available at the end of the article © 2012 Boyer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Boyer et al. BMC Family Practice 2012, 13:42 Boyer et al. BMC Family Practice 2012, 13:42 Boyer et al. BMC Family Practice 2012, 13:42 http://www.biomedcentral.com/1471-2296/13/42 Open Access Abstract Background: To our knowledge, no study has examined quality of life (QoL) among caregivers of individuals with schizophrenia between a developing and a developed country. The aim of this study was to assess QoL of the caregivers of individuals with schizophrenia in two countries characterized by different social, economic and cultural conditions, namely Chile and France. Methods: Data were collected from public mental health outpatient services in Arica (Chile), and in Marseille (France). QoL was measured with the short-form health survey scale - 36 items (SF36). QoL of 41 Chilean caregivers was firstly compared with 245 French caregivers. Univariate and multivariate analyses using linear regression were then performed to determine variables potentially related to QoL scores. Results: The caregivers were primarily mothers in the two groups, but Chilean caregivers were younger, and lived more frequently with the individual with schizophrenia than French caregivers. The SF36 scores were globally low in the two groups, especially on the mental QoL scores. Chilean caregivers reported lower physical SF36 scores than French caregivers. In the multivariate analysis, being mother and Chilean caregivers were the most regular features associating to a lower QoL. Conclusion: Despite differences between Chile and France, especially in terms of quality and quantity of mental health services and economic supports, caregivers’ QoL levels remain particularly low for both countries. Future support programmes should address the specific needs of caregivers. Keywords: Quality of life, Caregivers, Cross culture, Family support, Schizophrenia Quality of life among caregivers of patients with schizophrenia: a cross-cultural comparison of Chilean and French families Laurent Boyer1, Alejandra Caqueo-Urízar2, Raphaelle Richieri1, Christophe Lancon1, José Gutiérrez-Maldonado3 and Pascal Auquier1 Participants The inclusion criteria for the caregiver were as follows: (1) having a family member with a diagnosis of schizo- phrenia according to the DSM-IV criteria [18]; (2) being identified by the individual with schizophrenia as the main caregiver; (4) being 18 years of age or older; and (5) giving informed consent to participate in the study. In Chile, the study was approved centrally by the Ethics Committee of the University of Tarapacá and subse- quently by each participating mental health services. In France, the university research team received an agree- ment from the French Institute for Public Health Research with support from the Ministries of Health and Research to conduct QoL researches. This study was conducted in accordance with the Declaration of Helsinki and French Good Clinical Practices [19,20]. Data collection The aim of this study was thus to assess QoL of the caregivers of individuals with schizophrenia in two coun- tries characterized by different social, economic and cul- tural conditions, namely Chile and France, using the short-form health survey scale - 36 items (SF36). The data collected included the following: The data collected included the following: 1. Socio-demographic characteristics of the caregivers: Gender, age, and employment status. 2. Socio-demographic characteristics of the individuals with schizophrenia: Gender, and age. Background depressive symptoms [1,10]. Moreover, caregivers’ nega- tive experience may affect their ability to care for the patients [11,12]. Research on caregivers’ QoL is thus of importance both for the caregivers themselves and indir- ectly for patients’ health. Schizophrenia is a disabling and severe psychiatric dis- order which is a major cause of suffering for patients. Schizophrenia also affects functioning and health of family caregivers, mainly because the caregivers have assumed functions that were performed in the past by psychiatric institutions [1-5]. Caregivers supply the patient with care and support [1,2]. The impact of caregiving on caregivers’ quality of life (QoL) is substantial [3-7], especially when experiencing a significant burden [8,9], restricted roles and activities, and increased psychosomatic, anxious, or Caregivers of individuals with schizophrenia have received significant attention in the past few years. Previ- ous studies mainly documented association of burden with socio-demographic characteristics, coping and so- cial support of caregivers, and severity of symptoms and disabilities of patients [2,5,13-16]. More rarely, social, economic and cultural factors were studied although there is some evidence that these factors have a potential influence on caregiving consequences. Cross-cultural or cross-national comparisons of caregivers would help to Correspondence: laurent.boyer@ap-hm.fr 1Aix-Marseille University, EA 3279 Research Unit, 27 Boulevard Jean Moulin, Marseille 13284, France Full list of author information is available at the end of the article Boyer et al. BMC Family Practice 2012, 13:42 http://www.biomedcentral.com/1471-2296/13/42 Page 2 of 6 Page 2 of 6 interview. In Chile, 45 caregivers of patients with schizo- phrenia were initially approached and 41 agreed (accept- ance rate = 91%). In France, 265 caregivers were approached and 245 agreed to participate (acceptance rate = 93%). understand these factors, but only few studies to date have been performed, in particular in developed coun- tries [14,15,17]. Moreover, these studies were focused on caregivers’ burden. In this area, there is an interest in studying caregivers’ QoL from different social, economic and cultural contexts. Method Settings h d p , g 3. QoL questionnaire: the Short Form 36 (SF36) was used to assess QoL [21]. SF36 is a generic, self- administered, and worldwide-used questionnaire consisting of 36 items describing 8 dimensions: Physical Functioning (PF), Social Functioning (SF), Role-Physical Problems (RPP), Role-Emotional Problems (REP), Mental Health (MH), Vitality (VIT), Bodily Pain (BP), and General Health (GH). Each dimension is scored within a range of 0 (low QoL level) to 100 (high QoL level). Two component summary measures of SF36, namely physical and mental composite scores (PCS-SF36 and MCS-SF36) can be calculated. The use of SF-36 in caregivers of individuals with mental health problems was recommended in a recent review of the available instruments [22]. Moreover, the SF-36 allows comparisons between French and Chilean populations, cross-cultural validation showing that the Chilean and French versions of the SF-36 have the same subscales and structure as the original SF-36 and have demonstrated adequate psychometric properties. 3. QoL questionnaire: the Short Form 36 (SF36) was used to assess QoL [21]. SF36 is a generic, self- administered, and worldwide-used questionnaire consisting of 36 items describing 8 dimensions: Physical Functioning (PF), Social Functioning (SF), Role-Physical Problems (RPP), Role-Emotional Problems (REP), Mental Health (MH), Vitality (VIT), Bodily Pain (BP), and General Health (GH). Each dimension is scored within a range of 0 (low QoL level) to 100 (high QoL level). Two component summary measures of SF36, namely physical and mental composite scores (PCS-SF36 and MCS-SF36) can be calculated. The use of SF-36 in caregivers of individuals with mental health problems was recommended in a recent review of the available instruments [22]. Moreover, the SF-36 allows comparisons between French and Chilean populations, cross-cultural validation showing that the Chilean and French versions of the SF-36 have the same subscales and structure as the original SF-36 and have demonstrated adequate psychometric properties. The study was conducted in public mental health out- patient services in Arica (Chile), and in Marseille (France). Arica is a city in northern Chile, with a popula- tion of slightly under 195,000 inhabitants. Marseille is located in the south-eastern part of France with a popu- lation of nearly one million inhabitants. Chile and French Heath systems are described in Additional file 1. Statistical analysis Data were expressed in proportion or mean and stand- ard deviation. Characteristics of Chilean and French caregivers were compared using Chi-squared or Fisher exact tests for categorical variables and Student or Wilcoxon rank sum test for continuous variables. Procedure In Chile as in France, personnel from each public mental health outpatient service identified consecutive patients who had been given a diagnosis of schizophrenia and were between 18 and 64 years old on a period of one month. Each patient was asked by medical or nursing staff to name his or her main caregiver, i.e. the person who spends the most time supporting and taking care of the patient. We asked the patient if we could contact the caregiver. When the patient agreed and when the care- giver met the inclusion criteria, clinical psychologist explained the aim of the evaluation and invited care- givers to take part. After providing consent to participate in the study, caregivers were assessed individually in a private booth via a self-report questionnaire and an Associations between QoL scores and continuous vari- ables (caregiver: age; patient: age) or categorical variables (caregiver: relationship, living situation, employment sta- tus; patient: gender; country) were analyzed using Pear- son’s correlation and Chi-squared tests. Multivariate analyses using linear regression were performed to de- termine variables potentially related to QoL scores. The final models expressed the standardised beta coefficient. All the tests were two-sided. Statistical significance was defined as p < 0.05. Statistical analysis was performed using the SPSS version 17.0 software package (SPSS Inc, Chicago, IL, USA). Boyer et al. BMC Family Practice 2012, 13:42 http://www.biomedcentral.com/1471-2296/13/42 Boyer et al. BMC Family Practice 2012, 13:42 http://www.biomedcentral.com/1471-2296/13/42 Page 3 of 6 Page 3 of 6 Table 1 Characteristics of Chilean and French caregivers and individuals with schizophrenia Chilean (N = 41) French (N = 245) p-value N (%) N (%) 1. Caregivers Relationship (mother) 26 (63.4) 165 (67.1) 0.646 Age (years), M(SD) } 54.3 (15.1) 60.6 (9.5) 0.013 Living situation (with individual with schizophrenia) 37 (90.2) 156 (64.7) 0.001 Employment status (employed) 18 (43.9) 92 (37.4) 0.428 SF36+, M(SD) Physical functioning 63.4 (24.9) 78.3 (23.4) <0.001 Social functioning 55.8 (22.7) 62.5 (23.9) 0.095 Role physical 45.1 (40.4) 55.9 (40.1) 0.114 Role emotional 43.1 (38.9) 47.7 (41.2) 0.503 Mental health 44.5 (22.0) 51.4 (18.7) 0.034 Vitality 38.4 (17.7) 46.4 (19.7) 0.015 Bodily pain 51.0 (20.3) 57.2 (25.0) 0.082 General health 49.4 (13.4) 55.5 (21.6) 0.018 Mental composite score 35.5 (11.4) 37.4 (10.5) 0.287 Physical composite score 42.8 (8.2) 46.9 (10.0) 0.013 2. Comparisons of characteristics and SF36 scores between Chilean and French caregivers Comparisons of characteristics and SF36 scores between Chilean and French caregivers Comparisons of characteristics and SF36 scores between Chilean and French caregivers Table 1 presents the characteristics and SF36 scores of Chilean (N = 41) and French (N = 245) caregivers. The caregivers were primarily mothers, 63.4% for Chilean caregivers and 67.1% for French caregivers (p = 0. 646). Chilean caregivers were younger, and lived more fre- quently in the same home with the individual with schizophrenia than French caregivers (respectively p = 0.013, and p = 0.001). A majority of caregivers was unemployed (61.5%), but there was no statistically differ- ence between the two groups (p = 0.428). The SF36 scores were globally low in the two groups, especially on the mental scores. Whatever the country, caregivers reported a lower mental QoL level (MCS < 40) than physical QoL level (PCS > 40). There was no statistical significant difference in SF36 mental dimen- sion scores between the two groups, except for MH di- mension which was lower in Chilean caregivers. On the contrary, Chilean caregivers reported lower physical SF36 scores than French caregivers (PF, VIT, GH, and PCS). Procedure Individuals with schizophrenia Gender (male) 26 (63.4) 166 (67.8) 0.584 Age (years), M(SD) 33.2 (8.4) 31.7 (9.1) 0.315 } M+/−SD: mean +/−standard deviation. + range [0–100], 0 lowest and 100 highest level of QoL. Table 1 Characteristics of Chilean and French caregivers and individuals with schizophrenia Factors associated to caregivers’ QoL of individuals with BD and MD The results of the univariate and multivariate analyses are presented in Tables 2 and 3. In the multivariate ana- lysis, the relationship with the individual with schizo- phrenia and the country were the most regular features associating to QoL. Being mother was significantly asso- ciated to a lower QoL on all the SF36 dimensions and composite scores, and Chilean caregivers presented lower QoL level for 6 dimensions (PF, SF, RPP, MH, VIT) and PCS. In a lesser extent, living with the patient and being unemployed were associated to lower QoL levels (respectively RPP, REP and MCS; and BP, PCS). On the contrary, no relationship was found with socio- demographic characteristics of individuals with schizophrenia. psychiatric institutions [11]. However, the French health system has not sufficiently taking into account this change in the development of health programs and pol- icies. Hospital-based care has still an overwhelming im- portance, and community services are not sufficiently developed [23]. In Chile, although a communitarian ap- proach was implemented over the last decade [24], pub- lic health services approximately cover 80% of the population, psychiatric services are poorly staffed [25], and as a consequence care for patients with schizophre- nia is mainly taken by the caregivers [13]. Discussion To our knowledge, this is the first study to investigate QoL among caregivers of individuals with schizophrenia between a developing and a developed country. There are several important findings in our study. Second, Chilean caregivers reported lower physical SF36 scores than French caregivers. This difference may be explained by a better access to and higher availability of health and economic resources for care- givers in France [26]. In Chile, economic burden may be playing an important role in caregivers’ QoL. Lack of psychiatrist, day hospitals, access to drug treatments, among others, could generate a considerable concern in these relatives [2,25]. Beyond these explanations, it is important to note QoL levels remain low for the physical dimensions in France. First, caregivers of individuals with schizophrenia experienced particularly low QoL levels in Chile and France, across multiple domains of QoL including phys- ical and psychological dimensions. This finding confirms previous studies on the significant impact of being a caregiver, in particular in individuals with mental disor- ders [3]. In France as in other western countries, family has taken functions which were performed in the past by Boyer et al. BMC Family Practice 2012, 13:42 http://www.biomedcentral.com/1471-2296/13/42 Page 4 of 6 Table 2 Factors associated with SF36 scores: univariate analyses PFM (SD)} or R# SF RPP REP MH VIT BP GH PCS MCS 1. Caregiver Relationship Mother 73.1(24.8)* 57.3(23.7)** 48.5(40.1)* 42.9(39.5)* 47.5(18.9) 42.4(19.2) 53.4(24.7)* 52.5(20.6)* 45.2(9.9)* 35.6(10.2)* Other 82.1(21.5) 70.1(21.7) 65.8(38.1) 55.3(42.5) 56.1(18.9) 51.0(19.3) 62.3(23.0) 58.7(20.5) 48.5(9.4) 40.2(10.9) Age −0.22** −0.05 −0.13* −0.06 0.04 −0.02 −0.15* −0.15* −0.23** 0.05 Living situation with 74.5(24.8) 60.0(24.6) 50.3(40.5)* 44.2(41.0) 48.7(19.2)* 43.6(19.1)* 55.6(25.3) 53.8(21.3) 45.8(10.1) 36.3(10.4)* without 79.9(21.7) 65.5(22.1) 63.5(38.8) 54.0(39.8) 54.0(19.5) 48.9(20.3) 58.3(23.0) 56.5(19.1) 47.5(9.1) 39.2(11.0) Employment status Yes 82.9(19.2)** 65.0(23.0) 62.2(37.7)* 53.1(40.5) 50.3(18.0) 47.1(18.4) 62.8(23.3) 60.0(19.1) 49.6(8.4) 37.4 (10.5) No 71.9(25.8) 59.4(24.1) 49.4(41.1) 43.3(40.7) 50.4(20.1) 44.2(20.3) 52.4(24.3) 51.3(21.0) 44.3(10.1) 37.0(10.8) 2. Individual with schizophrenia Gender Male 77.0(23.1) 60.9(23.3) 51.8(40.8) 44.4(41.5) 49.0(19.1) 44.7(19.3) 56.7(24.3) 54.0(19.6) 46.6(9.5) 36.3(10.6) Female 74.0(26.1) 62.9(24.8) 58.9(38.8) 51.8(39.1) 52.8(19.5) 46.2(20.1) 55.2(24.7) 55.5(22.7) 45.6(10.6) 38.9(10.5) Age −0.21 −0.05 −0.12 −0.08 −0.07 −0.07 −0.22** −0.18* −0.23 0.02 3. Country Chile 63.4(24.9) 55.8(22.7) 45.1(40.4) 43.1(38.9) 44.5(22.0)* 38.4(17.7)* 51.0(20.3) 49.4(13.4)* 42.8(8.2)* 35.5(11.4) France 78.2(23.4) 62.5(23.9) 55.9(40.1) 47.7(41.2) 51.4(18.7) 46.4(19.7) 57.2(25.0) 55.4(21.6) 46.9(9.1) 37.4(10.5) SF-36—PF: Physical Functioning; SF: Social Functioning; RPP: Role—Physical Problems; REP: Role—Emotional Problems; MH: Mental Health; VIT: Vitality; BP: Bodily Pain; GH: General Health; PCS: Physical Composite Score; MCS: Mental Composite Score. Discussion } M+/−SD: mean +/−standard deviation; # R : Spearman’s correlation coefficient;* p ≤0.05; ** p ≤0.01. Third, caregivers’ QoL did not statistically differ be- tween Chile and France for mental and psychological QoL dimensions. This finding may appear paradoxical because Chile is characterized by a lower-quantity or quality mental health system than in France. We can hypothesize that emotional and psychological impact is more related to distressing notions such as shame, em- barrassment, stigma, rather than burden of care related to household such as taking care of daily tasks. This finding suggests that family interventions should focus on general coping strategies, on the improvement of the social network, on stigma reduction, and on the develop- ment of personal strength. Finally, consistent with prior research [3], the current analysis also find that mothers were generally the pri- mary caregivers and that they reported a lower QoL than other types of informal caregivers. Mothers may experience a higher burden because they are respon- sible for most aspects of the patients’ daily care [13]. Working life was significantly associated with better QoL in our study. For Magliano and colleagues [27], professional and social network supports represent cru- cial resources for reducing family burden in schizophre- nia. Lastly, the living situation of caregivers and their patients was also an important factor associated with QoL, and our results support the fact that opportunities Table 3 Factors associated with SF36 scores: multivariate analyses PFß# SF RPP REP MH VIT BP GH PCS MCS 1. Caregiver Relationship Mother/Other −0.20* −0.26** −0.21* −0.17* −0.23* −0.21* −0.20* −0.16* −0.17* −0.22** Age −0.23* −0.11 −0.15 −0.04 −0.02 −0.04 −0.03 −0.04 −0.15 0.05 Living situation With/without −0.08 −0.11 −0.19* −0.15* −0.11 −0.11 −0.08 −0.07 −0.08 −0.14* Employment status Yes/No 0.12 0.09 0.12 0.11 0.02 0.06 0.15* 0.14 0.18* 0.05 2. Individual with schizophrenia Gender Male/Female −0.03 0.04 0.10 0.07 0.08 0.05 −0.02 0.05 0.01 0.10 Age −0.02 0.01 −0.03 −0.06 −0.01 −0.02 −0.14 −0.10 −0.06 −0.08 3. Country Chile/France −0.27** −0.14* −0.11* −0.04 −0.13* −0.16* −0.09 −0.11 −0.18* −0.07 SF-36—PF: Physical Functioning; SF: Social Functioning; RPP: Role—Physical Problems; REP: Role—Emotional Problems; MH: Mental Health; VIT: Vitality; BP: Bodily Pain; GH: General Health; PCS: Physical Composite Score; MCS: Mental Composite Score. References 1. Awad AG, Voruganti LN: The burden of schizophrenia on caregivers: a review. Pharmacoeconomics 2008, 26:149–162. 2. Caqueo-Urizar A, Gutierrez-Maldonado J: Burden of care in families of patients with schizophrenia. Qual Life Res 2006, 15:719–724. 3. Caqueo-Urizar A, Gutierrez-Maldonado J, Miranda-Castillo C: Quality of life in caregivers of patients with schizophrenia: a literature review. Health Qual Life Outcomes 2009, 7:84. Discussion # ß: standardised beta coefficient (ß represents the change of the standard deviation in QoL score resulting from a change of one standard deviation in the independent variable); * p ≤0.05; ** p ≤0.01. Table 3 Factors associated with SF36 scores: multivariate analyses PFß# SF RPP REP MH SF-36—PF: Physical Functioning; SF: Social Functioning; RPP: Role—Physical Problems; REP: Role—Emotional Problems; MH: Mental Health; VIT: Vitality; BP: Bodily Pain; GH: General Health; PCS: Physical Composite Score; MCS: Mental Composite Score. # ß: standardised beta coefficient (ß represents the change of the standard deviation in QoL score resulting from a change of one standard deviation in the independent variable); * p ≤0.05; ** p ≤0.01. Page 5 of 6 Boyer et al. BMC Family Practice 2012, 13:42 http://www.biomedcentral.com/1471-2296/13/42 for residential care should in principal increase family QoL [15]. for psychiatric patients, a communitarian approach was implemented over the last decade. Patients may benefit from short-stay in public hospitals in the acute phase, and outpatient care especially by monthly delivering medication by nurses. Care for these patients is mainly taken by their relatives, also known as informal caregivers, who are generally women. However, there is no systematic support for families who rather turn to informal support networks such as church or self-help groups. The French health system combines universal coverage with a public– private mix of hospital and ambulatory care and a higher volume of service provision than in Chile. French public mental health services are organised in “sectors”, each sector catering for a mean population of 54,000 inhabitants. This organisation was aimed at insuring equal access to care whatever the place of residence. In France as in other western countries, psychiatric institutions began discharging mentally ill patients into the community and family has taken functions which were performed in the past by psychiatric institutions. Families of patients suffering from schizophrenia, especially mothers, were more and more involved in the therapeutic process. However, the French health system has not sufficiently taking into account this change in the development of health programs and policies. Hospital-based care has an overwhelming importance, and community services are not sufficiently developed. Author details 1 1Aix-Marseille University, EA 3279 Research Unit, 27 Boulevard Jean Moulin, Marseille 13284, France. 2Departamento de Filosofía y Psicología, Universidad de Tarapacá, 18 de Septiembre # 2222, Arica, Chile. 3Departamento de Personalidad, Evaluación y Tratamiento Psicológico, Universidad de Barcelona, Paseo Valle de Hebrón, Barcelona 08035, Spain. Competing interest h h h d The Authors have declared that there are no conflicts of interest in relation to the subject of this study. The Authors have declared that there are no conflicts of interest in relation to the subject of this study. Role of funding source In France: none. In France: none. In Chile, this research was funded by the Universidad de Tarapacá through Proyecto de Investigación Ciencia y Tecnología 3731–12. Received: 26 October 2011 Accepted: 28 May 2012 Published: 28 May 2012 Received: 26 October 2011 Accepted: 28 May 2012 Published: 28 May 2012 Acknowledgements h k ll f h Our thanks to all of the caregivers, patients and staff who helped with the study, and also to the Convenio de Desempeño Universidad de Tarapacá- Mineduc. Limitations and perspectives Some limitations have to be carefully considered. The sample may not be representative of the entire popula- tion of caregivers, especially in Chile. Confirmation is therefore needed on more diverse and large groups of patients. Moreover, although our models account for a large set of potentially relevant variables, other factors might have increased their explanatory power, such as patients’ characteristics (illness duration, symptom severity. . .). These factors should be studied in future studies. Finally, our study concerned a generic type of QoL instrument (SF-36). Although SF-36 has good psy- chometric properties among caregivers of people with mental disorders [22] and is suitable for use across caregivers of individuals with a broad range of mental health problems [28], an important limitation is its relevance when applied to a specific population. Future studies should be proposed using specific question- naires for caregivers of individuals with schizophrenia [6,29]. Finally, our findings should also be confirmed on objective aspects of caregiving, defined as any dis- turbance in the family environment due to the patient's disorder and which is potentially verifiable and observ- able. This analysis could fruitfully compare the findings between the uses of “subjective” or “objective” assess- ment instruments. overwhelming importance, and community services are not sufficiently developed. Author’s contribution Both authors contributed equally to this work. All authors read and approved the final manuscript. Despite differences between Chile and France, especially in terms of quality and quantity of mental health ser- vices and economic supports, caregivers’ QoL levels re- main particularly low for both countries. Our findings suggest that the specific needs of caregivers are not met, whatever the level of performance of the health care sys- tems. Our findings emphasize the need for service provi- ders and policy makers to engage families and service users in policy, planning and practice. There is a failure to use effective clinical approaches in working with fam- ilies as well as failures in communication in most ser- vices worldwide. Future support programmes should address the specific needs of caregivers. Additional file 4. Lua PL, Bakar ZA: Health-related quality of life profiles among family caregivers of patients with schizophrenia. Fam Community Health 2011, 34:331–339. Additional file 1: Chile’s health system is composed of a public health insurance (FONASA) covering about 69 percent of the population, and a private insurance plans (ISAPREs) covering 17 percent of the population. The remaining population is affiliated with other public agencies (such as Military Health Services) or is without coverage. Public health insurance (FONASA and other public agencies) and public health services approximately cover 80% of the population, and guaranty a relatively equitable access to health care. Concerning care 5. Möller-Leimkühler AM, Wiesheu A: Caregiver burden in chronic mental illness: the role of patient and caregiver characteristics. Eur Arch Psychiatry Clin Neurosci 2012, 262(2):157–166. 6. Richieri R, Boyer L, Reine G, Loundou A, Auquier P, Lancon C, Simeoni MC: The Schizophrenia Caregiver Quality of Life questionnaire (S-CGQoL): development and validation of an instrument to measure quality of life of caregivers of individuals with schizophrenia. Schizophr Res 2011, 126:192–201. Boyer et al. BMC Family Practice 2012, 13:42 http://www.biomedcentral.com/1471-2296/13/42 Boyer et al. BMC Family Practice 2012, 13:42 http://www.biomedcentral.com/1471-2296/13/42 Page 6 of 6 7. Zamzam R, Midin M, Hooi LS, Yi EJ, Ahmad SN, Azman SF, Borhanudin MS, Radzi RS: Schizophrenia in Malaysian families: a study on factors associated with quality of life of primary family caregivers. Int J Ment Health Syst 2011, 5:16. 29. Richieri R, Boyer L, Reine G, Loundou AD, Simeoni MC, Auquier P, Lancon C: A preliminary validation of a new French instrument to assess quality of life for caregivers of patients suffering from schizophrenia. Encéphale 2011, 37:425–432. 8. Glozman JM: Quality of life of caregivers. Neuropsychol Rev 2004, 14:183–196. doi:10.1186/1471-2296-13-42 Cite this article as: Boyer et al.: Quality of life among caregivers of patients with schizophrenia: a cross-cultural comparison of Chilean and French families. BMC Family Practice 2012 13:42. 9. Li J, Lambert CE, Lambert VA: Predictors of family caregivers' burden and quality of life when providing care for a family member with schizophrenia in the People's Republic of China. Nurs Health Sci 2007, 9:192–198. 10. Schulz R, Beach SR: Caregiving as a risk factor for mortality: the Caregiver Health Effects Study. JAMA 1999, 282:2215–2219. 11. Reine G, Lancon C, Simeoni MC, Duplan S, Auquier P: Caregiver burden in relatives of persons with schizophrenia: an overview of measure instruments. Encéphale 2003, 29:137–147. 12. Additional file Velligan DI, Weiden PJ, Sajatovic M, Scott J, Carpenter D, Ross R, Docherty JP: The expert consensus guideline series: adherence problems in patients with serious and persistent mental illness. J Clin Psychiatry 2009, 70(Suppl 4):1–46. quiz 47-8. Review. 13. Gutierrez-Maldonado J, Caqueo-Urizar A, Kavanagh DJ: Burden of care and general health in families of patients with schizophrenia. 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Ware JE Jr, Sherbourne CD: The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care 1992, 30:473–483. 22. Harvey K, Catty J, Langman A, Winfield H, Clement S, Burns E, White S, Burns T: A review of instruments developed to measure outcomes for carers of people with mental health problems. Acta Psychiatr Scand 2008, 117:164–176. 23. Verdoux H: The current state of adult mental health care in France. Eur Arch Psychiatry Clin Neurosci 2007, 257:64–70. 24. Boyer et al. BMC Family Practice 2012, 13:42 http://www.biomedcentral.com/1471-2296/13/42 Additional file Minoletti A, Zaccaria A: The national mental health plan in chile: 10 years of experience. Rev Panam Salud Publica 2005, 18:346–358. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit of experience. 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https://openalex.org/W3048695777	https://www.econjournals.com/index.php/ijeep/article/download/7774/5279/24718	English	null	FOREIGN DIRECT INVESTMENT, ELECTRICITY POWER SUPPLY AND ECONOMIC GROWTH IN NIGERIA	International journal of energy economics and policy	2,020	cc-by	4,113	Accepted: 05 February 2020 Received: 01 March 2019 ABSTRACT This study examined the stimulation of foreign direct investment (FDI) inflows through constant electricity power supply for economic growth in Nigeria, by engaging time series data sourced from the world development indicators (WDI) for the period 1986-2017 and employed the Autoregressive distribution lag econometric approach to co-integration. The gross domestic product growth rate per capita was the proxy for economic growth and the dependent variable, while the independent variables include FDI, labour force participation rate, gross fixed capital formation and electricity power supply. The result from the study showed that, in the long-run, increased FDI inflows, gross fixed capital formation, electricity power supply, have the potency of increasing economic growth by 30%, 20% and 6%, respectively. Therefore, based on the results obtained, the study recommended that there should be constant electricity power supply to keep pace with productivity for efficient economic growth in Nigeria. Keywords: Economic Growth, Electricity Power, Foreign Direct Investment JEL Classifications: O47, L94, H54 Keywords: Economic Growth, Electricity Power, Foreign Direct Investment JEL Classifications: O47, L94, H54 Serifat O. Onayemi1, Philip A. Olomola2, Philip O. Alege3, Oluwakemi O. Onayemi4* 1,3,4Covenant University, Ota, Nigeria, 2Obafemi Awolowo University, Ile-Ife, Nigeria. Email: oluwakemionayemi@yahoo.c DOI: https://doi.org/10.32479/ijeep.7774 DOI: https://doi.org/10.32479/ijeep.7774 This Journal is licensed under a Creative Commons Attribution 4.0 International License Foreign Direct Investment, Electricity Power Supply and Economic Growth in Nigeria Serifat O. Onayemi1, Philip A. Olomola2, Philip O. Alege3, Oluwakemi O. Onayemi4 1,3,4Covenant University, Ota, Nigeria, 2Obafemi Awolowo University, Ile-Ife, Nigeria. Email: oluwakemionayemi@yahoo.com 2. LITERATURE REVIEW Various studies have been conducted on the role of FDI on economic growth. Alam (2013) examined the comparative relationship between electricity power consumption, FDI and economic growth in India and Pakistan by employing co- integration and error correction methods of analyses in data running from1975-2008. In both countries, electricity consumption and FDI boost economic growth. Makiela and Quattara (2018) explored the transmission channels from FDI to growth on a sample of developed and developing countries from 1970-2007. The result showed that FDI affects growth through inputs accumulation and not through total factor productivity. This implies that factors other than FDI may cause increase in productivity in developing countries. Matthew et al. (2018) examined the nexus between institutions and economic growth by finding out the effect of human capital development, through the usage of electricity power for maximum productivity, in enhancing growth in Nigeria. The study found out that human capital development is insignificantly related to economic growth in Nigeria, while electricity consumption is significantly linked with economic growth. They argued that though institutional framework is good in stimulating FDI, foreign investors would like to invest in a country with constant supply of electricity power since the cost of generating power is high in developing countries The study implies that electricity power supply is statically significant in determining how effective human capital development is, in attaining economic growth in Nigeria. Alege and Ogundipe (2014) in their study employed the system generalised method of moments (SGMM) econometric approach to examine the rate at which national framework of Nigeria influences economic growth. The study found out that institutional factors such as political stability and absence of violence affect FDI inflow. Investors need a peaceful and feasible environment. Consequently, where there is absence of peace, investors will find it difficult to establish their businesses there. Ibrahiem (2015) investigated the effects of renewable electricity consumption and FDI on economic growth on a time series data from 1980-2011 in Egypt by employing Auto Regressive Distributed Lag (ARDL) bound testing approach. The result showed that renewable electricity consumption and FDI have a long run positive effect on economic growth. 1. INTRODUCTION The result showed that electricity consumption and FDI have positive impacts on economic growth in Vietnam, both in the short and long run. literature was reviewed while in section three we discussed the methodology. In section four we presented results obtained in the study while section five was the conclusion. Long et al. (2018) investigated the causal relationship between electricity consumption, FDI and economic growth in Vietnam during the period 1990-2015, by using Toda Yamamoto approach to causality and autoregressive distributive lag approach. The result showed that electricity consumption and FDI have positive impacts on economic growth in Vietnam, both in the short and long run. 2. LITERATURE REVIEW It should be noted that literature on the electricity consumption- FDI-economic growth nexus is scarce in Nigeria, therefore, this study is set out to contribute to knowledge by filling this gap; thus, policy to turn around the situation towards attaining the desired level of economic growth in Nigeria was recommended in the study. Alvarado and Ponce (2017) investigated the effect of FDI on economic growth in 19 Latin American countries, by employing panel data econometrics. The study found out that on the aggregate, the effect of FDI on economic growth is not significant. However, when the levels of development reached by the countries in the region were incorporated, a positive and significant relationship was observed between FDI and economic growth in high-income countries but uneven and non-significant result was obtained in upper-middle-income countries, however, the effect in lower-middle income countries was negative and statistically significant. 1. INTRODUCTION Asian tigers. This implies that, electricity consumption in the Asian regions accelerated the industrial development of China and other Asian countries. This is one of the reasons why FDI inflows have increased substantially in recent years across various countries of the world. Foreign direct investment (FDI) simply means the flow of capital between different countries of the world. This is referred to as the investment which is engaged towards acquiring lasting interest in organisations that operate outside the home country of the investor’s economy. There has been an argument as to the role of FDI in achieving economic growth in various studies in economics and other fields. In theory, FDI involves technology transfers, human capital development and generation of employment, resulting in economic growth in the host economy, as seen in Latin America (Alvarado and Ponce, 2017). Various approaches have been engaged to encourage FDI inflows in different regions of the world. Such approaches include regular supply of electricity which will enhance productivity to stimulate economic growth (Matthew et al., 2018). Other studies such as Sunde (2017) observed that in Asia, especially in China, the invention and application of the electric power technology promoted economic growth of the Irrespective of the various studies which have been conducted to examine the relationship between economic growth and FDI, conclusion on the subject matter has not been achieved. Among the studies reviewed, irrespective of the fact that it is understood that FDI increases economic growth, some have argued that institution is not a factor to enhance FDI inflows, others have argued that electricity supply is a major determinant of FDI inflow. Very few studies have investigated the impact of electricity supply on FDI towards attaining economic growth in Nigeria. This study is motivated to fill this gap, add to the frontiers of knowledge and to the extant literature. The study is structured into five sections. Section one is the introduction, while in section two, relevant This Journal is licensed under a Creative Commons Attribution 4.0 International License Onayemi, et al.: Foreign Direct Investment, Electricity Power Supply and Economic Growth in Nigeria Long et al. (2018) investigated the causal relationship between electricity consumption, FDI and economic growth in Vietnam during the period 1990-2015, by using Toda Yamamoto approach to causality and autoregressive distributive lag approach. 4.1. Trend Analysis The trend analysis as presented in Figure 1 shows the movement and fluctuations of the selected variables: gross domestic product growth rate per capita, FDIs, labour force participation rate and gross fixed capital formation. (3) From Figure 1 which shows the trend of the variables, it was observed that variables did not show a consistent trend from 1986 to 2016. The growth rate of the gross domestic product per capital was between −12% and 10% from 1986 to 2002 but increased to 30% between 2002 and 2004. However, it decreased to 0% in 2005. Similarly, other variables show inconsistencies in their trends from 1986 to 2016 as shown in Figure 1. Where: Δ is the change in operator and the ECMt–1 represents the correction mechanism of the white noise. γ means the speed of adjustment. The hypothesis is stated thus:s H0: β0 = β1 = β2 = β3 = β4 (No long run relationship exist) H : β ≠β ≠β ≠β ≠β (Long run relationship exist) H0: β0 = β1 = β2 = β3 = β4 (No long run relationship exist) H1: β0 ≠ β1 ≠ β2 ≠ β3 ≠ β4 (Long run relationship exist) H0: β0 = β1 = β2 = β3 = β4 (No long run relationship exist) H1: β0 ≠ β1 ≠ β2 ≠ β3 ≠ β4 (Long run relationship exist) 3. METHODOLOGY The model specification provides a general overview of the basic variables utilised during the analysis. Therefore, the baseline mode for the model is specified in its implicit form as presented in equation (1) GDPPCGRt= f (FDIt), GFCFt, LABFt, ELECTt) (1) (1) Sunde (2017) employed ARDL bound testing approach to co- integration to investigate the long run relationship between economic growth, FDI and exports in South Africa. Also, error correction model was used to examine the short run dynamics, while the VECM Granger causality approach was used to investigate the direction of causality. The result showed that both FDI and exports spur economic growth. The granger causality showed a unidirectional causality between economic growth and FDI, running from FDI to economic growth. Given the implicit form of the model as specified in equation (1), it can be predicted that the nexus existing among the exogenous and the endogenous variables are in their linear forms as explicitly specified in equation (2), thus: Given the implicit form of the model as specified in equation (1), it can be predicted that the nexus existing among the exogenous and the endogenous variables are in their linear forms as explicitly specified in equation (2), thus: GDPPCGRt =β0 + β1FDIt + β1FDIt+β2GFCFt + β3LABit + β4ELECTt + µit (2) (2) From above; GDPPCGR means gross domestic product per capita growth rate, FDI means FDI, GFCF means gross fixed capital formation, LAB means labour force participation rate and ELECT means electricity power. β0 is the constant term, β1 β2, β3, β4, are the coefficients of the explanatory variables, t represent time, while ε represents the error term. Zhang et al. (2017) examined the effect of electricity consumption on economic growth in China. The study found out that electricity provides the sustainable power for economic and social development because increase in electricity consumption promoted the progress of industrial economy in China. International Journal of Energy Economics and Policy \| Vol 10 • Issue 5 • 2020 Onayemi, et al.: Foreign Direct Investment, Electricity Power Supply and Economic Growth in Nigeria Onayemi, et al.: Foreign Direct Investment, Electricity Power Supply and Economic Growth in Nigeria 4. RESULTS The method employed for the analysis is the Autoregressive Distribution Lag (ARDL) approach to co-integration, which aimed at examining how FDI will be stimulated through constant electricity power supply towards achieving economic growth in Nigeria. Thus, the model which was adopted from the empirical work of Osabuhien, Osabuhien and Urhie (2018) is specified as This section of the study presents the results obtained from the analysis. The results presented in this study are of two folds: First, the trend analysis which shows the rate at which the selected variables in the study behave over the period of study. The second aspect of the result is the result obtained from ARDL econometric approach. 0 1 1 2 1 1 0 3 1 4 1 0 0 1 1        − − = = − = = = − − Δ = + Δ + Δ + Δ + Δ + + ∑ ∑ ∑ ∑ n n t t t t t n n t t t t t t GDPGRPC FDI GFCF LABF ELECT ECM (3) 4.2. Econometric Resultsi Similarly, Figure 1: Trend analysis of variables International Journal of Energy Economics and Policy \| Vol 10 • Issue 5 • 2020 245 Source: Authors compilation using data from world development indicators (2017) 53.5 54 54.5 55 55.5 56 56.5 57 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Labour Participatation Rate -20 -10 0 10 20 30 40 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Gross Domestic Product per capita 0 2 4 6 8 10 12 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Foreign Direct Investment 0 10 20 30 40 50 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Gross Fixed Capittal Formation Figure 1: Trend analysis of variables -20 -10 0 10 20 30 40 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Gross Domestic Product per capita 0 2 4 6 8 10 12 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Foreign Direct Investment Gross Fixed Capittal Formation 0 10 20 30 40 50 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Gross Fixed Capittal Formation 53.5 54 54.5 55 55.5 56 56.5 57 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 Labour Participatation Rate Labour Participatation Rate Source: Authors compilation using data from world development indicators (2017) Onayemi, et al.: Foreign Direct Investment, Electricity Power Supply and Economic Growth in Nigeria Onayemi, et al.: Foreign Direct Investment, Electricity Power Supply and Economic Growth in Nigeria FDI has a mean value of 7.6, standard deviation of 3226, with minimum and maximum of 33.19 and 86.28 respectively. Labour force participation rate has a mean value of 40.08, with the standard deviation of 18.88, minimum and maximum values of 2.5464 and 4.8765 respectively, while. Electricity power supply has a mean value of 4277, standard deviation of 94674.0 and minimum and maximum values of 2821 and 5991, respectively. significant. In the short run, FDI inflows increases gross domestic product by 3%, but in the long-run, 30%, at both lag 1 and lag 2. 4.2. Econometric Resultsi The implication of this is that increased inflow of FDI has the potency of increasing the level of growth in the economy. Labour force is also statistically significant in explaining the level of economic growth. In the long-run, labour force participation rate increases economic growth by 20%. The implication of this result is that, increased workforce through employment will invariably enhance the level of growth in the economy. In this regard, effort should be made to increase work force in order to enhance productivity in the Nigerian economic growth process. In line with the empirical study of Matthew et al. (2018), which was conducted to examine the effect of electricity supply on economic growth through the development of human capital, the study found that increased supply of electricity power enhances economic growth, this is validated in this study as seen the result obtained from the autoregressive distribution lag which shows that increase in the level of supply of electricity power will enhance economic growth. From the result, 1% increase in the supply of To ensure that the estimates are not spurious and that the results are BLUE- Best Linear Unbiased Estimators, the unit root test of stationarity was conducted on the selected variables using the ADF and the Philip-Peron (PP) criterion as presented in Table 2. From the result obtained, it was observed that the variables trend difference. That is, they have different order of integration. Given the above, the result from the Autoregressive distribution lag (ARDL) is presented in Table 3. From the ARLD result, it shows that FDI, labour force, gross fixed capital formation and electricity power supply are statistically Table 1: Summary statistics of variables Variable Mean Standard deviation Minimum Maximum Gross domestic product per capita 0.9076 7.3173 −15.4547 20.3565 Foreign direct investment 7.6017 32.259 33.1972 86.2819 Gross fixed capital formation 0.6687 0.08192 0.52934 0.8162 Labour force 40.0847 18.8786 2.5464 4.8765 Electricity power 4277 94674.10000 2821 5991 Source: Authors’ Computation, (2018). 4.2. Econometric Resultsi The a priori expectations are: β1 > 0, β1 > 0, β2 > 0, β3 > 0, and β4 > 0. That is, FDI, gross fixed capital formation, labour force participation rate and electricity power consumption should be positive, which means that, increase in the rate of the exogenous variables have positive effect on economic growth in Nigeria. The first aspect of the econometric results is the presentation of summary analysis in Table 1. From Table 1, the mean value of the gross domestic product per capita is approximately 0.91 and it ranges from −15.5 (minimum) to 20.4 (maximum), with a standard deviation of 7.32. 4.2. Econometric Resultsi Table 1: Summary statistics of variables Table 2: Unit root test for stationarity Variable ADF test statistics Critical values PP test statistics Critical values @ 1% @ 5% @ 10% Integration order Remark @ 1% @ 5% @ 10% Integration order Remark Gross domestic product per capita −4.53 −3.62 −3.78 −2.458 1(0) Level Stationary −2.15 −3.26 −2.45 −2.11 1 (0) Level Stationary Foreign direct investment −3.372 −3.67 −2.45 1(0) Level Stationary −3.86 −3.26 −2.45 −2.11 1 (0) Level Stationary Gross fixed capital formation −6.50 −4.32 −3.57 −3.23 1(1) at 1st difference Stationary −9.08 −4.43 −3.44 −3.04 1 (1) at 1st difference Stationary Labour force −5.44 −4.22 −3.07 −3.48 −3.07 1(1) at 1st difference Stationary −6.05 −4.43 −3.44 −3.04 1 (1) at 1st difference Stationary Electricity power supply −6.96 −4.23 −3.57 −3.12 1(1) at 1st difference Stationary −11.8 −4.62 −3.52 3.29 1 (1) at 1st difference Stationary Source: Authors using data from world development indicators with e views 10 Table 2: Unit root test for stationarity Source: Authors using data from world development indicators with e-views 10 Table 3: Result from ARDL: Dependent Variable: Gross domestic product per capita growth rate : Result from ARDL: Dependent Variable: Gross domestic product per capita growth rate Table 3: Result from ARDL: Dependent Variable: Gross domestic product per capita growth rate Long-run dynamics Foreign direct investment Gross capital formation Labour force Electricity 0.03 (0.00) [0.02] 0.21 (0.20) (0.07) 0.84 (0.18) [0.09] 0.18 (0.98) [0.22] Short run dynamics Foreign direct investment Gross fixed capital formation Labour force Electricity L1D L2D L1D L2D L1D L2D L1D L2D 0.06 (0.00) [0.00] 0.30 (0.79) [0.01] 1.06 (0.35) [0.02] 2.20 (0.80) [0.00] 0.11 (0.49) [0.191] 0.200 (0.63) [0.19] 0.07 (0.33) [0.55] 0.06 (0.14) [0.13] The standard error and the probability values are in parenthesis () and [] respectively. ,, means that variables are statically significant at 1%, 5% and 10% respectively, while LD shows that variables are lagged and differenced. Source: Authors’ using STATA12 The standard error and the probability values are in parenthesis () and [] respectively. ,, means that variables are statically significant at 1%, 5% and 10% respectively, while LD shows that variables are lagged and differenced. 5. CONCLUSION Alvarado, R., Ponce, P. (2017), Foreign direct investment and economic growth in Latin America. Economic Analysis and Policy, 56, 176- 187. This study investigated the rate at which FDI would be encouraged through the supply of electricity power towards achieving economic growth in Nigeria for the period 1986-2017. The study engaged the trend analysis and the autoregressive distribution lag on the four selected exogenous variables which are the inflows of FDI, labour force participation rate, gross fixed capital accumulation and electricity power supply. Ibrahiem, D.M. (2015), Renewable elecricity consumption, foreign direct investment and economic growth in Egypt: An ARDL approach. Procedia Economics and Finance, 30, 313-323. Long, D.L., Ngoc, B.H., My, D.T.H. (2018), Foreign direct investment,electricity consumption and economic growth in Vietnam. International Journal of Energy Economics and Policy, 8(3), 267-274. Makiela, K., Outtara, B. (2018), Foreign direct investment and economic growth: Exploring transmission channels. Economic Modelling, 72, 296-305. From the results obtained; the trend analysis showed that over the period under study, it was observed that the selected variables do not show a constant trend with the period. From the econometric result, using the autoregressive distribution lag, result showed that in the long run, FDI has the potency of increasing economic growth by 30%. Similarly, labour force increases economic growth by 10%, while electricity consumption increases economic growth by 6%. The result of the electricity consumption is similar to the findings of Matthew et al. (2018). Matthew, A.O., Ede, C., Osabohien, R., Ejemeyovwi, J., Fasina, F.F., Akinpelumi, D. (2018), Electricity consumption and human capital development: Exploring the implications for economic growth in Nigeria. International Journal of Energy Economics and Policy, 8(6), 1-8. Osabohien, R., Osabuohien, E., Urhie, E. (2018), Food security, institutional framework, and technology: Examining the nexus in Nigeria using ARDL approach. Current Nutrition and Food Science, 14(2), 154-163. Given the result obtained, the study recommended that electricity power should be frequently supplied in order to stimulate FDI inflows which will in turn enhance economic growth in Nigeria. This can be done by the Power Holding Company of Nigeria (PHCN) by providing constant supply of electric power in the company for efficient productivity. Other studies should be carried Given the result obtained, the study recommended that electricity power should be frequently supplied in order to stimulate FDI inflows which will in turn enhance economic growth in Nigeria. Sunde, T. 4.2. Econometric Resultsi Source: Authors’ using STATA12 246 Onayemi, et al.: Foreign Direct Investment, Electricity Power Supply and Economic Growth in Nigeria Table 4: Vector error correction model Endogenous D_GDPPCGR D_ FDI D_LABF D_ GFCF D_ELECT D_psav D_cc Exogenous ECterm −0.45 (0.00) −0.37* (0.02) −0.51* (0.04) −0.51** (0.03) −0.51* (0.45) −0.38 (0.42) −0.61* (0.00) GDPPCGR (LD) 0.26* (0.00) 6.26 (0.179) −0.41* (0.00) −212.73 (0.35) 0.109 (0.38) 0.73 (0.41) −9.24* (0.00) FDI (LD) −0.04 (0.04) 0.05 (0.56) −11.83 (0.09) −9.13 (0.60) 0.002 (0.30) 0.07*** (0.90) −0.16* (0.00) LABF (LD) 4.09* (0.00) 0.06* (0.20) −0.01 (0.809) 0.00 (0.35) −2.508 (0.53) −4.08 (0.05) −2.09* (0.001) GFCF (LD) −2.07* (0.00) 0.04(0.00) 1.60** (0.08) 1.04* (0.00) −1.407 (0.56) 1.41 (0.13) 0.01* (0.00) ELECT (LD) −0.87* (0.00) 6.32* (0.00) −293** (0.02) −95.15 (0.87) −0.39* (0.08) −0.04 (0.5) 4.68 (0.03) AIC: 7.97317 HQIC: 9.21476 SBIC: 52.14865 “,,** means that variables are statically significant at 1%, 5% and 10% respectively, while LD shows that variables were lagged and differenced based on Akaike information criterion (AIC), Hannan-Quinn information criterion (HQIC) and Schwarz’s Bayesian information criterion (SBIC)” Table 4: Vector error correction model “,,** means that variables are statically significant at 1%, 5% and 10% respectively, while LD shows that variables were lagged and differenced based on Akaike information criterion (AIC), Hannan-Quinn information criterion (HQIC) and Schwarz’s Bayesian information criterion (SBIC)” “,,** means that variables are statically significant at 1%, 5% and 10% respectively, while LD shows that variab criterion (AIC), Hannan-Quinn information criterion (HQIC) and Schwarz’s Bayesian information criterion (SBIC)” out in this area looking at other factors that can stimulate FDI apart from electricity in Nigeria. out in this area looking at other factors that can stimulate FDI apart from electricity in Nigeria. electricity power has the tendency of increasing economic growth by 6%. The policy implication for this is that effort should be made by the government and the power holding company of Nigeria to ensure constant production, distribution and supply of electricity to ensure productivity in the country. REFERENCES Alege, P.O., Ogundipe, A. (2014), Foreign direct investment and economic growth in ECOWAS: A System-GMM approach. Covenant Journal of Business and Social Sciences, 5(1), 1-22. The vector error correction mechanism was used to bring the system back to equilibrium in the long-run in the event of shock in the short-run as presented in Table 4. Alam, A. (2013), Electric power consumption, foreign direct investment and economic growth: A comparative study of India and Pakistan. World Journal of Science, Technology and Sustainable Development, 10(1), 55-65. 5. CONCLUSION (2017), Foreign direct investment, exports and economic growth: ADRL and causality analysis for South Africa. Research in International Business and Finance, 41C, 434-444. This can be done by the Power Holding Company of Nigeria (PHCN) by providing constant supply of electric power in the company for efficient productivity. Other studies should be carried Zhang, C., Zhou, K., Yang, S., Shao, Z. (2017), On electricity consumption and economic growth in China. Renewable and Sustainable Energy Reviews, 76, 353-368. International Journal of Energy Economics and Policy \| Vol 10 • Issue 5 • 2020

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